Olivier Féron

The endothelial isoform of nitric oxide synthase (eNOS) modulates cardiac myocyte function and is expressed in the particulate subcellular fraction. We have previously shown that eNOS is targeted to plasmalemmal caveolae in endothelial cells. Caveolae, specialized domains of the plasma membrane, may serve to sequester signaling proteins; a family of transmembrane proteins, the caveolins, form a key structural component of these microdomains. Caveolae in cardiac tissues contain the muscle-specific isoform caveolin-3, and caveolae in endothelial cells contain the widely expressed isoform caveolin-1, which shares limited sequence identity with caveolin-3. Our immunohistochemical analyses of rat cardiac muscle used isoform-specific caveolin antibodies to reveal prominent caveolin-3 staining in myocyte sarcolemmal membranes and at intercalated discs, whereas caveolin-1 staining was prominent in the vascular endothelium. Caveolin or eNOS antibodies were utilized to immunoprecipitate cardiac myocyte or cultured aortic endothelial cell lysates, which then were analyzed in immunoblots. In endothelial cells, we found that eNOS is quantitatively immunoprecipitated by antibodies to caveolin-1. In cardiac myocyte lysates, nearly all the eNOS is immunoprecipitated instead by antibodies to caveolin-3 and, conversely, eNOS antiserum immunoprecipitated primarily caveolin-3. These studies establish expression of eNOS in cardiac myocyte caveolae and document tissue-specific and quantitative associations of eNOS with caveolin. These findings may have important implications for the regulation of eNOS by caveolin isoforms and by other signaling proteins targeted to caveolae. The endothelial isoform of nitric oxide synthase (eNOS) modulates cardiac myocyte function and is expressed in the particulate subcellular fraction. We have previously shown that eNOS is targeted to plasmalemmal caveolae in endothelial cells. Caveolae, specialized domains of the plasma membrane, may serve to sequester signaling proteins; a family of transmembrane proteins, the caveolins, form a key structural component of these microdomains. Caveolae in cardiac tissues contain the muscle-specific isoform caveolin-3, and caveolae in endothelial cells contain the widely expressed isoform caveolin-1, which shares limited sequence identity with caveolin-3. Our immunohistochemical analyses of rat cardiac muscle used isoform-specific caveolin antibodies to reveal prominent caveolin-3 staining in myocyte sarcolemmal membranes and at intercalated discs, whereas caveolin-1 staining was prominent in the vascular endothelium. Caveolin or eNOS antibodies were utilized to immunoprecipitate cardiac myocyte or cultured aortic endothelial cell lysates, which then were analyzed in immunoblots. In endothelial cells, we found that eNOS is quantitatively immunoprecipitated by antibodies to caveolin-1. In cardiac myocyte lysates, nearly all the eNOS is immunoprecipitated instead by antibodies to caveolin-3 and, conversely, eNOS antiserum immunoprecipitated primarily caveolin-3. These studies establish expression of eNOS in cardiac myocyte caveolae and document tissue-specific and quantitative associations of eNOS with caveolin. These findings may have important implications for the regulation of eNOS by caveolin isoforms and by other signaling proteins targeted to caveolae.

The endothelial nitric-oxide synthase (eNOS) is a key determinant of vascular homeostasis. Like all known nitric-oxide synthases, eNOS enzyme activity is dependent on Ca2+-calmodulin. eNOS is dynamically targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem.271, 22810–22814). We now report that in endothelial cells the interaction between eNOS and caveolin is importantly regulated by Ca2+-calmodulin. Addition of calmodulin disrupts the heteromeric complex formed between eNOS and caveolin in a Ca2+-dependent fashion. In addition, overexpression of caveolin markedly attenuates eNOS enzyme activity, but this inhibition is reversed by purified calmodulin. Caveolin overexpression does not affect the activity of the other NOS isoforms, suggesting eNOS-specific inhibition of NO synthase by caveolin. We propose a model of reciprocal regulation of eNOS in endothelial cells wherein the inhibitory eNOS-caveolin complex is disrupted by binding of Ca2+-calmodulin to eNOS, leading to enzyme activation. These findings may have broad implications for the regulation of Ca2+-dependent signal transduction in plasmalemmal caveolae. The endothelial nitric-oxide synthase (eNOS) is a key determinant of vascular homeostasis. Like all known nitric-oxide synthases, eNOS enzyme activity is dependent on Ca2+-calmodulin. eNOS is dynamically targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem.271, 22810–22814). We now report that in endothelial cells the interaction between eNOS and caveolin is importantly regulated by Ca2+-calmodulin. Addition of calmodulin disrupts the heteromeric complex formed between eNOS and caveolin in a Ca2+-dependent fashion. In addition, overexpression of caveolin markedly attenuates eNOS enzyme activity, but this inhibition is reversed by purified calmodulin. Caveolin overexpression does not affect the activity of the other NOS isoforms, suggesting eNOS-specific inhibition of NO synthase by caveolin. We propose a model of reciprocal regulation of eNOS in endothelial cells wherein the inhibitory eNOS-caveolin complex is disrupted by binding of Ca2+-calmodulin to eNOS, leading to enzyme activation. These findings may have broad implications for the regulation of Ca2+-dependent signal transduction in plasmalemmal caveolae. Nitric oxide is a ubiquitous molecule implicated in diverse biological processes and is synthesized in mammalian cells by a family of Ca2+-calmodulin-dependent nitric-oxide synthase (NOS) 1The abbreviations used are: NOS, nitric-oxide synthase; NO, nitric oxide; eNOS, endothelial isoform of NOS; iNOS, inducible isoform of NOS; nNOS, neuronal isoform of NOS; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-holamidopropyl)dimethylammonio]-1-propanesulfonic acid. enzymes (1Nathan C. Xie Q.-W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar, 2Marletta M. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (822) Google Scholar, 3Sase K. Michel T Trends Cardiovasc. Med. 1997; 7: 28-37Crossref PubMed Scopus (84) Google Scholar). Endothelium-derived nitric oxide (NO), formed by the endothelial isoform of nitric-oxide synthase (eNOS), serves as an important determinant of blood pressure and platelet aggregation. In endothelial cells, increases in intracellular Ca2+ elicited by diverse extracellular signals lead to activation of eNOS. The three known mammalian nitric-oxide synthases share similar overall Ca2+-calmodulin-dependent catalytic pathways. However, the eNOS enzyme is unique among the three known NOS isoforms in being localized to the specialized cell surface signal-transducing domains termed plasmalemmal caveolae (4Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, 5Feron O. Belhassen L. Kobzick L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Plasmalemmal caveolae are small invaginations in the plasma membrane that may serve as sites for the sequestration of signaling proteins (6Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10909-10913Crossref PubMed Scopus (588) Google Scholar,7Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z. Hermanowski-Vosatka A. Tu Y.H. Cook R.F. Sargiacomo M. J. Cell. Biol. 1995; 126: 111-126Crossref Scopus (839) Google Scholar) including receptors, G proteins, and protein kinases, as well as eNOS. The principal protein in caveolae is the integral membrane protein caveolin, an oligomeric protein that serves as a structural “scaffold” within caveolae (8Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (489) Google Scholar). eNOS can be quantitatively immunoprecipitated by antibodies directed against caveolin; conversely, eNOS antiserum also immunoprecipitates caveolin (5Feron O. Belhassen L. Kobzick L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar), although it has not yet been established whether the interaction between these two proteins is direct. Moreover, a functional role of the eNOS-caveolin association, beyond its postulated role in subcellular targeting of the enzyme, remains to be determined. We document in this report that the interaction between eNOS and caveolin may be regulated by Ca2+-calmodulin, and we show that caveolin can specifically inhibit eNOS enzyme activity. A plasmid construct encoding eNOS has been described previously (9Busconi L. Michel T. J. Biol. Chem. 1993; 268: 8410-8413Abstract Full Text PDF PubMed Google Scholar, 10Lamas S. Marsden P.A. Li G. Tempst P. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6348-6352Crossref PubMed Scopus (922) Google Scholar). cDNA clones encoding iNOS (11Xie Q.-W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.M. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Crossref PubMed Scopus (1745) Google Scholar) and nNOS (12Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2290) Google Scholar) were kindly provided by Carl Nathan (Cornell University Medical College) and Solomon Snyder (Johns Hopkins University), respectively. Caveolin-1 cDNA (13Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar) in the eukaryotic expression vector pCB-7 was obtained from Michael Lisanti (Whitehead Institute). An irrelevant plasmid encoding β-galactosidase was used as a control to maintain identical amounts of DNA in each transfection. Cultures of bovine aortic endothelial cells (studied between passages 4 and 10) and COS-7 cells were performed as described previously (9Busconi L. Michel T. J. Biol. Chem. 1993; 268: 8410-8413Abstract Full Text PDF PubMed Google Scholar, 14Lee C. Robinson L.J. Michel T. J. Biol. Chem. 1995; 270: 27403-27406Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). COS-7 cells were transfected with 10 μg of total plasmid DNA in 100-mm cell culture plates using LipofectAMINE™ (Life Technologies, Inc.) according to the manufacturer's protocol. Endothelial cells were lysed and solubilized either with: 1) a previously described Ca2+-free CHAPS buffer (5Feron O. Belhassen L. Kobzick L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar, 14Lee C. Robinson L.J. Michel T. J. Biol. Chem. 1995; 270: 27403-27406Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) supplemented with 1 mm EGTA/1 mmEDTA or 2) an otherwise identical CHAPS buffer containing 1 mm CaCl2 and no EDTA/EGTA. CHAPS-solubilized bovine aortic endothelial cell lysates were incubated either with a polyclonal caveolin antibody (Transduction Laboratories) at a final concentration of 4 μg/ml or with a previously characterized polyclonal antiserum directed against eNOS (9Busconi L. Michel T. J. Biol. Chem. 1993; 268: 8410-8413Abstract Full Text PDF PubMed Google Scholar) used at a final dilution of 1:100. Immunoprecipitated complexes were then recovered, denatured in Laemmli sample buffer, separated on 12% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (5Feron O. Belhassen L. Kobzick L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Monoclonal antibodies directed against eNOS or caveolin-1 (Transduction Laboratories) were then used to detect eNOS and caveolin-1 using chemiluminescence, as described previously (5Feron O. Belhassen L. Kobzick L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Calmodulin was detected using a previously characterized monoclonal calmodulin antibody (15Sacks D.B. Porter S.E. Ladenson J.H. McDonald J.M. Anal. Biochem. 1991; 194: 369-377Crossref PubMed Scopus (82) Google Scholar). Expression of immunoblotted proteins was quantitated by laser densitometric analysis of x-ray films following chemiluminescence detection. NO synthase activity in lysates prepared from transfected COS-7 cell was determined by measuring conversion of [3H]l-arginine to [3H]l-citrulline as described previously (9Busconi L. Michel T. J. Biol. Chem. 1993; 268: 8410-8413Abstract Full Text PDF PubMed Google Scholar,14Lee C. Robinson L.J. Michel T. J. Biol. Chem. 1995; 270: 27403-27406Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In some experiments, this NO synthase activity assay was performed in washed membrane fractions prepared from endothelial cell lysates as described previously (9Busconi L. Michel T. J. Biol. Chem. 1993; 268: 8410-8413Abstract Full Text PDF PubMed Google Scholar), except that calmodulin concentrations were varied by the addition of purified bovine brain calmodulin (Sigma) as indicated. In exploring the factors governing eNOS-caveolin association, we discovered that the co-immunoprecipitation of eNOS and caveolin was markedly attenuated when endothelial cell lysates were prepared in buffers containing excess free Ca2+ (Fig.1). Under no conditions did the presence of Ca2+ affect the recovery of caveolin or eNOS from endothelial cell lysates when the proteins were directlyimmunoprecipitated by their cognate antibody. Onlyco-immunoprecipitation was abrogated by Ca2+, as shown in Fig. 1. We next investigated the role of Ca2+ in modulating the association of eNOS and calmodulin. This is particularly important because calmodulin, a ubiquitous Ca2+-binding protein, plays a central role in nitric-oxide synthase catalysis (16Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 90: 10769-10772Crossref Scopus (416) Google Scholar). Co-immunoprecipitation experiments using eNOS antiserum to explore eNOS-calmodulin interactions in endothelial cell lysates are shown in Fig. 2. We could detect co-immunoprecipitation of eNOS and calmodulin only when free Ca2+ was present. This result is in striking contrast to the loss of eNOS-caveolin co-immunoprecipitation observed in the presence of Ca2+(Fig. 1). Taken together, these data suggest that Ca2+differentially modulates the association of eNOS with caveolinversus calmodulin in endothelial cell lysates. We next used antibodies against caveolin to co-immunoprecipitate eNOS in Ca2+-free buffers and then washed the immune complexes extensively to remove residual Ca2+ and calmodulin. Subsequent addition of either Ca2+ or calmodulin alone had no effect on the subsequent recovery of co-immunoprecipitated eNOS from the eNOS-caveolin complex (Fig. 3, upper panel). However, when Ca2+ and calmodulin were added together, eNOS was entirely lost from the caveolin immune complex. This “lost” eNOS could be completely recovered in the supernatant of the immune complex (Fig. 3, middle panel), indicating that the eNOS molecule had been released and not degraded following treatment of the caveolin-eNOS complex with Ca2+ plus calmodulin. None of these treatments affected the recovery of caveolin itself from the immune complex (Fig. 3, lower panel). The extensive washing of these immune complexes, required to remove the endogenous calmodulin, likely led to the loss of some eNOS (because the affinity of the eNOS-caveolin interaction is undoubtedly less than the affinity of the antibody for caveolin), leading to the detection of a relatively faint but highly reproducible (n = 4) signal for eNOS released from these complexes by the combination of Ca2+plus calmodulin. Diverse experimental approaches have shown that agonist activation of eNOS in endothelial cells is dependent on Ca2+-calmodulin (1Nathan C. Xie Q.-W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar, 2Marletta M. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (822) Google Scholar). The striking effects of Ca2+-calmodulin on the interactions of eNOS and caveolin therefore suggested to us that caveolin may influence eNOS enzyme activity. Indeed, caveolin has recently been shown in vitro to interact with other signaling proteins, including H-ras and G protein α subunits, preferentially associating with the “inactive” forms of these proteins (17Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar), but the cellular regulation of these interactions is less well understood. We explored the functional consequences of the interaction between eNOS and caveolin using transient transfection experiments in COS-7 cells and analyzed eNOS enzyme activity in transfected cells by assaying the conversion of [3H]l-arginine to [3H]l-citrulline in cell lysates, as shown in Fig. 4. In three separate experiments, each conducted in triplicate, we found that the co-transfection of a plasmid cDNA construct encoding caveolin with eNOS cDNA led to a marked attenuation of NOS activity (3.4 ± 0.3 versus 1.6 ± 0.1 pmol citrulline/min·mg protein in the absence or the presence of caveolin co-expression, respectively; see Fig. 4 A). There was no change in the abundance of eNOS protein associated with caveolin co-transfection, as assessed in immunoblots of these cellular lysates analyzed in each experiment (data not shown). Importantly, caveolin co-transfection failed to attenuate the enzyme activity expressed by transfected iNOS or nNOS cDNA, shown in Fig. 4 B. As for eNOS, the enzyme activity of iNOS and nNOS is calmodulin-dependent (although important differences in the Ca2+ dependence of the different NOS isoforms have been noted). In further contrast to eNOS, the other NOS isoforms are not targeted to caveolae. To explore the specificity of the inhibitory effect of caveolin co-expression on eNOS enzyme activity, we performed activity assays in the presence of varying concentrations of purified calmodulin added to washed membrane fractions prepared from transfected COS-7 cells. As shown in Fig. 4 C, in cells transfected with eNOS cDNA alone, there is a robust NOS activity even in the absence of added calmodulin (presumably due the presence of endogenous calmodulin in these membranes); enzyme activity increases only slightly with the addition of exogenous calmodulin. By contrast, caveolin co-expression markedly inhibits eNOS activity (by >90%) in the absence of added calmodulin; addition of increasing concentrations of exogenous calmodulin relieves this enzyme inhibition in a dose-dependent fashion, documenting that the caveolin inhibitory effect may be specifically overcome by purified calmodulin. Taken together, these studies suggest that the interaction between eNOS and caveolin is dynamically and specifically regulated by Ca2+-calmodulin and may serve as an important point of control in NO-dependent signaling. A direct interaction of caveolin with calmodulin appears unlikely to us because there was no influence of caveolin on the activity of other calmodulin-binding proteins (iNOS and nNOS) closely related to eNOS. This hypothesis is consistent with our failure to detect co-immunoprecipitation of calmodulin with caveolin (Fig. 2), under conditions in which eNOS was shown to associate with either one or the other protein. Furthermore, the amino acid sequence of caveolin isoforms show no obvious sequence or structural homologies to the known NOS isoforms (10Lamas S. Marsden P.A. Li G. Tempst P. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6348-6352Crossref PubMed Scopus (922) Google Scholar, 11Xie Q.-W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.M. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Crossref PubMed Scopus (1745) Google Scholar, 12Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2290) Google Scholar) nor to any known calmodulin-binding protein sequences. Caveolin can attenuate the tyrosine kinase activity of c-src, an enzyme that bears no structural homology to eNOS, and is not known to be regulated by calmodulin (13Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar). We speculate that there is a common higher order structure assumed by the inactive conformation of diverse caveolae-targeted signaling proteins that forms the basis for their common interaction with caveolin. The targeting of eNOS to caveolae is likely to facilitate the interactions of eNOS with other co-localized signaling and regulatory molecules (3Sase K. Michel T Trends Cardiovasc. Med. 1997; 7: 28-37Crossref PubMed Scopus (84) Google Scholar). Formation of an inhibitory eNOS-caveolin heteromeric complex may serve to ensure the latency of the NO signal until calcium-mobilizing extracellular stimuli destabilize this complex and activate the enzyme. This close control of enzyme activity may be particularly important for eNOS in caveolae, where calmodulin, the enzyme's key allosteric activator, also is localized (4Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar) and where even subtle increases in intracellular Ca2+ could thus lead to enzyme activation if the interactions of caveolin with eNOS were not keeping the system in check. Because nitric oxide has cytotoxic as well as signaling functions (1Nathan C. Xie Q.-W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar, 2Marletta M. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (822) Google Scholar), attenuation of basal enzyme “leakiness” by caveolin may be of particular importance. The reciprocal regulation of eNOS by caveolin and calmodulin may represent a novel mechanism for the concerted control of NO production in the vascular wall. We are grateful to John Joyal for helpful discussions and for assistance performing calmodulin immunoblots.

Hypercholesterolemia is causally associated with defects of endothelial nitric oxide (NO)-dependent vasodilation. Increased uptake of cholesterol by endothelial cells (ECs) upregulates the abundance of the structural protein caveolin-1 and impairs NO release through the stabilization of the inhibitory heterocomplex between caveolin-1 and endothelial NO synthase (eNOS). Therefore, we examined whether the hydroxy-methylglutaryl-coenzyme A reductase inhibitor atorvastatin modulates caveolin abundance, eNOS activity, and NO release through a reduction in endogenous cholesterol levels.ECs were incubated with increasing doses of atorvastatin in the absence or in the presence of human LDL cholesterol (LDL-Chol) fractions in the presence of antioxidants. Our results show that atorvastatin (10 nmol/L to 1 micromol/L) reduced caveolin-1 abundance in the absence (-75%) and in the presence (-20% to 70%) of LDL-Chol. This was paralleled by a decreased inhibitory interaction between caveolin-1 and eNOS and a restoration and/or potentiation of the basal (+45%) and agonist-stimulated (+107%) eNOS activity. These effects were observed in the absence of changes in eNOS abundance and were reversed with mevalonate. In the presence of LDL-Chol, atorvastatin also promoted the agonist-induced association of eNOS and the chaperone Hsp90, resulting in the potentiation of eNOS activation.We provide biochemical and functional evidence that atorvastatin promotes NO production by decreasing caveolin-1 expression in ECs, regardless of the level of extracellular LDL-Chol. These findings highlight the therapeutic potential of inhibiting cholesterol synthesis in peripheral cells to correct NO-dependent endothelial dysfunction associated with hypercholesterolemia and possibly other diseases.

Hypercholesterolemia is a central pathogenic factor of endothelial dysfunction caused in part by an impairment of endothelial nitric oxide (NO) production through mechanisms that remain poorly characterized. The activity of the endothelial isoform of NO synthase (eNOS) was recently shown to be modulated by its reciprocal interactions with the stimulatory Ca2+-calmodulin complex and the inhibitory protein caveolin. We examined whether hypercholesterolemia may reduce NO production through alteration of this regulatory equilibrium. Bovine aortic endothelial cells were cultured in the presence of serum obtained from normocholesterolemic (NC) or hypercholesterolemic (HC) human volunteers. Exposure of endothelial cells to the HC serum upregulated caveolin abundance without any measurable effect on eNOS protein levels. This effect of HC serum was associated with an impairment of basal NO release paralleled by an increase in inhibitory caveolin-eNOS complex formation. Similar treatment with HC serum significantly attenuated the NO production stimulated by the calcium ionophore A23187. Accordingly, higher calmodulin levels were required to disrupt the enhanced caveolin-eNOS heterocomplex from HC serum-treated cells. Finally, cell exposure to the low-density lipoprotein (LDL) fraction alone dose-dependently reproduced the inhibition of basal and stimulated NO release, as well as the upregulation of caveolin expression and its heterocomplex formation with eNOS, which were unaffected by cotreatment with antioxidants. Together, our data establish a new mechanism for the cholesterol-induced impairment of NO production through the modulation of caveolin abundance in endothelial cells, a mechanism that may participate in the pathogenesis of endothelial dysfunction and the proatherogenic effects of hypercholesterolemia.

Nitric oxide production in the vascular endothelium is promoted by diverse agonists that transiently increase intracellular Ca2+ concentration and activate the endothelial nitric-oxide synthase (eNOS), a Ca2+/calmodulin-dependent enzyme. eNOS is acylated by the fatty acids myristate and palmitate and is targeted thereby to plasmalemmal signal-transducing domains termed caveolae. eNOS enzyme activity is markedly attenuated by its interactions with caveolin, the structural scaffolding protein of caveolae. We have discovered that in living cells, the eNOS-caveolin heteromeric complex undergoes cycles of dissociation and re-association modulated by Ca2+-mobilizing agonists. Calcium ionophore A23187 and the muscarinic cholinergic agonist carbachol both promote the dissociation of eNOS from caveolin in cultured cells, associated with translocation of eNOS from caveolae. As [Ca2+]i returns to basal levels, eNOS re-associates with caveolin, and the inhibited enzyme complex is then restored to caveolae, a process accelerated by palmitoylation of the enzyme. These data establish an eNOS-caveolin regulatory cycle, wherein enzyme activation is modulated by reversible protein-protein interactions controlled by Ca2+/calmodulin and by enzyme palmitoylation. Alterations in this cycle are likely to have an important influence on nitric oxide-dependent signaling in the vascular wall. Nitric oxide production in the vascular endothelium is promoted by diverse agonists that transiently increase intracellular Ca2+ concentration and activate the endothelial nitric-oxide synthase (eNOS), a Ca2+/calmodulin-dependent enzyme. eNOS is acylated by the fatty acids myristate and palmitate and is targeted thereby to plasmalemmal signal-transducing domains termed caveolae. eNOS enzyme activity is markedly attenuated by its interactions with caveolin, the structural scaffolding protein of caveolae. We have discovered that in living cells, the eNOS-caveolin heteromeric complex undergoes cycles of dissociation and re-association modulated by Ca2+-mobilizing agonists. Calcium ionophore A23187 and the muscarinic cholinergic agonist carbachol both promote the dissociation of eNOS from caveolin in cultured cells, associated with translocation of eNOS from caveolae. As [Ca2+]i returns to basal levels, eNOS re-associates with caveolin, and the inhibited enzyme complex is then restored to caveolae, a process accelerated by palmitoylation of the enzyme. These data establish an eNOS-caveolin regulatory cycle, wherein enzyme activation is modulated by reversible protein-protein interactions controlled by Ca2+/calmodulin and by enzyme palmitoylation. Alterations in this cycle are likely to have an important influence on nitric oxide-dependent signaling in the vascular wall. The endothelial isoform of nitric-oxide synthase (eNOS) 1The abbreviations used are: eNOS: endothelial isoform of nitric-oxide synthase; palm−, palmitoylation-deficient eNOS mutant; OG, octyl glucoside; mAchR, muscarinic acetylcholine receptor; IP, immunoprecipitation(s). is robustly expressed in the vascular endothelium and in cardiac myocytes, and the cellular regulation of eNOS may represent an important determinant of cardiovascular homeostasis (reviewed in Ref. 1Sase K. Michel T. Trends Cardiovasc. Med. 1997; 7: 25-34Crossref Scopus (84) Google Scholar). In endothelial cells and in cardiac myocytes, eNOS is targeted to specialized invaginations of the plasmalemma termed caveolae (2Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). Plasmalemmal caveolae serve as sites for the sequestration of signaling proteins and are further characterized by the presence of caveolin, an intrinsic membrane protein that forms a structural “scaffold,” organizing both proteins and lipids within this key membrane organelle (3Couet J. Li S. Okamoto T. Scherer P.E. Lisanti M.P. Trends Cardiovasc. Med. 1997; 4: 103-110Crossref Scopus (111) Google Scholar, 4Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). Caveolin directly interacts with several structurally distinct signaling proteins in caveolae, including G proteins and cellular oncogenes (4Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar) as well as eNOS (2Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 5Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 6Ju H. Zou R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: 18522-18525Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 7Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 8Garcia-Cardena G. Martasek P. Masters B.S.M. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar, 9Feron O. Michel J.B. Sase K. Michel T. Biochemistry. 1998; 37: 193-200Crossref PubMed Scopus (116) Google Scholar). The activity of purified eNOS, a Ca2+/calmodulin-dependent enzyme (10Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (815) Google Scholar, 11Nathan C. Xie Q.-W. Cell. 1994; 78: 915-918Abstract Full Text PDF PubMed Scopus (2753) Google Scholar), is markedly attenuated by its interaction with caveolin (5Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 6Ju H. Zou R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: 18522-18525Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 7Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 8Garcia-Cardena G. Martasek P. Masters B.S.M. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar, 9Feron O. Michel J.B. Sase K. Michel T. Biochemistry. 1998; 37: 193-200Crossref PubMed Scopus (116) Google Scholar). We have also shown that purified Ca2+/calmodulin can overcome the inhibitory interaction between eNOS and caveolin in vitro(5Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 7Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 9Feron O. Michel J.B. Sase K. Michel T. Biochemistry. 1998; 37: 193-200Crossref PubMed Scopus (116) Google Scholar), but the relevance of these observations to the dynamic regulation of eNOS in endothelial cells is less well understood. In vascular endothelial cells and in cardiac myocytes, the cycle of eNOS activation and deactivation is intimately coupled to the changes in intracellular Ca2+ that are promoted by stimulation of diverse G protein-coupled receptors (12Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 13Vanhoutte P.M. Hypertension. 1989; 13: 658-667Crossref PubMed Scopus (610) Google Scholar). In this report, we describe a series of experiments that have explored the relationships between intracellular Ca2+ regulation and the dynamics of eNOS-caveolin interactions in living cells. We also document the role of eNOS palmitoylation in the reversible caveolar targeting of the eNOS-caveolin complex following muscarinic cholinergic stimulation. cDNA constructs encoding wild-type eNOS and the palmitoylation-deficient eNOS mutant (palm−) have previously been described (14Robinson L.J. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11776-11780Crossref PubMed Scopus (131) Google Scholar). A plasmid construct encoding the muscarinic m2 mAchR cDNA was obtained from T. I. Bonner (National Institute of Mental Health, Bethesda, MD) (15Bonner T.I. Buckley N.J. Young A.C. Brann M.R. Science. 1987; 237: 527-532Crossref PubMed Scopus (1220) Google Scholar). Bovine aortic endothelial cell and COS-7 cell culture conditions and cDNA transfection protocols were as described previously (2Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 5Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 9Feron O. Michel J.B. Sase K. Michel T. Biochemistry. 1998; 37: 193-200Crossref PubMed Scopus (116) Google Scholar). The recombinant expression of eNOS and of the m2 mAchR was verified by Western blot and specific muscarinic radioligand binding, respectively, as reported (16Feron O. Smith T.W Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Transfected COS-7 cells or endothelial cells were extensively washed with phosphate-buffered saline, harvested, pelleted by centrifugation, resuspended in OG buffer (60 mmol/liter OG, 50 mm Tris-HCl, pH 7.4, 125 mm NaCl, 2 mm dithiothreitol, 50 μm EGTA, and protease inhibitors (1 mg/ml leupeptin, 1 mg/ml pepstatin, and 1 mmphenylmethylsulfonyl fluoride)) and sonicated as described previously (2Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 5Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 9Feron O. Michel J.B. Sase K. Michel T. Biochemistry. 1998; 37: 193-200Crossref PubMed Scopus (116) Google Scholar). When cell fractionation was performed, cells were first lysed by sonication in a detergent-free hypotonic buffer and separated into soluble and particulate fractions by ultracentrifugation (100,000 × g, 1 h) (9Feron O. Michel J.B. Sase K. Michel T. Biochemistry. 1998; 37: 193-200Crossref PubMed Scopus (116) Google Scholar, 14Robinson L.J. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11776-11780Crossref PubMed Scopus (131) Google Scholar). Aliquots of cell homogenates were incubated with a rabbit caveolin-1 polyclonal antibody (lot 5, Transduction Labs) at a final concentration of 4 μg/ml; antibody titration experiments (not shown) documented that this concentration led to quantitative immunoprecipitation (IP) of caveolin from cell lysates. The isoform specificity and lack of cross-reactivity of these antibodies have been previously established (2Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 5Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 9Feron O. Michel J.B. Sase K. Michel T. Biochemistry. 1998; 37: 193-200Crossref PubMed Scopus (116) Google Scholar). After 1 h at 4 °C, protein G-Sepharose beads (50 μl of a 50% slurry) were added to the supernatant for a further 1-h incubation at 4 °C. Bound immune complexes were washed three times with OG buffer and then once with 50 mm Tris-HCl, pH 7.4, 150 mm NaCl. In some experiments, the supernatant fraction (remaining following pelleting of the protein G-Sepharose immune complexes) was precipitated by addition of trichloroacetic acid and buffered with a Tris-HCl solution, pH 7.4. The immunoprecipitates and/or the corresponding supernatant precipitates were then eluted by boiling in Laemmli sample buffer. SDS-polyacrylamide gel electrophoresis on 7.5% polyacrylamide gels, immunoblotting with eNOS or caveolin antibodies (Transduction Labs), and chemiluminescent detection protocols were performed as described previously (2Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). As shown in Fig. 1, treatment of endothelial cells with the Ca2+ ionophore A23187 leads to the dissociation of caveolin from eNOS. The fraction of eNOS that is liberated from caveolin by Ca2+ ionophore treatment can be recovered in its entirety from the supernatant fraction following the caveolin immunoprecipitation; there is no change in the recovery of caveolin in these or the other drug treatments (Fig. 1, lower panel). Following addition of the Ca2+ chelator EGTA to the ionophore-activated cells, the eNOS-caveolin complex re-forms, as shown by the quantitative immunoprecipitation of eNOS by the caveolin antibody after Ca2+ chelation (Fig. 1, lanes labeled 75). The re-formation of the inhibitory eNOS-caveolin complex may represent a mechanism whereby the enzyme can become de-activated following the return of intracellular Ca2+ to basal levels. Prolonged agonist treatment of endothelial cells leads also to the translocation of eNOS from caveolae to a soluble subcellular compartment (17Michel T. Li G.K. Busconi L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6252-6256Crossref PubMed Scopus (305) Google Scholar, 18Dudek R. Wildhirt S. Suzuki H. Winder S. Bing R.J. Pharmacology. 1995; 50: 257-260Crossref PubMed Scopus (5) Google Scholar, 19Fukuda S.-I. Takaichi S. Naritomi H. Hashimoto N. Nagata I. Nozaki K. Kikuchi H. Brain Res. 1995; 696: 30-36Crossref PubMed Scopus (19) Google Scholar), but the role of caveolin in this enzyme translocation is entirely unknown. It seems plausible that this eNOS translocation represents a means for the desensitization of the enzyme upon prolonged agonist stimulation. We therefore examined the subcellular distribution of the eNOS-caveolin complex following treatment of endothelial cells with the Ca2+ ionophore. In resting endothelial cells, nearly all (>95%) of the eNOS is in the particulate subcellular fraction, and the enzyme can be almost quantitatively immunoprecipitated by antibodies directed against caveolin (Fig. 2). Following treatment of endothelial cells with Ca2+ ionophore and the consequent dissociation of the caveolin-eNOS complex, the newly liberated caveolin-free eNOS is now detected in both particulate and soluble subcellular fractions following differential ultracentrifugation. When the Ca2+ chelator EGTA is subsequently added, eNOS located in both the particulate and soluble subcellular fractions re-associates with caveolin, and the soluble complex is then re-targeted to caveolae, as shown in Fig. 2. Taken together, these data suggest a regulatory cycle in which agonist stimulation initially leads to eNOS activation by the Ca2+/calmodulin-dependent disruption of the eNOS-caveolin heteromeric complex, followed later by enzyme translocation and re-formation of the inhibitory caveolin-eNOS heteromer; finally, this inactive complex is re-targeted to caveolae, ready for another round of agonist activation. Although the agonist-evoked modulation of caveolin-eNOS binding affinity may account for changes in the hydrophobicity of eNOS and explain, in part, the reversible translocation of eNOS to and from caveolae, such a mechanism also probably involves cycles of de-palmitoylation/re-palmitoylation of the enzyme. We have indeed previously reported that eNOS is targetedto plasmalemmal caveolae by palmitoylation (20Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar) but also that agonists promote enzyme de-palmitoylation leading to eNOS translocation from caveolae (21Robinson L.J. Busconi L. Michel T. J. Biol. Chem. 1995; 270: 995-998Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Palmitoylation is a reversible post-translational modification characteristic of diverse signaling proteins targeted to caveolae and involves the addition of the 16-carbon fatty acid palmitate to specific cysteine residues within the protein. Palmitate is attached to signaling proteins via a labile thioester bond, and for eNOS as well as some other signaling proteins, agonist activation promotes de-palmitoylation and protein translocation from caveolae (21Robinson L.J. Busconi L. Michel T. J. Biol. Chem. 1995; 270: 995-998Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 22Milligan G. Parenti M. Magee A.I. Trends Biochem. Sci. 1995; 20: 181-187Abstract Full Text PDF PubMed Scopus (284) Google Scholar, 23Wedegaertner P.B. Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 503-506Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). eNOS undergoes palmitoylation at two cysteine residues (Cys15 and Cys26); mutagenesis of these residues to alanine reduces the overall affinity of the enzyme for biological membranes and attenuates the selective targeting of eNOS to caveolae (14Robinson L.J. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11776-11780Crossref PubMed Scopus (131) Google Scholar, 20Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). However, the membrane-associated palm−mutant is still able to bind caveolin (9Feron O. Michel J.B. Sase K. Michel T. Biochemistry. 1998; 37: 193-200Crossref PubMed Scopus (116) Google Scholar), suggesting that this post-translational modification does not affect the eNOS-caveolin interaction per se, even if selective targeting of the palm− eNOS mutant to caveolae is impaired. We devised a series of experiments to explore further the relationships between eNOS palmitoylation, caveolin binding, and enzyme recycling following agonist activation and exploited a heterologous expression system in transiently transfected COS cells. To reconstitute the palm− eNOS mutant with a receptor-coupled eNOS pathway in COS cells, we co-transfected cDNA encoding either wild-type or palm− eNOS mutant, along with a cDNA construct encoding the m2 muscarinic cholinergic receptor (mAchR). We chose to study the m2 mAchR because (a) this receptor is well known to function in a physiologically important pathway that regulates eNOS activation in several cell types (12Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 13Vanhoutte P.M. Hypertension. 1989; 13: 658-667Crossref PubMed Scopus (610) Google Scholar,16Feron O. Smith T.W Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 24Kelly R.A. Balligand J.-L. Smith T.W. Circ. Res. 1996; 79: 363-380Crossref PubMed Scopus (629) Google Scholar) and (b) this recombinant receptor has been extensively validated as an activator of phospholipase C (25Zhu X. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 2827-2831Crossref Scopus (90) Google Scholar, 26Katz A. Wu D. Simon M.I. Nature. 1992; 360: 686-689Crossref PubMed Scopus (419) Google Scholar) leading to transient increases in intracellular Ca2+ levels (27Dell'Acqua M.L. Carroll R.C. Peralta E.G. J. Biol. Chem. 1993; 268: 5676-5685Abstract Full Text PDF PubMed Google Scholar, 28Ishizaka N. Noda M. Kimura Y. Hashii M. Fukuda K. Katayama M. Brown D.A. Higashida H. Pfluegers Arch. Eur. J. Physiol. 1995; 429: 426-433Crossref PubMed Scopus (13) Google Scholar). We found that the muscarinic agonist carbachol induces the dissociation of wild-type eNOS from caveolin in COS cells co-transfected with eNOS and m2 mAchR cDNAs; this process is agonist-dependent and is blocked by the cholinergic antagonist atropine (Fig. 3 A). The carbachol-induced dissociation of the eNOS-caveolin complex shows an appropriate agonist dose dependence, with an EC50 of ∼1 μm (Fig. 3, B and C). It must be emphasized that the eNOS released from the heteromeric immune complex with caveolin is recovered in the supernatant of the immunoprecipitations, indicating that the enzyme is released and not degraded following drug treatments (as shown in Figs. Figure 1, Figure 2, Figure 3). Having validated the pharmacological characteristics of this response, we next turned to a series of time course experiments exploring the agonist-regulated association and dissociation of caveolin from wild-type and palm− eNOS using co-immunoprecipitation protocols. For the wild-type enzyme, treatment of co-transfected COS cells with carbachol led to the rapid dissociation of eNOS from caveolin; the wild-type enzyme became fully dissociated from caveolin within 5 min following the addition of carbachol, after which time the proteins were found to re-associate, with the heteromeric complex entirely reformed by 7–10 min (Fig. 4 A). Although this time course parallels that seen with receptor-mediated nitric oxide release from the vascular endothelium studied in situ (29Malinski T Taha Z. Nature. 1992; 358: 676-678Crossref PubMed Scopus (1029) Google Scholar), it is difficult to directly compare the temporal sequence of these events because of the important differences in experimental conditions. For example, the NO-dependent transient hypotensive response seen following the infusion of acetylcholine in vivo is much less rapid than the cellular responses elicited by muscarinic cholinergic activation in vitro, although there is a generally similar temporal pattern (27Dell'Acqua M.L. Carroll R.C. Peralta E.G. J. Biol. Chem. 1993; 268: 5676-5685Abstract Full Text PDF PubMed Google Scholar, 28Ishizaka N. Noda M. Kimura Y. Hashii M. Fukuda K. Katayama M. Brown D.A. Higashida H. Pfluegers Arch. Eur. J. Physiol. 1995; 429: 426-433Crossref PubMed Scopus (13) Google Scholar). We next performed a series of identically configured time course experiments, now studying the palm− eNOS mutant instead of the wild-type enzyme. In contrast to the wild-type enzyme, even prior to agonist addition a significant fraction of the palm−mutant is found unassociated with caveolin, consistent with the mutant's impaired targeting to caveolae in the resting cell. However, after the addition of carbachol, the entire fraction of the palm− mutant that had been complexed to caveolin rapidly dissociates, and within 5 min, no heteromeric caveolin-palm− eNOS complex could be found, just as for the wild-type enzyme, and the entirety of the eNOS could be recovered in the post-immunoprecipitation supernatant (Fig. 4 B). However, at this point, the palm− eNOS mutant shows a dramatic divergence from the wild-type enzyme: the re-association of the caveolin-palm− complex is markedly delayed. In contrast to the wild-type eNOS, in which the entirety of the enzyme is recovered in a heteromeric complex within 7–10 min, the palm− mutant shows only a sluggish re-formation of the heteromer, a process delayed in its onset and barely at completion a full hour after the addition of drug. There is no substantive change in agonist-mediated Ca2+ transients (assessed using fura-2 epifluoresence) between COS cells transfected with the wild-typeversus palm− mutant eNOS (data not shown). We therefore interpret this marked divergence in the kinetics of re-formation of the heteromeric palm− eNOS-caveolin complex to reflect the essential role for re-palmitoylation in facilitating the re-targeting of eNOS to caveolae, thereby facilitating the protein-protein interactions between eNOS and caveolin that permit the heteromeric complex to re-form. In summary, we have shown for the first time in cells that the eNOS-caveolin complex can be rapidly disrupted and subsequently restored following agonist activation, associated with the reversible translocation of the enzyme. We therefore postulate the existence of a dynamic cycle of eNOS-caveolin interactions initiated by agonist-promoted increases in [Ca2+]i that disrupt the caveolin-eNOS complex, leading to enzyme activation. Following more prolonged agonist stimulation, eNOS is de-palmitoylated (21Robinson L.J. Busconi L. Michel T. J. Biol. Chem. 1995; 270: 995-998Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar) and is no longer selectively sequestered in caveolae. The translocated enzyme probably partitions both into noncaveolar plasma membrane and into more hydrophilic regions of the cell, the precise identity of which has not been established. Subsequent to the enzyme's translocation into this more “soluble” cell compartment and following the decline in [Ca2+]i to basal levels, caveolin may once again interact with eNOS, leading to enzyme inhibition. The re-association of eNOS with caveolin may occur either at the membrane level or in the cytosol through which caveolin complexes may shuttle between caveolae and an internalized caveolar vesicle/trans-Golgi network (3Couet J. Li S. Okamoto T. Scherer P.E. Lisanti M.P. Trends Cardiovasc. Med. 1997; 4: 103-110Crossref Scopus (111) Google Scholar, 30Kurzchalia T.V. Dupree P. Parton R.G. Kellner R. Virta H. Lehnert M. Simons K. J. Cell Biol. 1992; 118: 1003-1014Crossref PubMed Scopus (464) Google Scholar, 31Conrad P.A. Smart E. Ying Y.-S. Anderson R.G.W. Bloom G. J. Cell Biol. 1995; 131: 1421-1433Crossref PubMed Scopus (218) Google Scholar). The re-association and re-targeting of the heteromeric eNOS-caveolin complex appears to be accelerated (or stabilized) by enzyme palmitoylation, which takes place either within caveolae or en route to this organelle. The re-palmitoylation of eNOS facilitates rapid and efficient stabilization of the inactivated enzyme in the caveolar environment ready for another cycle of stimulation by agonists. This dynamic cycle of eNOS intracellular regulation adds another level of complexity to the post-translational life history of this vital signaling protein and may represent an important control point for the modulation of NO-dependent signaling in the vascular wall.

<h2>Summary</h2> Tumor acidosis promotes disease progression through a stimulation of fatty acid (FA) metabolism in cancer cells. Instead of blocking the use of FAs by acidic cancer cells, we examined whether excess uptake of specific FAs could lead to antitumor effects. We found that n-3 but also remarkably n-6 polyunsaturated FA (PUFA) selectively induced ferroptosis in cancer cells under ambient acidosis. Upon exceeding buffering capacity of triglyceride storage into lipid droplets, n-3 and n-6 PUFA peroxidation led to cytotoxic effects in proportion to the number of double bonds and even more so in the presence of diacylglycerol acyltransferase inhibitors (DGATi). Finally, an n-3 long-chain PUFA-rich diet significantly delayed mouse tumor growth when compared with a monounsaturated FA-rich diet, an effect further accentuated by administration of DGATi or ferroptosis inducers. These data point out dietary PUFA as a selective adjuvant antitumor modality that may efficiently complement pharmacological approaches.

Abstract Acidosis, a common characteristic of the tumor microenvironment, is associated with alterations in metabolic preferences of cancer cells and progression of the disease. Here we identify the TGF-β2 isoform at the interface between these observations. We document that acidic pH promotes autocrine TGF-β2 signaling, which in turn favors the formation of lipid droplets (LD) that represent energy stores readily available to support anoikis resistance and cancer cell invasiveness. We find that, in cancer cells of various origins, acidosis-induced TGF-β2 activation promotes both partial epithelial-to-mesenchymal transition (EMT) and fatty acid metabolism, the latter supporting Smad2 acetylation. We show that upon TGF-β2 stimulation, PKC-zeta-mediated translocation of CD36 facilitates the uptake of fatty acids that are either stored as triglycerides in LD through DGAT1 or oxidized to generate ATP to fulfill immediate cellular needs. We also address how, by preventing fatty acid mobilization from LD, distant metastatic spreading may be inhibited.

Abstract Angiogenic endothelial cells and tumour cells can survive under hypoxic conditions and even proliferate and migrate in a low‐oxygen environment. In both cell types, high rates of glycolysis (i.e. conversion of glucose to lactate) and glutaminolysis provide most of the required biosynthetic intermediates and energy to support sprouting and cell division without coupling to oxidative phosphorylation. This metabolic preference is observed under hypoxic conditions, but also in situations in which oxygen is present. In the case of tumour cells, this is known as the Warburg effect and is largely governed by oncogenes. In endothelial cells lining tumour blood vessels, the option of respiration‐independent metabolism allows the neovasculature to resist the hostile environment of fluctuating oxygen tension (ranging from severe hypoxia to quasi‐normal levels of oxygen). In addition, accumulation in tumours of lactate, the end‐product of glycolysis, largely contributes to the angiogenic phenotype through inhibition of prolyl hydroxylase 2 and the activation of HIF 1α and NF κB. Activation of the latter in a hypoxia‐independent manner leads to the increased production of interleukin‐8/ CXCL 8 which drives the autocrine stimulation of endothelial cell proliferation and maturation of neovessels. In conclusion, the addiction of proliferating endothelial cells for glucose and glutamine as fuels and the driving force of lactate to promote angiogenesis provide novel potential treatment options without the disadvantages of conventional anti‐angiogenic drugs.

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca²⁺-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997)<i>J. Biol. Chem.</i> 272, 15583–15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82–101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in <i>Escherichia coli</i>. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca²⁺-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

Contrary to beta(1)- and beta(2)-adrenoceptors, beta(3)-adrenoceptors mediate a negative inotropic effect in human ventricular muscle. To assess their functional role in heart failure, our purpose was to compare the expression and contractile effect of beta(3)-adrenoceptors in nonfailing and failing human hearts.We analyzed left ventricular samples from 29 failing (16 ischemic and 13 dilated cardiomyopathic) hearts (ejection fraction 18.6+/-2%) and 25 nonfailing (including 12 innervated) explanted hearts (ejection fraction 64.2+/-3%). beta(3)-Adrenoceptor proteins were identified by immunohistochemistry in ventricular cardiomyocytes from nonfailing and failing hearts. Contrary to beta(1)-adrenoceptor mRNA, Western blot analysis of beta(3)-adrenoceptor proteins showed a 2- to 3-fold increase in failing compared with nonfailing hearts. A similar increase was observed for Galpha(i-2) proteins that couple beta(3)-adrenoceptors to their negative inotropic effect. Contractile tension was measured in electrically stimulated myocardial samples ex vivo. In failing hearts, the positive inotropic effect of the nonspecific amine isoprenaline was reduced by 75% compared with that observed in nonfailing hearts. By contrast, the negative inotropic effect of beta(3)-preferential agonists was only mildly reduced.Opposite changes occur in beta(1)- and beta(3)-adrenoceptor abundance in the failing left ventricle, with an imbalance between their inotropic influences that may underlie the functional degradation of the human failing heart.

In cardiac myocytes, as well as specialized conduction and pacemaker cells, agonist binding to muscarinic acetylcholine receptors (mAchRs) results in the activation of several signal transduction cascades including the endothelial isoform of nitric-oxide synthase (eNOS) expressed in these cells. Recent evidence indicates that, as in endothelial cells, eNOS in cardiac myocytes is localized to plasmalemma caveolae, specialized lipid microdomains that contain caveolin-3, a muscle-specific isoform of the scaffolding protein caveolin. In this report, using a detergent-free method for isolation of sarcolemmal caveolae from primary cultures of adult rat ventricular myocytes, we demonstrated that the muscarinic cholinergic agonist carbachol promotes the translocation of mAchR into low density gradient fractions containing most myocyte caveolin-3 and eNOS. Following isopycnic centrifugation, the different gradient fractions were exposed to the muscarinic radioligand [3H]quinuclidinyl benzilate (QNB), and binding was determined after membrane filtration or immunoprecipitation. In a direct radioligand binding assay, we found that [3H]QNB binding can be detected in caveolin-enriched fractions only when cardiac myocytes have been previously exposed to carbachol. Furthermore, most of this [3H]QNB binding can be specifically immunoprecipitated by an antibody to the m2 mAchR, indicating that the translocation of this receptor subtype is responsible for the [3H]QNB binding detected in the low density fractions. Moreover, the [3H]QNB binding could be quantitatively immunoprecipitated from the light membrane fractions with a caveolin-3 antibody (but not a control IgG1 antibody), confirming that the m2 mAchR is targeted to caveolae after carbachol treatment. Importantly, atropine, a muscarinic cholinergic antagonist, did not induce translocation of m2 mAchR to caveolae and prevented receptor translocation in response to the agonist carbachol. Thus, dynamic targeting of sarcolemmal m2 mAchR to caveolae following agonist binding may be essential to initiate specific downstream signaling cascades in these cells. In cardiac myocytes, as well as specialized conduction and pacemaker cells, agonist binding to muscarinic acetylcholine receptors (mAchRs) results in the activation of several signal transduction cascades including the endothelial isoform of nitric-oxide synthase (eNOS) expressed in these cells. Recent evidence indicates that, as in endothelial cells, eNOS in cardiac myocytes is localized to plasmalemma caveolae, specialized lipid microdomains that contain caveolin-3, a muscle-specific isoform of the scaffolding protein caveolin. In this report, using a detergent-free method for isolation of sarcolemmal caveolae from primary cultures of adult rat ventricular myocytes, we demonstrated that the muscarinic cholinergic agonist carbachol promotes the translocation of mAchR into low density gradient fractions containing most myocyte caveolin-3 and eNOS. Following isopycnic centrifugation, the different gradient fractions were exposed to the muscarinic radioligand [3H]quinuclidinyl benzilate (QNB), and binding was determined after membrane filtration or immunoprecipitation. In a direct radioligand binding assay, we found that [3H]QNB binding can be detected in caveolin-enriched fractions only when cardiac myocytes have been previously exposed to carbachol. Furthermore, most of this [3H]QNB binding can be specifically immunoprecipitated by an antibody to the m2 mAchR, indicating that the translocation of this receptor subtype is responsible for the [3H]QNB binding detected in the low density fractions. Moreover, the [3H]QNB binding could be quantitatively immunoprecipitated from the light membrane fractions with a caveolin-3 antibody (but not a control IgG1 antibody), confirming that the m2 mAchR is targeted to caveolae after carbachol treatment. Importantly, atropine, a muscarinic cholinergic antagonist, did not induce translocation of m2 mAchR to caveolae and prevented receptor translocation in response to the agonist carbachol. Thus, dynamic targeting of sarcolemmal m2 mAchR to caveolae following agonist binding may be essential to initiate specific downstream signaling cascades in these cells. The activation of a muscarinic acetylcholine receptor (mAChR) 1The abbreviations used are: mAchR, muscarinic acetylcholine receptor(s); GPR, G protein-coupled receptor; β-AR, β-adrenergic receptor; eNOS, endothelial isoform of nitric-oxide synthase; NO, nitric oxide; ARVM, adult rat ventricular myocytes; QNB, 1-quinuclidinyl benzilate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Mes, 4-morpholineethanesulfonic acid; MBS, Mes-buffered saline; PVDF, polyvinylidene difluoride; TBST, Tris-buffered saline with Tween 20; PAGE, polyacrylamide gel electrophoresis. triggers a number of signal transduction pathways that, in the heart, may elicit both positively and negatively inotropic and chronotropic effects (1Korth M. Kühlkamp V. Pfluegers Arch. Eur. J. Physiol. 1985; 403: 266-272Crossref PubMed Scopus (47) Google Scholar, 2Eglen R.M. Montgomery W.W. Whiting R.L. J. Pharmacol. Exp. Ther. 1988; 247: 911-917PubMed Google Scholar). Recent studies have shown that, of the five mAchR subtypes identified to date, only the m1 and m2 subtypes are expressed in adult mammalian cardiac tissues (3Gallo M.P. Alloatti G. Eva C. Oberto A. Levi R.C. J. Physiol. 1993; 471: 41-60Crossref PubMed Scopus (73) Google Scholar, 4Sharma V.K. Colecraft H.M. Wang D.X. Levey A.I. Grigorenko E.V. Yeh H.H. Sheu S.-S. Circ. Res. 1996; 79: 86-93Crossref PubMed Scopus (67) Google Scholar). According to these reports, the m2 mAchR, which is expressed at a much higher level than the m1 mAchR, triggers the inhibitory response while m1 receptor activation elicits, when stimulated by higher concentrations of agonist, a compensatory excitatory effect on heart function. Therefore, distinct downstream signaling cascades must be involved following m1 and m2 mAchR activation. Both m1 and m2 receptor subtypes also have been reported to undergo translocation into specific subcompartments derived from the plasma membrane (5Harden T.K. Petch L.A. Traynelis S.F. Waldo G.L. J. Biol. Chem. 1985; 260: 13060-13066Abstract Full Text PDF PubMed Google Scholar, 6Raposo G. Dunia I. Marullo S. André Guillet J.-G. Strosberg A.D. Benedetti E.L. Hoebeke J. Biol. Cell. 1987; 60: 117-124Crossref PubMed Scopus (72) Google Scholar, 7Ho A.K.S. Zhang Y.-J. Duffield R. Zheng G.-M. Cell. Signalling. 1991; 3: 587-598Crossref PubMed Scopus (6) Google Scholar, 8Svoboda P. Milligan G. Eur. J. Biochem. 1994; 224: 455-462Crossref PubMed Scopus (38) Google Scholar, 9Goldman P.S. Schlador M.L. Shapiro R.A. Nathanson N.M. J. Biol. Chem. 1996; 271: 4215-4222Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 10Tolbert L.M. Lameh J. J. Biol. Chem. 1996; 271: 17335-17342Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), a characteristic of many G protein-coupled receptors (GPR) following agonist binding. To date, two major pathways for GPR clustering and sequestration have been reported, which involve plasma membrane modifications that lead to the formation of either clathrin-coated or non-coated vesicles (11Sandvig K. van Deurs B. Trends Cell Biol. 1994; 4: 275-277Abstract Full Text PDF PubMed Scopus (73) Google Scholar). While the human muscarinic cholinergic receptor Hm1 has been shown to internalize via clathrin-coated vesicles (10Tolbert L.M. Lameh J. J. Biol. Chem. 1996; 271: 17335-17342Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), mAchR have also been shown to be internalized through non-clathrin-coated vesicles in human fibroblasts, although the identity of these vesicular structures has not been defined (6Raposo G. Dunia I. Marullo S. André Guillet J.-G. Strosberg A.D. Benedetti E.L. Hoebeke J. Biol. Cell. 1987; 60: 117-124Crossref PubMed Scopus (72) Google Scholar). Recently, a clathrin-independent sequestration pathway has received attention with the characterization of a population of plasmalemmal vesicles termed caveolae. Caveolae are small flask-shaped invaginations of the plasma membrane characterized by high levels of cholesterol and glycosphingolipids (12Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar), the principal scaffolding protein of which are the caveolins, 20–24 kDa integral membrane proteins that undergo homo-oligomerization (13Monier S. Parton R.G. Vogel F. Behlke J. Henske A. Kurzchalia T.V. Mol. Biol. Cell. 1995; 6: 911-927Crossref PubMed Scopus (401) Google Scholar). These specialized lipid microdomains have been shown to play a role in the compartmentation of a number of plasma membrane-linked signal transduction pathways, including those mediated by receptor tyrosine kinases (14Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 15Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). In addition, a recent report by Parton et al. (16Parton R.G. Way M. Zorzi N. Stang E. J. Cell Biol. 1997; 136: 137-154Crossref PubMed Scopus (299) Google Scholar) provides additional evidence that coalescence and fission of caveolae may be essential for the development of the T-tubular system that is essential for normal intracellular calcium homeostasis and excitation-contraction coupling in cardiac and skeletal muscle. The specific mechanisms involved in receptor sequestration may differ among distinct cellular phenotypes. For example, several reports have proposed the involvement of clathrin-coated pits in the mechanism of internalization of β-adrenergic receptors (β-AR) (17Muntz K.H. Trends Cell Biol. 1994; 6: 356Google Scholar), and yet a recent report indicated that in epidermoid A431 cells, β-AR are clustered within caveolae in response to agonist stimulation (18Dupree P. Parton R.G. Raposo G. Kurzhalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar). The recent development of antibodies directed against different tissue-specific isoforms of caveolin has permitted a better characterization of caveolar microdomains. Using these antibodies in immunoprecipitation experiments, we have recently shown that eNOS, the constitutively expressed isoform of nitric-oxide synthase in cardiac myocytes, is targeted to sarcolemmal caveolae in cardiac myocytes and endothelial cells (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). Interestingly, reports from our laboratory and by others have shown that the generation of nitric oxide (NO) is an obligate intermediate step in the signal transduction cascade involved in the m2 mAchR-mediated inhibitory responses of the heart, particularly following β-adrenergic stimulation (20Balligand J.-L. Kelly R.A. Marsden P.A. Smith T.W. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 347-351Crossref PubMed Scopus (632) Google Scholar, 21Balligand J.-L. Kobzik L. Han X. Kaye D.M. Belhassen L. O'Hara D.S. Kelly R.A. Smith T.W. Michel T. J. Biol. Chem. 1995; 270: 14582-14586Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 22Han X. Shimoni Y. Giles W.R. J. Gen. Physiol. 1995; 106: 45-65Crossref PubMed Scopus (144) Google Scholar, 23Han X. Kobzik L. Balligand J.-L. Kelly R.A. Smith T.W. Circ. Res. 1996; 78: 998-1008Crossref PubMed Scopus (116) Google Scholar). Caveolae may, therefore, constitute the structural framework within which this signaling cascade operates. Thus, the dynamic targeting of agonist-stimulated muscarinic cholinergic receptors to caveolae in cardiac myocytes could facilitate the activation of eNOS, which we have shown to be quantitatively and specifically associated with caveolin-3, the muscle-specific isoform of caveolin (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 24Way M. Parton R.G. FEBS Lett. 1996; 378: 108-112Crossref PubMed Scopus (59) Google Scholar, 25Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar, 26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar). The co-localization in caveolae of this Ca2+/calmodulin-dependent NOS isoform with proteins known to regulate Ca2+ homeostasis, including a Ca2+-ATPase and InsP3 receptor-like proteins (27Fujimoto T. J. Cell Biol. 1993; 120: 1147-1157Crossref PubMed Scopus (360) Google Scholar), as well as with heterotrimeric G proteins (12Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, 26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar, 28Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), suggest that these plasmalemmal microdomains may constitute a platform for the recruitment and regulation of the signaling proteins involved in the NO-mediated muscarinic cholinergic pathway in heart muscle. In this report, we describe experiments designed to explore the hypothesis that m2 mAchR are targeted to plasmalemmal caveolae upon agonist stimulation in adult rat ventricular myocytes. Using a detergent-free method for caveolae isolation followed by isopycnic centrifugation, we provide evidence that the m2 mAchR, after agonist stimulation, co-purifies with caveolin-3 and eNOS. Furthermore, we show that the radioliganded m2 mAchR can be specifically immunoprecipitated from these caveolin-enriched fractions using antibodies directed against caveolin-3. Purified adult rat ventricular myocyte (ARVM) primary cultures were plated on laminin and cultured for 24 h in a defined medium as reported previously (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). ARVM were incubated either with or without carbachol (100 μm, 15 min), lysed, and fractionated on sucrose gradients; in some experiments (see “Results and Discussion”), myocytes were preincubated in the presence of 1 μm atropine (15 min) or 5 mm acetic acid (5 min) before carbachol treatment. Before harvesting, cells were washed extensively with ice-cold phosphate-buffered saline to ensure complete removal of drugs. This was validated by the lack of any detectable difference in specific [3H]quinuclidinyl benzylate (QNB) binding levels (see below) in total lysates of ARVM, whether treated or not with a muscarinic agonist or antagonist. ARVM were scraped in a freshly prepared solution of 200 mmNa2CO3 and lysed by sonication (three 5-s bursts, minimal output power) using a Branson sonifier 450 (Branson Ultrasonic Corp., Danbury, CT), according to a method modified from Song et al. (26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar). The cell lysate was then adjusted to 45% sucrose by addition of a sucrose stock solution prepared in MBS (25 mm Mes, pH 6.5, 150 mm NaCl) and placed at the bottom of a 5–15-25–35% discontinuous sucrose gradient (in MBS containing 100 mm Na2CO3) for an overnight ultracentrifugation (150,000 g). The gradient was fractionated in nine fractions corresponding to sucrose concentrations 5, 15, 25, 35, and 45%, and the four intermediate interfaces. Each fraction was neutralized with HCl before further analysis. Heat-denatured proteins were loaded and separated on 12% SDS-polyacrylamide gels (Mini Protean II, Bio-Rad) and transferred to a PVDF membrane (Bio-Rad). After blocking with 5% non-fat dry milk in Tris-buffered saline with 0.1% (v/v) Tween 20 (TBST), membranes were incubated with the specified primary antibody (Transduction Labs) for 1 h in TBST containing 1% non-fat dry milk. After six washes (10 min each), the membranes were incubated for 1 h with a horseradish peroxidase-labeled goat anti-mouse immunoglobulin secondary antibody (Jackson ImmunoResearch Labs) at a 1:10,000 dilution in TBST containing 1% non-fat dry milk. After five additional washes, the membranes were rinsed once in TBST, incubated with a chemiluminescent reagent according to the manufacturer protocols (Renaissance, NEN Life Science Products), and exposed to x-ray film. Mannosidase II activity was determined by hydrolysis ofp-nitrophenyl-α-d-mannopyranoside (Sigma) with volumes reduced to facilitate the assay in 96-well plates, as described previously (29Denker S.P. McCaffery J.M. Palade G.E. Insel P.A. Farquhar M.G. J. Cell Biol. 1996; 133: 1027-1040Crossref PubMed Scopus (116) Google Scholar). After incubation at 37 °C for 1 h followed by quenching with 100 mm NaOH, absorbance was measured at 405 nm using a Microplate Reader (SLT Lab Instruments). [3H]ouabain (NEN Life Science Products) binding was determined as described (30Feron O. Wibo M. Christen M.-O. Godfraind T. Br. J. Pharmacol. 1992; 105: 480-484Crossref PubMed Scopus (23) Google Scholar); nonspecific binding was estimated in the presence of 1 mm ouabain (Sigma). Membranes were collected on Whatman GF/B fiber filters, washed twice with chilled Tris-HCl, pH 7.4, and the radioactivity was determined in a scintillation counter. Protein amounts, mannosidase activity and [3H]ouabain binding are expressed as percent of total protein, of total activity, and of total specific [3H]ouabain binding, respectively. The gradient fractions (buffered at pH 7.4) were adjusted to 5 mm MgCl2, 1 mm EGTA, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mmphenylmethylsulfonyl fluoride, and aliquots of the different fractions were incubated with 2 nm [3H]QNB (NEN Life Science Products) at 30 °C for 60 min; nonspecific binding was determined in the presence of 1 μm atropine. Assays were performed in triplicate and terminated by rapid filtration on Whatman GF/B filters or followed by an immunoprecipitation protocol (adapted from those in Refs. 31Luthin G.R. Harkness J. Artymyshyn R.P. Wolfe B.B. Mol. Pharmacol. 1988; 34: 327-333PubMed Google Scholar and 32Levey A.I. Kitt C.A. Simonds W.F. Price D.L. Brann M.R. J. Neurosci. 1991; 11: 3218-3226Crossref PubMed Google Scholar). For these immunoprecipitation experiments, the binding buffer also contained 1% digitonin and 0.2% CHAPS; nonspecific [3H]QNB binding was determined by performing all the steps of the immunoprecipitation protocol in the presence of 1 μm atropine. After sequential incubations of the [3H]QNB-bound receptors with an antibody directed against the m2 mAchR (4 h, 4 °C, Chemicon) and agarose-conjugated protein-G (1–2 h, 4 °C), immunocomplexes were precipitated by centrifugation, washed four times with 25 mm Mes buffer containing 1% digitonin and 0.2% CHAPS, and resuspended in 1% SDS. A similar protocol was used for the immunoprecipitation with the caveolin-3 antibody (Transduction Labs) except that binding and washing buffers did not contain digitonin. The isoform specificity and lack of cross-reactivity of the caveolin (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 24Way M. Parton R.G. FEBS Lett. 1996; 378: 108-112Crossref PubMed Scopus (59) Google Scholar, 25Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar, 26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar) and muscarinic (32Levey A.I. Kitt C.A. Simonds W.F. Price D.L. Brann M.R. J. Neurosci. 1991; 11: 3218-3226Crossref PubMed Google Scholar) antibodies have been established previously. Moreover, the specificity of the caveolin-3 immunoprecipitation was established by comparing the [3H]QNB binding detected from immunoprecipitates performed using a non-immune idiotype-specific purified mouse myeloma IgG1 (Zymed). In all the experiments described here above, samples were transferred in counting vials containing 10 ml of scintillant, and the radioactivity was determined in a liquid scintillation counter. Caveolin-enriched membranes have been historically isolated on the basis of their insolubility in Triton due to their specialized lipid composition (12Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, 33Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (495) Google Scholar). However, it has been reported recently that the inclusion of detergent can result in the loss of proteins normally associated with caveolae (26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar, 34Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar), as well as in apparent redistribution of mitochondrial and endoplasmic reticulum proteins into caveolae (35Kurzchalia T.V. Hartmann E. Dupree P. Trends Cell Biol. 1995; 5: 187-189Abstract Full Text PDF PubMed Scopus (75) Google Scholar). Therefore, for isolating caveolae from cardiac myocytes, we have optimized a detergent-free purification method based on the resistance to extraction of caveolin complexes by sodium carbonate and on the fine disruption of cellular membrane by sonication (18Dupree P. Parton R.G. Raposo G. Kurzhalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar, 26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar). Thus, after homogenization of ARVM in a sodium carbonate buffer, the lysate was adjusted to 45% sucrose and placed at the bottom of a 5–15-25–35% discontinuous gradient for an overnight ultracentrifugation. Aliquots of the different fractions collected were separated by SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with anti-caveolin-3 or anti-eNOS antibodies. The immunoblots presented in Fig. 1 A show that the majority of caveolin-3 and eNOS in ventricular myocytes appears in fractions 2 and 3, which correspond to the 5–15% sucrose equilibrium densities. This co-purification of eNOS and caveolin-3 is in agreement with our previous data on the co-immunoprecipitation of these two proteins from CHAPS-solubilized cardiac myocyte lysates (19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar) and on the co-isolation of eNOS and caveolin-1 in endothelial cells (36Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). The gradient fractions were also analyzed for their protein content as well as for the presence of mannosidase II, as a Golgi marker (29Denker S.P. McCaffery J.M. Palade G.E. Insel P.A. Farquhar M.G. J. Cell Biol. 1996; 133: 1027-1040Crossref PubMed Scopus (116) Google Scholar), and for the level of specific [3H]oubain binding (30Feron O. Wibo M. Christen M.-O. Godfraind T. Br. J. Pharmacol. 1992; 105: 480-484Crossref PubMed Scopus (23) Google Scholar), as a specific marker of (Na+, K+)-ATPase, a relatively evenly distributed enzyme at the sarcolemmal surface of cardiac myocytes. As shown by the pattern of distribution of these markers across the gradient (Fig. 1 B), the bulk of cellular protein that equilibrates at the high sucrose density (fractions 7–9), corresponds to Golgi and sarcolemmal membranes. The small amount of caveolin-3 and eNOS associated with these high density fractions (Fig.1 A) is probably due to some association of both proteins with the trans-Golgi network (37Belhassen L. Feron O. Kaye D.M. Michel T. Smith T.W. Kelly R.A. J. Biol. Chem. 1997; 272: 11198-11204Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) or to incomplete cell lysis prior to sucrose density gradient centrifugation. We next explored the effects of carbachol, a muscarinic cholinergic agonist, on the distribution of mAchR using the centrifugation protocol described above, to determine if a change in receptor subcellular localization was induced by agonist binding. The following experiments were performed on primary cultures of ARVM exposed to 100 μm carbachol for 15 min. After extensive washing, myocytes were lysed and submitted to isopycnic centrifugation on a sucrose gradient. Aliquots of the different fractions obtained were incubated with [3H]QNB, a muscarinic antagonist radioligand, at 30 °C for 60 min. In a first set of experiments, membranes were directly filtered on Whatman GF/B glass filters. As shown in Fig. 2 A, in lysates prepared from untreated myocytes, the binding of [3H]QNB is only detected in the high-density fractions. In contrast, following carbachol treatment, 27.4 ± 3.3% of the [3H]QNB binding (n = 6) can be recovered in the low-density fractions 2 and 3, which correspond to the caveolin-enriched membranes (Fig. 1 A). The rest of the [3H]QNB binding remains concentrated in fractions 7–9 and likely represents binding to non-caveolar sarcolemmal muscarinic receptors.Figure 2Agonist-induced translocation of muscarinic receptors in cardiac myocytes. The presence of muscarinic receptors in each fraction is determined by the amount of specific [3H]QNB binding detected by harvesting membranes on Whatman glass filters (A) or by immunoprecipitation with anti-m2 antibodies (B, C); control and carbachol (100 μm, 15 min) conditions are symbolized by open (○) and closed (•, ▴, ▪) symbols, respectively. In panel C, the incubation in presence of carbachol (100 μm, 15 min) was preceded by a 15-min incubation with atropine 1 μm(▴) or a 5-min incubation with 5 mm acetic acid, pH 5.0 (▪). For each condition, nonspecific binding was determined in the presence of 1 μm atropine. The data are expressed as the percent of total specific [3H]QNB binding and are representative of those obtained in three to six experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In a second series of experiments, we used a complementary approach to explore the carbachol-induced shift in [3H]QNB binding. The different fractions collected after isopycnic centrifugation were immunoprecipitated using an m2 mAchR antibody, and the amount of specific [3H]QNB binding in each immunoprecipitate was determined. As shown in Fig. 2 B, the pattern of distribution of m2 mAchR is similar to that directly deduced from the [3H]QNB binding to each fraction (Fig.2 A) with, however, a more accentuated shift of [3H]QNB bound m2 mAchR toward the low density fractions when myocytes have been exposed to carbachol. 34.6 ± 3.9% (n = 6) of the [3H]QNB binding is now detected in the caveolar fractions 2 and 3. Importantly, when ARVM are preincubated with 1 μm atropine before carbachol treatment (Fig. 2 C), the enrichment of the m2 mAchR in fractions 2 and 3 is no longer observed, thereby indicating the specificity of the agonist-mediated clustering process. Interestingly, in a previous study, Raposo et al. (6Raposo G. Dunia I. Marullo S. André Guillet J.-G. Strosberg A.D. Benedetti E.L. Hoebeke J. Biol. Cell. 1987; 60: 117-124Crossref PubMed Scopus (72) Google Scholar) reported that treatment of human fibroblasts, either with a muscarinic cholinergic agonist or with the muscarinic cholinergic antagonist atropine, triggered the redistribution of the Hm1 mAchR into specific regions of the plasma membrane, presumably caveolae, and that only longer exposures with the agonist lead to the receptor endocytosis. Furthermore, Tolbert and Lameh (10Tolbert L.M. Lameh J. J. Biol. Chem. 1996; 271: 17335-17342Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) showed, using immunofluorescence confocal microscopy, that the Hm1 mAchR, after agonist stimulation, are internalized via clathrin-coated vesicles in HEK cells stably transfected with the epitope-tagged Hm1 receptors. Together with the data reported here, these results suggest that the extent and the mode of receptor compartmentation in response to agonist stimulation may be governed by both the receptor subtype and the cell type in which it is expressed. In our experimental conditions, it is unlikely that clustering of the m2 mAchR into coated pits can explain the shift in mAchR into lower density sucrose gradients. Indeed, evidence from the literature indicates that the equilibrium density of clathrin-coated pits is higher than that of caveolae (38Woodward M.P. Roth T.F. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4394-4398Crossref PubMed Scopus (90) Google Scholar) and therefore would not match the pattern of distribution of carbachol-stimulated muscarinic receptors obtained in Fig. 2, A and B. Furthermore, when myocytes are pre-incubated with 5 mm acetic acid, pH 5.0, a treatment known to disrupt clathrin-mediated endocytosis (39Sandvig K. Olsnes S. Petersen O.W. Deurs B.V. J. Cell Biol. 1987; 105: 679-689Crossref PubMed Scopus (252) Google Scholar), a movement of m2 mAchR into caveolin-enriched fractions is still detected (Fig. 2 C). To confirm the dynamic targeting of muscarinic receptors to caveolae in cardiac myocytes, we used a caveolin-3 antibody to immunoprecipitate caveolar membranes and identify the m2 mAchR by radioligand binding assays. In these studies, cardiac myocytes preincubated either with or without carbachol were lysed and fractionated on sucrose gradients, and the fractions corresponding to caveolae were pooled and incubated with [3H]QNB. After subsequent incubation with either an anti-caveolin-3 antibody or a nonspecific IgG1 antibody and agarose-conjugated protein-G, immunocomplexes were collected by centrifugation, and radioactivity was determined in a scintillation counter. As summarized in Fig. 3, in the absence of carbachol treatment, there was no significant immunoprecipitation of [3H]QNB binding by caveolin-3 antibodies since the level of [3H]QNB binding was similar to that obtained when using the nonspecific IgG1 for the immunoprecipitation. In contrast, following agonist treatment, a substantial fraction of specific [3H]QNB binding can be immunoprecipitated by anti-caveolin-3 antibodies (Fig. 3); no change in caveolin-3 expression was observed after carbachol treatment (not shown). In fact, 73 ± 5% (n = 3) of the [3H]QNB binding originally present in pooled fractions 2 and 3 (determined by direct filtration on Whatman GF/B glass filters) could be recovered after anti-caveolin-3 immunoprecipitation. Similar experiments (not shown) performed on fractions 7–9, which correspond to the bulk of plasma membrane (80–95% of total protein when pooled together), did not reveal any specific [3H]QNB binding in the caveolin-3 immunoprecipitate, in agreement with the low abundance of caveolin-3 in these fractions (see Fig. 1 A). Importantly, in myocytes incubated with carbachol in the presence of the muscarinic antagonist atropine, the [3H]QNB binding immunoprecipitated by anti-caveolin-3 antibodies remained at the level detected in a control immunoprecipitation performed with a nonspecific IgG1. This is in agreement with the data shown in Fig. 2 C in which no significant binding was detected in the anti-m2 mAchR immunoprecipitates from caveolar fractions of myocytes incubated with carbachol in the presence of atropine. Taken together, these data establish that the m2 mAchR redistributes to plasmalemmal caveolae of cardiac myocytes following agonist binding. The dynamic targeting of the m2 mAchR to caveolae has important implications for muscarinic receptor biology as well as for the regulation of eNOS activation. Although several laboratories have reported evidence for the translocation to low density gradient fractions of the muscarinic receptors upon agonist stimulation (5Harden T.K. Petch L.A. Traynelis S.F. Waldo G.L. J. Biol. Chem. 1985; 260: 13060-13066Abstract Full Text PDF PubMed Google Scholar, 6Raposo G. Dunia I. Marullo S. André Guillet J.-G. Strosberg A.D. Benedetti E.L. Hoebeke J. Biol. Cell. 1987; 60: 117-124Crossref PubMed Scopus (72) Google Scholar, 7Ho A.K.S. Zhang Y.-J. Duffield R. Zheng G.-M. Cell. Signalling. 1991; 3: 587-598Crossref PubMed Scopus (6) Google Scholar), there are, to our knowledge, no data in the literature that address the specific nature of these “light membranes.” The co-purification and co-immunoprecipitation (this study, and also see Refs. 19Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar and 36Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar) of caveolin, eNOS, and the agonist-stimulated m2 mAchR in isopycnic centrifugation fractions, which together represent less than 5% of the total amount of protein, indicate that caveolae are the common structural platform for these proteins. Together with immunoelectron microscopy data showing that, in A431 cells, β-AR are sequestrated within caveolae in response to agonist stimulation (18Dupree P. Parton R.G. Raposo G. Kurzhalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar), our data indicate that clathrin-coated pit formation can no longer be considered as the exclusive pathway for clustering G protein-coupled receptors within specialized plasmalemmal microdomains. The fate of caveolar β-AR and mAchR is uncertain, since it is not clear whether caveolae pinch off from the plasma membrane and lead to early endosomes. If this is the case, it suggests that dual pathways of receptor internalization may exist in some cells. While numerous studies present the sequestration of G protein-coupled receptors after agonist stimulation as a key event for initiating a process of desensitization (for review, see Ref. 40Eva C. Gamalero S.R. Genazzani E. Costa E. J. Pharmacol. Exp. Therap. 1990; 253: 257-265PubMed Google Scholar), the data in this manuscript support the hypothesis that, following stimulation by agonist, cardiac m2 mAchR translocation to caveolae may be necessary to initiate specific downstream signaling cascades. Interestingly, several recent studies have shown that internalization of the m2 and m4 mAchR is mediated by mechanisms distinct from the phosphorylation by the G protein-coupled receptor kinase (GRK) family known to lead to receptor desensitization (41Pals-Rylaarsdam R. Xu Y. Witt-Enderby P. Benovic J.L. Hosey M.M. J. Biol. Chem. 1995; 270: 29004-29011Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 42Bogatkewitsch G.S. Lenz W. Jakobs K.H. van Koppen C.J. Mol. Pharmacol. 1996; 50: 424-429PubMed Google Scholar). The translocation of muscarinic receptors within caveolae should allow their interaction with the heterotrimeric G protein complexes known to be concentrated within these plasmalemmal microdomains (12Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, 26Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar, 28Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar) and lead, after recruitment of co-factors and intermediate effector proteins, to the activation of eNOS, a resident caveolar protein in cardiac myocytes. Analysis of caveolin-enriched fractions to identify additional signaling molecules involved in the muscarinic cholinergic stimulation of the NO pathway in cardiac myocytes is ongoing in our laboratory. The caveolar compartmentation described here for the muscarinic cholinergic pathway may serve as a paradigm for other G protein receptor-mediated signaling cascades that are targeted to caveolae.

3-Hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitors or statins exert direct beneficial effects on the endothelium in part through an increase in nitric oxide (NO) production. Here, we examined whether posttranslational modifications of the endothelial NO synthase (eNOS) could account for the proangiogenic effects of statins. We used endothelial cells (ECs) isolated from cardiac microvasculature, aorta, and umbilical veins, as well as dissected microvessels and aortic rings, that were cultured on reconstituted basement membrane matrix (Matrigel). Tube or precapillary formation was evaluated after statin treatment, in parallel with immunoblotting and immunoprecipitation experiments. Atorvastatin stimulated NO-dependent angiogenesis from both isolated and outgrowing (vessel-derived) ECs, independently of changes in eNOS expression. We found that in macro- but not microvascular ECs, atorvastatin stabilized tube formation through a decrease in caveolin abundance and its inhibitory interaction with eNOS. We also identified the chaperone protein hsp90 as a key target for the proangiogenic effects of statins. Using geldanamycin, an inhibitor of hsp90 function, and overexpression of recombinant hsp90, we documented that the statin-induced phosphorylation of eNOS on Ser1177 was directly dependent on the ability of hsp90 to recruit Akt in the eNOS complex. Finally, we showed that statin promoted the tyrosine phosphorylation of hsp90 and the direct interaction of hsp90 with Akt, which further potentiated the NO-dependent angiogenic processes. Our study provides new mechanistic insights into the NO-mediated angiogenic effects of statins and underscores the potential of these drugs and other modulators of hsp90 and caveolin abundance to promote neovascularization in disease states associated or not with atherosclerosis.

Vascular endothelial growth factor (VEGF) exerts its angiogenic effects partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca²⁺-dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca²⁺/calmodulin disruption of the caveolin-eNOS complex and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the VEGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca²⁺]_i, this late stage of eNOS activation is strictly conditional on the initial VEGF-induced Ca²⁺-dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90.

The endothelial isoform of nitric oxide synthase (eNOS) is dually acylated and thereby targeted to signal-transducing microdomains termed caveolae. In endothelial cells, eNOS interacts with caveolin-1, which represses eNOS enzyme activity. In cardiac myocytes, eNOS associates with the muscle-specific caveolin-3 isoform, but whether this interaction affects NO production and regulates myocyte function is unknown. We isolated neonatal cardiac myocytes from mutant mice with targeted disruption of the eNOS gene and transfected them with wild-type (WT) eNOS or myristoylation-deficient (myr⁻) eNOS mutant cDNA. In myocytes expressing WT eNOS, the muscarinic cholinergic agonist carbachol completely abrogated the spontaneous beating rate and induced a 4-fold elevation of the cGMP level. By contrast, in the myr⁻ eNOS myocytes, carbachol failed to exert its negative chronotropic effect and to increase cGMP levels. We then used a reversible permeabilization protocol to load intact neonatal rat myocytes with an oligopeptide corresponding to the caveolin-3 scaffolding domain. This peptide completely and specifically inhibited the carbachol-induced negative chronotropic effect and the accompanying cGMP elevation. Thus, our results suggest that acylated eNOS may couple muscarinic receptor activation to heart rate control and indicate a key role for eNOS/caveolin interactions in the cholinergic modulation of cardiac myocyte function.

Decreased heart rate variability (HRV) and increased blood pressure variability (BPV), determined in part by nitric oxide (NO)-dependent endothelial dysfunction, are correlated with adverse prognosis in cardiovascular diseases. We examined potential alterations in BPV and HRV in genetically dyslipidemic, apolipoprotein (apo) E-/-, and control mice and the effect of chronic statin treatment on these parameters in relation to their NO synthase (NOS)-modifying properties.BP and HR were recorded in unrestrained, nonanesthetized mice with implanted telemetry devices with or without rosuvastatin. Cardiac and aortic expression of endothelial NOS and caveolin-1 were measured by immunoblotting. Both systolic BP and HR were elevated in apoE-/- mice, with abolition of their circadian cycles. Spectral analysis showed an increase in their systolic BPV in the very-low-frequency (+17%) band and a decrease in HRV in the high-frequency (-57%) band, reflecting neurohumoral and autonomic dysfunction. Decreased sensitivity to acute injection of atropine or an NOS inhibitor indicated basal alterations in both parasympathetic and NOS regulatory systems in apoE-/- mice. Aortic caveolin-1 protein, an inhibitor of endothelial NOS, was also increased in these mice by 2.0-fold and correlated positively with systolic BPV in the very-low-frequency band. Rosuvastatin treatment corrected the hemodynamic and caveolin-1 expression changes despite persisting elevated plasma cholesterol levels.Rosuvastatin decreases caveolin-1 expression and promotes NOS function in apoE-/-, dyslipidemic mice in vivo, with concurrent improvements in BPV and HRV. This highlights the beneficial effects of rosuvastatin on cardiovascular function beyond those attributed to lipid lowering.

Abstract Purpose: The aim of this work was to study changes in the tumor microenvironment early after an antiangiogenic treatment using thalidomide (a promising angiogenesis inhibitor in a variety of cancers), with special focus on a possible “normalization” of the tumor vasculature that could be exploited to improve radiotherapy. Experimental Design: Tumor oxygenation, perfusion, permeability, interstitial fluid pressure (IFP), and radiation sensitivity were studied in an FSAII tumor model. Mice were treated by daily i.p. injection of thalidomide at a dose of 200 mg/kg. Measurements of the partial pressure of oxygen (pO2) were carried out using electron paramagnetic resonance oximetry. Three complementary techniques were used to assess the blood flow inside the tumor: dynamic contrast-enhanced magnetic resonance imaging, Patent Blue staining, and laser Doppler imaging. IFP was measured by a “wick-in-needle” technique. Results: Our results show that thalidomide induces tumor reoxygenation within 2 days. This reoxygenation is correlated with a reduction in IFP and an increase in perfusion. These changes can be attributed to extensive vascular remodeling that we observed using CD31 labeling. Conclusions: In summary, the microenvironmental changes induced by thalidomide were sufficient to radiosensitize tumors. The fact that thalidomide radiosensitization was not observed in vitro, and that in vivo radiosensitization occurred in a narrow time window, lead us to believe that initial vascular normalization by thalidomide accounts for tumor radiosensitization.

Virtually all cell types within the myocardium express caveolae, where cell-specific isoforms of caveolin both maintain the structural organisation of these cholesterol-rich of the plasmalemma and serve as scaffolds for the dynamic constitution of "signalosomes", or hubs concentrating numerous transmembrane signaling proteins and their effectors. Analysis of the phenotype of mice with genetic deletion or overexpression of specific caveolin isoforms has provided key evidence for the importance of caveolins and caveolae in several aspects of the cardiovascular biology, including vascular contractility, lipid metabolism, angiogenesis, or the control of cardiac hypertrophy. Among specific protein-protein interactions involving caveolins in cardiac tissue, these genetic models unequivocally confirmed the functional importance of the dynamic association of the endothelial isoform of nitric oxide synthase (eNOS) for its post-translational regulation in endothelial cells and cardiac myocytes, which bears on the enzyme's capacity to modulate nitric oxide (NO)-dependent endothelial function, angiogenesis, and excitation-contraction coupling. We will review the current understanding of this regulation of eNOS (and potentially other NOS isoforms) through protein-protein interactions involving several G-protein-coupled receptors and other allosteric modulators in the context of emerging paradigms on the regulation of cardiac function by NO.

This phase II study assessed the safety and efficacy of everolimus, an oral mammalian target of rapamycin inhibitor in advanced transitional carcinoma cell (TCC) after failure of platinum-based therapy.Thirty-seven patients with advanced TCC received everolimus 10 mg/day until progressive disease (PD) or unacceptable toxicity. The primary end point was the disease control rate (DCR), defined as either stable disease (SD), partial response (PR), or complete response at 8 weeks. Angiogenesis-related proteins were detected in plasma and changes during everolimus treatment were analyzed. PTEN expression and PIK3CA mutations were correlated to disease control.Two confirmed PR and eight SD were observed, resulting in a DCR of 27% at 8 weeks. Everolimus was well tolerated. Compared with patients with noncontrolled disease, we observed in patients with controlled disease a significant higher baseline level of angiopoietin-1 and a significant early plasma decrease in angiopoietin-1, endoglin, and platelet-derived growth factor-AB. PTEN loss was observed only in patients with PD.Everolimus showed clinical activity in advanced TCC. The profile of the plasma angiogenesis-related proteins suggested a role of the everolimus antiangiogenic properties in disease control. PTEN loss might be associated with everolimus resistance.

Among all molecules developed for anticancer therapies, photodynamic therapeutic agents have a unique profile. Their maximal activity is specifically triggered in tumors by light, and toxicity of even systemically delivered drug is prevented in nonilluminated parts of the body. Photosensitizers exert their therapeutic effect by producing reactive oxygen species via a light-activated reaction with molecular oxygen. Consequently, the lowering of pO2 deep in solid tumors limits their treatment and makes essential the design of oxygen-independent sensitizers. In this perspective, we have recently developed Ir(III)-based molecules able to oxidize biomolecules by type I processes under oxygen-free conditions. We examine here their phototoxicity in relevant biological models. We show that drugs, which are mitochondria-accumulated, induce upon light irradiation a dramatic decrease of the cell viability, even under low oxygen conditions. Finally, assays on 3D tumor spheroids highlight the importance of the light-activation step and the oxygen consumption rate on the drug activity.

The multidisciplinary field of photodynamic therapy (PDT) is a combination of photochemistry and photophysics sciences, which has shown tremendous potential for cancer therapy application. PDT employs a photosensitizing agent (PS) and light to form cytotoxic reactive oxygen species and subsequently oxidize light-exposed tissue. Despite numerous advantages of PDT and enormous progress in this field, common PSs are still far from ideal treatment because of their poor permeability, non-specific phototoxicity, side effects, hydrophobicity, weak bioavailability, and tendency to self-aggregation. To circumvent these limitations, PS can be encapsulated in liposomes, an advanced drug delivery system that has demonstrated the ability to enhance drug permeability into biological membranes and loading both hydrophobic and lipophilic agents. Moreover, liposomes can also be coated by targeting agents to improve delivery efficiency. The present review aims to summarize the principles of PDT, various PS generations, PS-loaded nanoparticles, liposomes, and their impact on PDT, then discuss recent photodynamic cancer therapy strategies using liposomes as PS-loaded vectors, and highlight future possibilities and perspectives.

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (ICa-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on ICa-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOSnull mice, with targeted disruption of the eNOS gene. Although both eNOSnull and wild-type (WT) ventricular myocytes exhibited similar increases in ICa-L in response to ISO, there was no measurable suppression of ICa-L by CCh in cells from eNOSnull mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOSnull myocytes. Also, unlike myocytes from WT animals, eNOSnull myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented ICa-L in eNOSnull cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K+ channel by CCh was unaffected in atrial and ventricular eNOSnull myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of ICa-L in cardiac myocytes.

N-Terminal myristoylation and thiopalmitoylation of the endothelial isoform of nitric oxide synthase (eNOS) are required for targeting the enzyme to specialized signal-transducing microdomains of plasma membrane termed caveolae. We have previously documented that the subcellular localization of eNOS is dynamically regulated by agonists such as bradykinin, which promotes enzyme depalmitoylation and translocation from caveolae. More recently, we have shown that association of eNOS with caveolin, the principal structural protein in caveolae, leads to enzyme inhibition, in a reversible process modulated by Ca2+-calmodulin (CaM). We now report studies of the respective roles of acylation and caveolin interaction for regulating eNOS activity. Using eNOS truncation and deletion mutants expressed in COS-7 cells, we have identified an obligatory role for the N-terminal half of eNOS in stabilizing its association with caveolin. By exploring the differential effects of detergents (CHAPS vs octyl glucoside), we have shown that this direct interaction between both proteins is facilitated by, but does not require, eNOS acylation, and, importantly, that treatment of intact aortic endothelial cells with the calcium ionophore A23187 leads to the rapid disruption of the eNOS-caveolin complexes. Finally, using transiently transfected COS-7 cells, we have observed that the myristoylation-deficient cytosol-restricted eNOS mutant (myr-) as well as the cytosolic fraction of the palmitoylation-deficient eNOS mutant (palm-) may both interact with caveolin; this association also leads to a marked inhibition of enzyme activity, which is completely reversed by addition of calmodulin. We conclude that the regulatory eNOS-caveolin association is independent of the state of eNOS acylation, indicating that agonist-evoked Ca2+/CaM-dependent disruption of the caveolin-eNOS complex, rather than agonist-promoted depalmitoylation of eNOS, relieves caveolin's tonic inhibition of enzyme activity. We therefore propose that caveolin may serve as an eNOS chaperone regulating NO production independently of the enzyme's residence within caveolae or its state of acylation.

Auranofin (AF) is an anti-arthritic drug considered for combined chemotherapy due to its ability to impair the redox homeostasis in tumor cells. In this study, we asked whether AF may in addition radiosensitize tumor cells by targeting thioredoxin reductase (TrxR), a critical enzyme in the antioxidant defense system operating through the reductive protein thioredoxin. Our principal findings in murine 4T1 and EMT6 tumor cells are that AF at 3-10 μM is a potent radiosensitizer in vitro, and that at least two mechanisms are involved in TrxR-mediated radiosensitization. The first one is linked to an oxidative stress, as scavenging of reactive oxygen species (ROS) by N-acetyl cysteine counteracted radiosensitization. We also observed a decrease in mitochondrial oxygen consumption with spared oxygen acting as a radiosensitizer under hypoxic conditions. Overall, radiosensitization was accompanied by ROS overproduction, mitochondrial dysfunction, DNA damage and apoptosis, a common mechanism underlying both cytotoxic and antitumor effects of AF. In tumor-bearing mice, a simultaneous disruption of the thioredoxin and glutathione systems by the combination of AF and buthionine sulfoximine was shown to significantly improve tumor radioresponse. In conclusion, our findings illuminate TrxR in cancer cells as an exploitable radiobiological target and warrant further validation of AF in combination with radiotherapy.

Caspases are well known for their role in apoptosis. Recently, nonapoptotic roles of caspases have been identified, however, these noncanonical roles are not well documented and the mechanisms involved are not fully understood. Here, we studied the role of cleaved caspase-3 using human- and mouse-proficient caspase-3 cancer cell lines and human-deficient caspase-3 cancer cells. Cleaved caspase-3 functioned as a transcription factor and directly bound to DNA. A DNA-binding domain was identified in the small subunit of caspase-3 and an active conformation was essential for caspase-3 transcriptional activity. Caspase-3 DNA binding enhanced angiogenesis by upregulating the expression of proangiogenic genes and by activating pathways that promoted endothelial cell activation. Some proapoptotic genes were downregulated in caspase-3-proficient cells. Inhibiting caspase-3 increased the efficacy of chemotherapy and decreased spontaneous tumor development. These data highlight a novel nonapoptotic role of caspase-3 and suggest that cleaved caspase-3 could be a new therapeutic target in cancer. SIGNIFICANCE: These findings report a noncanonical function of caspase-3 by demonstrating its ability to transcriptionally regulate the VEGFR pathway.

Recent studies have highlighted the potential of photodynamic therapy (PDT) to induce immunogenic cell death (ICD). The clinical use of photosensitizers (PS) to stimulate an anticancer immune response, and not to sterilize tumor cells, may however require some optimizations. Here, we examined how the dose of PS and the scheduling of PDT influence the generation of danger-associated molecular patterns proteins (DAMPs) and favor T cell antitumor activity. We found that upon photoactivation, a low dose of the non-porphyrinic PS OR141 was more prone than higher doses to induce DAMPs in vitro and to inhibit squamous cell carcinoma growth in mice. We further used PDT-killed cancer cells to prime dendritic cells (DC) and stimulate their maturation to evaluate whether the timing of their injection could influence the antitumor effects of radiotherapy. While PDT-based DC vaccination administered before radiotherapy failed to increase tumor growth inhibition, DC injection in the peri-radiotherapy period led to significant tumor growth delay, emphasizing the importance of the coincidence of T cell activation and alterations of the tumor bed. In conclusion, the use of OR141 as a bona fide ICD inducer led us to unravel both the non-linear relationship between PS concentration and PDT-induced antitumor immune response, and the value of an optimal timing of PDT when co-administered with conventional anticancer therapies. This study therefore stresses the necessity of adapting the clinical use of PDT when the goal is to promote an immune response and identifies PDT-based DC vaccination as a suitable modality to reach such objective.

Immunogenic cell death (ICD) is a rapidly growing research area representing one of the emerging therapeutic strategies of cancer immunotherapy. ICD is an umbrella term covering several cell death modalities including apoptosis, necroptosis, ferroptosis and pyroptosis, and is the product of a balanced combination of adjuvanticity (damage-associated molecular patterns and chemokines/cytokines) and antigenicity (tumor associated antigens). Only a limited number of anti-cancer therapies are available to induce ICD in experimental cancer therapies and even much less is available for clinical use. To overcome this limitation, nanomaterials can be used to increase the immunogenicity of cancer cells killed by anti-cancer therapy, which in themselves are not necessarily immunogenic. In this review, we outline the current state of knowledge of ICD modalities and discuss achievements in using nanomaterials to increase the immunogenicity of dying cancer cells. The emerging trends in modulating the immunogenicity of dying cancer cells in experimental and translational cancer therapies and the challenges facing them are described. In conclusion, nanomaterials are expected to drive further progress in their use to increase efficacy of anti-cancer therapy based on ICD induction and in the future, it is necessary to validate these strategies in clinical settings, which will be a challenging research area.

The ultimate goal of radiotherapy is to induce irreversible damages in genetically unstable, fast-growing cancer cells while minimizing the cytotoxic effects on host tissues. The satus of the tumor vasculature is particular because it is located within the tumor but mostly arises from host cells. The aim of this study was to characterize the effects of low-dose irradiation on the function of endothelial cells lining tumor vessels. Using isolated arterioles mounted on a pressure myograph, we first documented that the nitric oxide (NO)-mediated vasorelaxation that was defective in tumor vessels was completely restored following local tumor irradiation. Immunoblot analyses revealed that this was attributable to an increase in the abundance of the endothelial NO synthase while the expression of its physiological inhibitor, caveolin-1, was reduced. We further showed that the potentiation of the NO-dependent pathway induced a marked increase in tumor blood flow and oxygenation that determined the higher sensitivity of the tumor to further irradiation. Finally, we documented that the NO-mediated effects of irradiation on the tumor vasculature increased the delivery and expression of a reporter gene into the tumor. Thus, low-dose irradiation of endothelial cells within a tumor is a key determinant of the effectiveness of radiotherapy and may offer a new strategy to increase gene and/or drug delivery to the tumor.

The combination of radiotherapy and antiangiogenic strategies has been shown to increase the tumor response in various experimental models. The rationale for this cotherapy was initially related to the expected gain in efficacy by acting on two different targets, e.g., tumor cells and endothelial cells (ECs). However, recent studies have documented more than additive effects due to apparent mutual potentiation of these approaches. In this study, we tested the hypothesis that these synergistic effects could stem from the stimulatory effects of ionizing radiations on angiogenesis, which would then need to be restrained to avoid tumor regrowth after irradiation. We found that irradiation dose-dependently induced the activation of the proangiogenic NO pathway in ECs through increases in endothelial nitric oxide synthase abundance and phosphorylation. Using 2- and 3-dimensional cultures of ECs and isolated mouse tumor arterioles, we documented that the irradiation-induced enhanced production of NO accounted for EC migration and sprouting. Irradiation was also shown to stimulate the colonization of Matrigel plugs implanted in mouse by ECs, where they formed capillary-like structures in a NO-dependent manner. These findings were confirmed by documenting the NO-mediated infiltration of CD31-positive ECs after local irradiation of Lewis lung carcinoma tumor-bearing mice. Finally, we measured a consistent increase in endothelial nitric oxide synthase mRNA by real-time PCR experiments in human biopsies of head and neck squamous cell carcinoma after low-dose irradiation. In conclusion, we have demonstrated that the potentiation of the NO signaling pathway after irradiation induces profound alterations in the EC phenotype leading to tumor angiogenesis. Moreover, our demonstration that the inhibition of NO production suppresses these provascular effects of irradiation highlights new potentials for the coordinated use of antiangiogenic strategies and radiotherapy in clinical practice.

Deubiquitinating enzymes (DUBs) are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors. Through a proteomic approach, we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight (MW) complexes. Activity-based proteome profiling identified CIAPIN1 as a submicromolar covalent target of VLX1570, and further analysis demonstrated that high MW complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce nonspecific protein aggregation, providing molecular explanation for general cellular toxicity.

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α-dependent microRNA (miR)-210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210-based therapeutic and diagnostic strategies.

HomeCirculation ResearchVol. 88, No. 2The Caveolar Paradox: Suppressing, Inducing, and Terminating eNOS Signaling Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessEditorialPDF/EPUBThe Caveolar Paradox: Suppressing, Inducing, and Terminating eNOS Signaling Olivier Feron and Ralph A. Kelly Olivier FeronOlivier Feron From the Unit of Pharmacology and Therapeutics (O.F.), Department of Medicine, University of Louvain Medical School, Brussels, Belgium and Genzyme Corporation (R.A.K.), Cambridge, Mass. Search for more papers by this author and Ralph A. KellyRalph A. Kelly From the Unit of Pharmacology and Therapeutics (O.F.), Department of Medicine, University of Louvain Medical School, Brussels, Belgium and Genzyme Corporation (R.A.K.), Cambridge, Mass. Search for more papers by this author Originally published2 Feb 2001https://doi.org/10.1161/01.RES.88.2.129Circulation Research. 2001;88:129–131It is now established that specialized plasmalemmal lipid microenvironments, termed lipid rafts by Simons and Toomre,1 take part in various signal transduction processes. One subset of lipid rafts (which contain mostly cholesterol and sphingolipids) is found in plasmalemmal vesicles termed caveolae. The term caveolae (“little caves”) was introduced more than 40 years ago to describe plasma membrane invaginations identified by electron microscopy in a wide variety of cell types. Originally, these 50- to 100-nm plasmalemmal vesicles were shown to participate in the transcellular transport of macromolecules (transcytosis) and in the uptake of small molecules (potocytosis).2 However, it is only recently, with the identification of caveolins as the structural coat component of caveolae, that it has been recognized that caveolae are involved in signal transduction by ensuring the compartmentation of signaling molecules, such as G protein and tyrosine kinase–associated receptors, as well as endothelial nitric oxide synthase (eNOS). The identification of such distinct roles raises the question of how the same organelle can participate in these apparently quite different functions simultaneously. However, in the case of eNOS, recent data suggest that both of these functions (ie, as signaling platforms and intracellular trafficking modules) are, in fact, intimately related and complementary.Repressing Basal Activity of eNOSAlthough both eNOS and caveolins have several consensus sequences that have been proposed to participate in protein-protein interactions, evidence for a functional association between eNOS and caveolins exists only for the caveolin scaffolding domain (CSD), a juxtamembrane region of 20 amino acids in the C-terminal moiety of caveolin.4 Like other modular protein domains, the scaffolding domain of caveolin facilitates the generation of preassembled oligomeric proteins and, in addition, maintains these various signaling proteins in their off state.234 Note, however, that the presence of a CSD consensus–binding sequence does not necessarily imply that a given protein will interact with one or more caveolins; for example, several of the toll-like receptors contain CSD-binding domains, but there is no evidence to date of either caveolar targeting or functional regulation of toll-like receptor signaling by caveolins (S. Franz, T. Bourcier, R.A. Kelly, unpublished observations).The physiological relevance of the inhibitory interaction of caveolar targeting on basal NO production was recently provided in a study on intact endothelial cells exposed to high levels of LDL cholesterol.5 As originally identified by Fielding and Fielding,6 caveolae also participate in reverse cholesterol transport by increasing caveolin abundance to promote cholesterol trafficking and efflux. The consequence for eNOS function of this cholesterol-induced increase in caveolin abundance is a marked decline in basal NO release, suggesting that the equilibrium between eNOS bound to caveolin and caveolin-free eNOS determines the basal component of eNOS-dependent NO release in endothelial cells. This interaction may be required to protect the cell from undesired, potentially cytotoxic, or nonphysiological bursts of NO in response to small fluctuations in intracellular calcium ([Ca2+]i).Promoting Stimulation of eNOSIn the presence of increased [Ca2+]i, calmodulin binding to caveolin antagonizes the blockade (or slowing) of electron transfer attributable to the binding of the scaffold protein to eNOS.4 In addition, transient increases in [Ca2+]i attributable to agonist activation7 or increases in vascular flow8 have been shown to promote the dissociation of eNOS from caveolin in endothelial cells. Therefore, conversely, an increase in caveolin abundance in endothelial cells could account for alterations in agonist or flow-mediated stimulation of NO production. In addition to hypercholesterolemia, mentioned above,5 data from 2 other studies provide support for this hypothesis. Shah et al9 recently reported that a decrease in perfusion pressure in cirrhotic liver correlated with a marked increase in caveolin abundance and stabilization of the caveolin/eNOS inhibitory complex. In addition, Pelligrino et al10 documented that in ovariectomized rats, a decrease in acetylcholine-induced pial arteriolar vasodilation was associated with both a decrease in eNOS abundance and an increase in caveolin-1 abundance.Importantly, persuasive evidence has also been provided that eNOS localization in caveolae per se (versus other intracellular locales of the enzyme) is key for agonist-stimulated NO release. Blair et al,11 for example, reported that endothelial cell exposure to oxidized LDL, which results in caveolar cholesterol depletion, rapidly caused the translocation of both eNOS and caveolin from caveolae, thereby leading to a marked decline in acetylcholine-induced eNOS activation. Together with a previous study documenting the obligatory caveolar location of eNOS for the muscarinic cholinergic stimulation of eNOS in cardiac myocytes,12 these data point to the key role of eNOS localization within plasmalemmal caveolae for its functional coupling to specific agonists. This paradigm must be modified in part, however, because of the known direct inhibitory interactions between caveolin and receptors such as the sphingosine 1-phosphate receptor EDG-1.13 Igarashi and Michel,13 for example, reported that caveolin overexpression markedly attenuates sphingosine 1-phosphate–mediated eNOS activation but not basal rates of NO production, suggesting a more proximal inhibitory effect of caveolins on EDG-1 receptor activation or coupling than on eNOS activity per se.Terminating eNOS SignalingIn this issue of Circulation Research, Li et al14 propose that eNOS-dependent signaling can be terminated by a NO/cGMP-dependent process that leads to the disruption of caveolin oligomers. The hypothesis put forward by these authors is that NO disrupts caveolar signaling by distancing elements of the cascade. In their study, they document that exposing endothelial cells or smooth muscle cells to NO donors alters the coupling of caveolar resident proteins, such as bradykinin or endothelin receptors, with downstream signal-transduction cascades that result in calcium release from intracellular stores. This interpretation is potentially confounded by the relatively high and noncompartmentalized release of concentrations of NOx by NO donors, which could, for example, affect [Ca2+]i transients induced by receptors or ion channels not located in caveolae. The authors also propose that NO signaling itself can be terminated by a decrease in plasmalemmal L-arginine transport through NO-mediated disruption of caveolae. However, the authors provide no evidence that L-arginine–induced NO production, which presumably results in a physiologically relevant increase in NO production, leads to the degree of caveolin redistribution that was observed in their NO donor experiments. Other caveats should also be noted. First, none of the data provided causally associates the change in the oligomeric status of caveolin with any of the functional alterations in intracellular signaling tested. Second, the proportion of disrupted caveolin complexes on NO donor exposure remains limited when compared with the total cellular pool of caveolins. Third, the data documenting caveolin deoligomerization may not be relevant to the activity of physiological, compartmentalized release of NO by eNOS.Nevertheless, the NO/cGMP-evoked decrease in the relative abundance of high molecular weight complexes of caveolins has been well documented by independent methodological approaches and certainly emphasizes the plasticity of function of lipid rafts and caveolar microdomains. Accordingly, the biophysical changes observed by Li et al14 may be related to alterations in detergent solubility of the caveolin heterocomplex on agonist stimulation, as recently reviewed by Fleming and Busse.15 Intracellular trafficking of caveolin and lipid microdomains may correspond to plasmalemmal caveolae after budding from the plasmalemmal membrane. Indeed, the GTPase dynamin, which is known to play an essential regulatory role in the process of endocytosis through clathrin-coated pits, was recently found to promote cholera toxin B chain internalization within caveolae after budding from the plasmalemmal membrane.16 Therefore, both caveolar fission and apparent caveolin deoligomerization could be the same phenomenon. Li et al,14 for example, also used the cholera toxin B subunit as a marker for caveolae and demonstrated its reversible translocation from the plasma-lemmal membrane after exposure to sodium nitroprusside. This is consistent with the observation that the M2 muscarinic acetylcholine receptor also follows this mode of sequestration and internalization through budded caveolae,17 thereby leading to a desensitization of downstream NO signaling after exposure to muscarinic agonists. Although dynamin was documented to be necessary for caveolar-plasmalemmal fission, it was not sufficient. Additional, presently unidentified mechanisms dependent on agonist-receptor binding apparently are also required for caveolar budding.Caveolae as eNOS OrganellesAlthough controversy remains regarding the subcellular location of all eNOS moieties within cells, the caveolar location of eNOS can be considered a major locale, at least for the activatable pool of the enzyme. Indeed, 2 studies have reported that increasing caveolin abundance resulted in a marked decline in agonist-stimulated NO generation, mimicking the phenotype observed in cells isolated from eNOS knockout animals. CSD peptides, for example, when introduced into cardiac myocytes by reversible permeabilization, mimic the inhibitory effects of full-length caveolin (caveolin-3 in the case of cardiac myocytes) and lead to a decrease in cGMP production on agonist stimulation.12 Moreover, muscarinic agonist regulation of myocyte beating rate could be inhibited by suppressing eNOS activation with 20-residue CSD caveolin surrogates. Recently, Bucci et al18 confirmed in mice that the activity of eNOS is highly sensitive to inhibition by CSD peptides. They showed that systemic administration of CSD peptides (fused to the homeodomain of antennapedia) suppressed acute inflammation and vascular leak to the same extent as that observed with an NOS inhibitor. Together, these studies demonstrate that expression of CSD peptides in either cardiac myocytes or endothelial cells clearly targets eNOS-dependent signaling.Considering the many potential targets with which caveolin has been proposed to interact, plasmalemmal caveolae seem to be a well-suited intracellular locale for eNOS and its complex regulation. Tonic repression of basal activity, facilitation of agonist-evoked stimulation, and termination of the signal are 3 apparently paradoxical tasks that seem to be effectively managed in the context of the caveolar/eNOS interaction.The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.FootnotesCorrespondence to Olivier Feron, University of Louvain Medical School, Pharmacology and Therapeutics Unit, UCL-FATH 5349, 53, Avenue E. Mounier, B-1200 Brussels, Belgium. E-mail [email protected] References 1 Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev.2000; 1:31–39.Google Scholar2 Anderson RG. The caveolae membrane system. Annu Rev Biochem.1998; 67:199–225.CrossrefMedlineGoogle Scholar3 Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, Lisanti MP. Caveolins, liquid-ordered domains, and signal transduction. Mol Cell Biol.1999; 19:7289–7304.CrossrefMedlineGoogle Scholar4 Feron O, Michel T. Cell and molecular biology of nitric oxide synthases. In: Loscalzo J, Vita JA, eds. Nitric Oxide and the Cardiovascular System. Totowa, NJ: Humana Press; 2000.Google Scholar5 Feron O, Dessy C, Moniotte S, Desager JP, Balligand JL. Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase. J Clin Invest.1999; 103:897–905.CrossrefMedlineGoogle Scholar6 Fielding CJ, Fielding PE. Cholesterol and caveolae: structural and functional relationships. Biochem Biophys Acta.2000; 1529:210–222.CrossrefMedlineGoogle Scholar7 Feron O, Saldana F, Michel JB, Michel T. The endothelial nitric-oxide synthase-caveolin regulatory cycle. J Biol Chem.1998; 273:3125–3128.CrossrefMedlineGoogle Scholar8 Rizzo V, McIntosh DP, Oh P, Schnitzer JE. In situ flow activates endothelial nitric oxide synthase in luminal caveolae of endothelium with rapid caveolin dissociation and calmodulin association. J Biol Chem.1998; 273:34724–34729.CrossrefMedlineGoogle Scholar9 Shah V, Toruner M, Haddad F, Cadelina G, Papapetropoulos A, Choo K, Sessa WC, Groszmann RJ. Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat. Gastroenterology.1999; 117:1222–1228.CrossrefMedlineGoogle Scholar10 Pelligrino DA, Ye S, Tan F, Santizo RA, Feinstein DL, Wang Q. Nitric-oxide-dependent pial arteriolar dilation in the female rat: effects of chronic estrogen depletion and repletion. Biochem Biophys Res Commun;.2000; 269:165–171.CrossrefMedlineGoogle Scholar11 Blair A, Shaul PW, Yuhanna IS, Conrad PA, Smart EJ. Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation. J Biol Chem.1999; 274:32512–32519.CrossrefMedlineGoogle Scholar12 Feron O, Dessy C, Opel DJ, Arstall MA, Kelly RA, Michel T. Modulation of the endothelial nitric-oxide synthase-caveolin interaction in cardiac myocytes. Implications for the autonomic regulation of heart rate. J Biol Chem.1998; 273:30249–30254.CrossrefMedlineGoogle Scholar13 Igarashi J, Michel T. Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae: eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction. J Biol Chem.2000; 275:32363–32370.CrossrefMedlineGoogle Scholar14 Li H, Brodsky S, Basco M, Romanov V, De Angelis DA, Goligorsky MS. Nitric oxide attenuates signal transduction: possible role in dissociating caveolin-1 scaffold. Circ Res..2001; 88:229-236.CrossrefMedlineGoogle Scholar15 Fleming I, Busse R. Signal transduction of eNOS activation. Cardiovasc Res.1999; 43:532–541.CrossrefMedlineGoogle Scholar16 Henley JR, Cao H, McNiven MA. Participation of dynamin in the biogenesis of cytoplasmic vesicles. FASEB J. 1999;13(suppl 2):S243–S247.Google Scholar17 Dessy C, Kelly RA, Balligand JL, Feron O. Dynamin mediates caveolar sequestration of muscarinic cholinergic receptors and alteration in NO signaling EMBO J.2000; 19:4272–4280.CrossrefMedlineGoogle Scholar18 Bucci M, Gratton JP, Rudic RD, Acevedo L, Roviezzo F, Cirino G, Sessa WC. In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation. Nat Med.2000; 6:1362–1367.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails February 2, 2001Vol 88, Issue 2Article InformationMetrics © 2001 American Heart Association, Inc.https://doi.org/10.1161/01.RES.88.2.129 Originally publishedFebruary 2, 2001 Keywordssignal transductionnitric oxidemuscarinic cholinergic signalingcaveolinPDF download Advertisement

The microstructure and the deposition rate of pyrocarbon deposited from propane have both been studied as a function of gas phase residence time (tr) and reactor temperature (T) in the vicinity of 1000°C and at a constant pressure of 2 kPa. Transitions between two different types of laminar microstructures, smooth laminar (SL) and rough laminar (RL), have been observed. Simultaneously, transitions between several kinetic regimes have been seen. The kinetic results permit us to interpret changes in carbon microstructure and to develop a qualitative chemical model for the formation of laminar pyrocarbons. This model could explain both kinetic and textural transitions by the occurrence of two parallel routes to carbon formation involving two different families of ultimate carbon precursors.

CHO cells expressing the α 1C‐a subunit (cardiac isoform) and the α 1C‐b subunit (vascular isoform) of the voltage‐dependent L‐type Ca 2+ channel were used to investigate whether tissue selectivity of Ca 2+ channel blockers could be related to different affinities for α 1C isoforms. Inward current evoked by the transfected α 1 subunit was recorded by the patch‐clamp technique in the whole‐cell configuration. Neutral dihydropyridines (nifedipine, nisoldipine, (+)‐PN200‐110) were more potent inhibitors of α 1C‐b ‐subunit than of α 1C‐a ‐subunit. This difference was more marked at a holding potential of −100 mV than at −50 mV. SDZ 207‐180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms. Pinaverium (ionized non‐dihydropyridine derivative) was 2 and 4 fold more potent on α 1C‐a than on α 1C‐b subunit at Vh of −100 mV and −50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages. [ 3 H]‐(+)‐PN 200‐110 binding experiments showed that neutral dihydropyridines had a higher affinity for the α 1C‐b than for the α 1C‐a subunit. SDZ 207‐180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the α 1C‐a subunit than for the α 1C‐b subunit. These results indicate marked differences among Ca 2+ channel blockers in their selectivity for the α 1C‐a and α 1C‐b subunits of the Ca 2+ channel. British Journal of Pharmacology (1998) 125 , 1005–1012; doi: 10.1038/sj.bjp.0702162

Receptor-coupled contraction of smooth muscle involves recruitment to the plasma membrane of downstream effector molecules PKCα and rhoA but the mechanism of this signal integration is unclear. Caveolins, the principal structural proteins of caveolar plasma membrane invaginations, have been implicated in the organization and regulation of many signal transducing molecules. Thus, using laser scanning confocal immunofluorescent microscopy, we tested the hypothesis that caveolin is involved in smooth muscle signaling by investigating caveolin isoform expression and localization, together with the effect of a peptide inhibitor of caveolin function, in intact differentiated smooth muscle cells. All three main caveolin isoforms were identified in uterine, stomach, and ileal smooth muscles and assumed a predominantly plasma membranous localization in myometrial cells. Cytoplasmic introduction of a peptide corresponding to the caveolin-1 scaffolding domain—an essential region for caveolin interaction with signaling molecules—significantly inhibited agonist-induced translocation of both PKCα and rhoA. Translocation was unimpaired by a scrambled peptide and was unaltered in sham-treated cells. The membranous localization of caveolins, and direct inhibition of receptor-coupled PKCα and rhoA translocation by the caveolin-1 scaffolding domain, supports the concept that caveolins can regulate the integration of extracellular contractile stimuli and downstream intracellular effectors in smooth muscle.

This paper is devoted to the problem of sampling Gaussian distributions in high dimension. Solutions exist for two specific structures of inverse covariance: sparse and circulant. The proposed algorithm is valid in a more general case especially as it emerges in linear inverse problems as well as in some hierarchical or latent Gaussian models. It relies on a perturbation-optimization principle: adequate stochastic perturbation of a criterion and optimization of the perturbed criterion. It is proved that the criterion optimizer is a sample of the target distribution. The main motivation is in inverse problems related to general (nonconvolutive) linear observation models and their solution in a Bayesian framework implemented through sampling algorithms when existing samplers are infeasible. It finds a direct application in myopic/unsupervised inversion methods as well as in some non-Gaussian inversion methods. An illustration focused on hyperparameter estimation for super-resolution method shows the interest and the feasibility of the proposed algorithm.

Abstract —Two of the neuregulins (NRG1 and NRG2) and their receptors (erbB2 and erbB4) are essential for normal cardiac development and can mediate hypertrophic growth and enhance survival of embryonic, postnatal, and adult rat ventricular myocytes. The expression of erbB4, the predominant NRG receptor in postnatal rat ventricular muscle, declines after midembryogenesis, and its expression is limited to cardiac myocytes. A full-length erbB4 rat cDNA isolated from neonatal ventricular muscle was found to be highly homologous to human erbB4 and contained a caveolin binding motif within the cytoplasmic kinase domain. Using the complementary techniques of detergent-free density-gradient ultracentrifugation of myocyte lysates and coimmunoprecipitation of erbB4 and caveolin-3, the caveolin isoform expressed in cardiac myocytes, erbB4 could be localized (using both approaches) to caveolar microdomains. Moreover, addition of a soluble NRG1, recombinant human glial growth factor 2, resulted in rapid (2-minute) translocation of erbB4 out of caveolar microdomain in cardiac myocytes. Thus, erbB4 is dynamically targeted to caveolar microdomains within cardiac myocytes. Its rapid translocation after NRG1 binding may contribute to receptor desensitization in the continuous presence of ligand.

Expression of survivin, a member of the inhibitor of apoptosis protein family, is elevated in human cancers and considered as a new therapeutic target. Mechanism upregulating survivin expression in tumour cells is poorly understood. In this study, we show that breast cancer patients harbouring a polymorphism G235A in the survivin promoter present a higher level of survivin expression. This polymorphism creates a binding site for the transcription factor GATA-1 inducing a second GATA-1-binding site in survivin promoter. At the mRNA level, GATA-1 was present in breast carcinomas and adjacent normal tissues, whereas the protein was only detected in carcinomas by western blot and immunohistochemistry. Transfection of wild-type and different constitutively active GATA-1 mutants (serine 26, 178 or 310) showed that only phospho-serine 26 GATA-1 was able to increase survivin expression. This increase was higher in G235A than in G235G cell lines. Phospho-serine 26 GATA-1 bound directly survivin promoter, with a stronger interaction in G235A than in G235G polymorphism indicating that both GATA-1-binding sites are functional. These data identify GATA-1 as a key feature in tumour aggressiveness by enhancing survivin expression and delineate its targeting as a possible new therapeutic strategy in breast carcinomas.

The fundamental unit of chromatin is the nucleosome which is composed of a histone octamer and the DNA that wraps around it. Histones are globular proteins subject to various reversible covalent modifications that primarily occur on their flexible N-terminal ends (the so-called tail). Histone acetylation is one of these major alterations that may influence the chromatin conformation and consecutively influence gene expression (1). Histone acetylation is usually associated with an increase in transcriptional activity. Indeed, since acetylation occurs on positively charged lysines residues, the addition of an acetyl group on these residues changes the overall charge of the histone tail thereby leading to weaker binding of the nucleosomal components (2). As a direct consequence of histone acetylation, DNA becomes more accessible to transcription factors. Enzymes named histone acetyltransferases (HATs) catalyzed this mode of post-translational modifications (3). Other enzymes termed histone deacetylases (HDACs) are involved in the reverse process of histone deacetylation that restores the ionic interactions between positively charged histones and negatively charged DNA, thereby yielding a more compact chromatin structure (making it harder for transcription factors to bind to the DNA) (2,3).

Metabolic plasticity in cancer cells makes use of metabolism-targeting agents very challenging. Drug-induced metabolic rewiring may, however, uncover vulnerabilities that can be exploited. We report that resistance to glycolysis inhibitor 3-bromopyruvate (3-BrPA) arises from DNA methylation in treated cancer cells and subsequent silencing of the monocarboxylate transporter MCT1. We observe that, unexpectedly, 3-BrPA-resistant cancer cells mostly rely on glycolysis to sustain their growth, with MCT4 as an essential player to support lactate flux. This shift makes cancer cells particularly suited to adapt to hypoxic conditions and resist OXPHOS inhibitors and anti-proliferative chemotherapy. In contrast, blockade of MCT4 activity in 3-BrPA-exposed cancer cells with diclofenac or genetic knockout, inhibits growth of derived spheroids and tumors in mice. This study supports a potential mode of collateral lethality according to which metabolic adaptation of tumor cells to a first-line therapy makes them more responsive to a second-line treatment.

Because nitric oxide (NO) regulates cardiac and vessel contraction, we compared the expression and activity of the endothelial NO synthase (eNOS) and caveolin, which tonically inhibits eNOS in normal and hypertrophic cardiomyopathic hearts. NOS activity (L-[(3)H]citrulline formation), eNOS immunostaining, and caveolin abundance were measured in heart tissue of 23 mongrel dogs before and at 3 and 7 wk of perinephritic hypertension (PHT). Hemodynamic parameters in vivo and endothelial NO-dependent relaxation of macro- and coronary microvessels in vitro were assessed in the same animals. eNOS immunostaining and total calcium-dependent NOS activity decreased at 7 wk in all four heart cavities (in left ventricle, from 17.0 +/- 1.3 to 0.2 +/- 0.2 fmol. min(-1). mg protein(-1), P < 0.001). Caveolin-1 and -3 also decreased in PHT dog hearts. Accordingly, basal vascular tone was preserved, but maximal endothelial NO-dependent relaxation was impaired in all vessels from 7-wk PHT dogs. The latter had preserved systolic function but impaired diastolic relaxation [relaxation time constant (T(1)), 25.1 +/- 0.9 vs. 22.0 +/- 1 ms in controls; P < 0.05]. Peripheral infusion of the NOS inhibitor N(G)-nitro-L-arginine methyl ester increased mean aortic pressure in both groups and reduced diastolic (T(1), 31.9 +/- 1.4 ms) and systolic function in PHT dogs (DP40, 47.5 +/- 2.5 vs. 59.4 +/- 3.8 s(-1) in control animals). In conclusion, both eNOS and caveolin proteins are decreased in the hypertrophic hearts of PHT dogs. This is associated with altered maximal (but not basal) vascular relaxation and impaired diastolic function. Further degradation of cardiac function after NOS inhibition suggests a critical role of residual NOS activity, probably supported by the concurrent downregulation of caveolin.

We deal with an electromagnetic inverse scattering problem where the goal is to characterize unknown objects from measurements of the scattered fields that result from their interaction with a known interrogating wave in the microwave frequency range. This nonlinear and ill-posed inverse problem is tackled from experimental data collected in a laboratory-controlled experiment led at the Institut Fresnel (Marseille, France), which consist of the time-harmonic scattered electric field values measured at several discrete frequencies. The modelling of the wave–object interaction is carried out through a domain integral representation of the fields in a 2D-TM configuration. The inverse scattering problem is solved by means of an iterative algorithm tailored for objects made of a finite number of different homogeneous dielectric and/or conductive materials. The latter a priori information is introduced via a Gauss–Markov field for the distribution of the contrast with a hidden Potts–Markov field for the class of materials in the Bayesian estimation framework. In this framework, we first derive the posterior distributions of all the unknowns and, then, an appropriate Gibbs sampling algorithm is used to generate samples and estimate them. The proposed Bayesian inversion method is applied to both a linear case derived from diffraction tomography and the full nonlinear problem.

Endothelial nitric oxide synthase (eNOS) phosphorylation increases nitric oxide formation, for example, after VEGF stimulation. We investigated whether nitric oxide formed after overexpression of VEGF or of phosphomimetic eNOS (S1177D) affects PMN-induced myocardial detriment after ischemia and reperfusion. Pigs (n=8 per group) were subjected to percutaneous liposome-based gene transfer by retroinfusion of the anterior interventricular vein 48 h before LAD occlusion (60 min) and reperfusion (24 h). Thereafter, regional myocardial function was assessed as subendocardial segment shortening (SES), and infarct size was determined. Tissue from the infarct region, the noninfarcted area at risk, and a control region was analyzed for NF-kappaB activation (EMSA), tumor necrosis factor (TNF)-alpha, and E-selectin mRNA and infiltration of polymorphonuclear neutrophils (PMN). L-NAME was applied in one group of VEGF-transfected animals. NF-kappaB activition, PMN infiltration in the infarct region, and AAR were reduced after transfection of VEGF or eNOS S1177D, but not after VEGF+L-NAME coapplication. Infarct size decreased, whereas SES improved after either VEGF or eNOS S1177D transfection, an effect inhibited by L-NAME coapplication. Retroinfusion of liposomal VEGF cDNA reduces NF-kappaB-dependent postischemic inflammation and subsequent myocardial reperfusion injury, an effect mediated at least in part by enhanced eNOS phosphorylation.

The effects of insulin on tumor oxygenation, perfusion, oxygen consumption,and radiation sensitivity were studied on two different mouse tumor models (TLT, a liver tumor, and FSAII, a fibrosarcoma). Anesthetized mice were infused with insulin i.v. at a rate of 16 milliUnits/kg/min for 25 min. Local tumor oxygenation measurements were carried out using two independent techniques: electron paramagnetic resonance oximetry and a fiber-optic device (OxyLite). Two complementary techniques were also used to assess the blood flow inside the tumor: a laser Doppler system (OxyFlo) and contrast-enhanced magnetic resonance imaging. The oxygen consumption rate of tumor cells after in vivo insulin infusion was measured using high frequency electron paramagnetic resonance oximetry. To know if insulin was able to enhance radiation-induced tumor regrowth delay, tumor-bearing mice were treated with 16 Gy of 250 kV radiation dose after insulin infusion. We provide evidence that insulin increases the local pressure of oxygen of tumors (from 0-3 mm Hg to 8-11 mm Hg) as well as the tumor response to irradiation (increasing regrowth delay by a factor of 2.11). We found that the insulin-induced increase of tumor pressure of oxygen: (a) is not caused by an increase in the tumor blood flow, which is even decreased after insulin infusion; (b) is because of a decrease in the tumor cell oxygen consumption (in vivo insulin consumed oxygen three times slower than control cells); and (c) is inhibited by a nitric oxide (NO) synthase inhibitor, Nomega-nitro-L-arginine methyl ester, when injected i.p. at 15 micromol/kg(-1), 1 h before insulin infusion. We demonstrate by immunoblotting that the NO pathway involves a phosphorylation of endothelial NO synthase and showed a concomitant increase in the cyclic GMP tumor level. These findings provide unique insights into biological processes in tumors, new possible management for treating cancer patients, and raise major questions about the role of insulin secretion (fasting status and diabetes) in the clinical response of tumors to radiation therapy.

Amlodipine is a calcium channel blocker (CCB) known to stimulate nitric oxide production from endothelial cells. Whether this ancillary property can be related to the capacity of amlodipine to concentrate and alter the structure of cholesterol-containing membrane bilayers is a matter of investigation. Here, we reasoned that since the endothelial nitric oxide synthase is, in part, expressed in cholesterol-rich plasmalemmal microdomains (e.g., caveolae and rafts), amlodipine could interfere with this specific locale of the enzyme and thereby modulate NO production in endothelial cells.Using a method combining lubrol-based extraction and subcellular fractionation on sucrose gradient, we found that amlodipine, but not verapamil or nifedipine, induced the segregation of endothelial NO synthase (eNOS) from caveolin-enriched low-density membranes (8+/-2% vs. 42+/-3% in untreated condition; P<0.01). We then performed co-immunoprecipitation experiments and found that amlodipine dose-dependently disrupted the caveolin/eNOS interaction contrary to other calcium channel blockers, and potentiated the stimulation of NO production by agonists such as bradykinin and vascular endothelial growth factor (VEGF) (+138+/-28% and +183+/-27% over values obtained with the agonist alone, respectively; P<0.01). Interestingly, we also documented that the dissociation of the caveolin/eNOS heterocomplex induced by amlodipine was not mediated by the traditional calcium-dependent calmodulin binding to eNOS and that recombinant caveolin expression could compete with the stimulatory effects of amlodipine on eNOS activity. Finally, we showed that the amlodipine-triggered, caveolin-dependent mechanism of eNOS activation was independent of other pleiotropic effects of the CCB such as superoxide anion scavenging and angiotensin-converting enzyme (ACE) inhibition.This study unravels the modulatory effects of amlodipine on caveolar integrity and the capacity of caveolin to maintain eNOS in its vicinity in the absence of any detectable changes in intracellular calcium levels. The resulting increase in caveolin-free eNOS potentiates the NO production in response to agonists including VEGF and bradykinin. More generally, this work opens new avenues of treatment for drugs able to structurally alter signaling pathways concentrated in caveolae.

Endothelium plays a crucial role in the regulation of cardiovascular homeostasis through the release of vasoactive factors. Nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHF) are the two major actors controlling the vasomotor tone. The endothelial nitric oxide synthase (eNOS) was reported in the mid 90ies to be under the control of caveolin, the structural protein of caveolae. Nowadays, a large body of evidence has confirmed that the caveolin/eNOS interaction was needed to prevent inadequate NO production under basal conditions but also to facilitate the integration of extracellular stimuli to intracellular NO signals. Compartmentation of key actors in the EDHF signaling pathway is now also proposed to take place into caveolae. Accordingly, caveolin-deficient animals revealed both an unopposed NO production promoting vessel dilation and a lack of EDHF-driven vasorelaxation. The transient receptor potential (TRP) channels are the link between caveolae and EDHF. Different TRP channels involved in the capacitative calcium entry were found to directly interact with caveolin-1 in endothelial cells. TRPC1 and TRPC4 form a complex with the endoplasmic reticulum IP3 receptor thereby optimizing calcium signaling. EDHF-driven vasodilation was documented to be altered in a TRPV4-deficient mouse model. The close vicinity between TRPV4 and SKCa channels in caveolae together with the gap-junctions subunits connexins support a role of these microdomains in the generation and propagation of EDHF to vascular smooth muscle cells. In conclusion, caveolae and caveolin are important control points in the control of blood pressure by the endothelium. This also highlights how any alteration in the caveolae integrity or caveolin abundance may lead to and/or exacerbate endothelial dysfunction and associated cardiovascular diseases.

This paper focuses on Bayesian shrinkage methods for covariance matrix estimation. We examine posterior properties and frequentist risks of Bayesian estimators based on new hierarchical inverse-Wishart priors. More precisely, we give the conditions for the existence of the posterior distributions. Advantages in terms of numerical simulations of posteriors are shown. A simulation study illustrates the performance of the estimation procedures under three loss functions for relevant sample sizes and various covariance structures.

Cardiac myocytes express the nitric-oxide synthase isoform originally identified in endothelial cells, termed eNOS or NOS3, where it plays a role in regulating myocyte responsiveness to both adrenergic and muscarinic cholinergic autonomic nervous system agonists. eNOS in endothelial cells has been shown to undergo extensive post-translational processing, and in cardiac myocytes as well as endothelial cells, eNOS has been shown to be targeted to plasmalemmal caveolae, a process that is dependent on myristoylation and palmitoylation. Other post-translational modifications essential for the correct subcellular targeting of eNOS have not been described previously. We demonstrate, using [35S]methionine pulse-chase experiments, that native eNOS in adult rat ventricular myocytes is initially translated as a nonpalmitoylated 150-kDa isoform, which is associated with cytosolic and intracellular membrane-enriched fractions. This is subsequently processed to a palmitoylated 135-kDa isoform, which is found only in a sarcolemma-enriched membrane fraction. Forskolin, an agent that elevates intracellular cAMP, rapidly inhibited processing of the 150-kDa isoform to the 135-kDa isoform and transport of eNOS to the sarcolemma, effects paralleled by protein kinase A-dependent phosphorylation of the larger eNOS isoform. Forskolin also decreased palmitoylation of the 135-kDa isoform, although it did not accelerate depalmitoylation of sarcolemmal eNOS, as determined by pulse-chase experiments with [3H]palmitate. Thus, post-translational processing of a 150-kDa isoform of myocyte eNOS appears to be necessary for intracellular trafficking of the enzyme to sarcolemmal caveolae. Both the post-translational processing and subcellular targeting of eNOS appear to be modified by changes in intracellular cAMP, an effect that may have important implications for cardiac myocyte responsiveness to autonomic agonists in vivo. Cardiac myocytes express the nitric-oxide synthase isoform originally identified in endothelial cells, termed eNOS or NOS3, where it plays a role in regulating myocyte responsiveness to both adrenergic and muscarinic cholinergic autonomic nervous system agonists. eNOS in endothelial cells has been shown to undergo extensive post-translational processing, and in cardiac myocytes as well as endothelial cells, eNOS has been shown to be targeted to plasmalemmal caveolae, a process that is dependent on myristoylation and palmitoylation. Other post-translational modifications essential for the correct subcellular targeting of eNOS have not been described previously. We demonstrate, using [35S]methionine pulse-chase experiments, that native eNOS in adult rat ventricular myocytes is initially translated as a nonpalmitoylated 150-kDa isoform, which is associated with cytosolic and intracellular membrane-enriched fractions. This is subsequently processed to a palmitoylated 135-kDa isoform, which is found only in a sarcolemma-enriched membrane fraction. Forskolin, an agent that elevates intracellular cAMP, rapidly inhibited processing of the 150-kDa isoform to the 135-kDa isoform and transport of eNOS to the sarcolemma, effects paralleled by protein kinase A-dependent phosphorylation of the larger eNOS isoform. Forskolin also decreased palmitoylation of the 135-kDa isoform, although it did not accelerate depalmitoylation of sarcolemmal eNOS, as determined by pulse-chase experiments with [3H]palmitate. Thus, post-translational processing of a 150-kDa isoform of myocyte eNOS appears to be necessary for intracellular trafficking of the enzyme to sarcolemmal caveolae. Both the post-translational processing and subcellular targeting of eNOS appear to be modified by changes in intracellular cAMP, an effect that may have important implications for cardiac myocyte responsiveness to autonomic agonists in vivo.

Pyrocarbon deposition through propane pyrolysis is studied in a 1-D hot-wall CVD furnace. The gas-phase pyrolysis is modeled with a partially reduced kinetic mechanism leading to polycyclic aromatic compounds (PAHs). The C2-C4 and C3 reaction paths are in competition for benzene formation. There is also an independent C3-C5 path leading to naphthalene. The gas-phase concentrations are correlated with experimental data including in situ Fourier transform infrared spectra intensities, pyrocarbon deposition rates, and pyrocarbon nanotextures. Rough laminar pyrocarbon deposition appears to be more related to PAHs than smooth laminar pyrocarbon. © 2001 The Electrochemical Society. All rights reserved.

cDNA fragments encoding a representative region of the L‐type calcium channel α‐1 subunit of rabbit intestine smooth muscle were amplified by polymerase chain reaction (PCR). The nucleotide sequences of these intestine clones shared a high similarity with aorta, lung and heart calcium channels. However, in the extracellular loop between the third and fourth segments of domain IV and in the transmembrane IVS3 segment itself, we observed primary sequence variations corresponding to alternative splicing phenomenons. Since structural differences of L‐type calcium channel α‐1 subunits could result in functional variations, the respective expression frequency of these isoforms was determined in various tissues and species, and in the embryonic A7r5 cell line. The ontogeny of these splicing events was also examined from tissues of different ages. From this quantitative study, carried out by PCR of reverse‐transcribed mRNA, it clearly appears that the observed splicing processes in the IVS3–IVS4 region are not only tissue‐dependent but also regulated during development.

When chemically vapour deposited from propane within fibrous preforms, pyrocarbon does not always exhibit the same microstructure in the whole preform. Texture profiles (or extinction angle profiles measured by polarized light optical microscopy) depend markedly on the residence time of the reactive gas in the hot zone and to a lesser extent on the infiltration temperature. For low residence times, a transition is observed from a smooth laminar (weakly anisotropic) texture at the external surface to an in-depth rough laminar (highly anistropic) texture. This transition can be explained on the basis of maturation of the gas phase within the preform. For high residence times, the reverse transition occurs, with a decrease of pyrocarbon anisotropy from the surface to the internal part of the preform. This opposite behavior can be associated to an external gas phase maturation followed by an internal depletion.

Oxygen deficiency in tumors reduces the efficacy of nonsurgical treatment modalities. We tested the hypothesis that electrical stimulation of the sciatic nerve could modify the oxygenation status and the blood flow of tumors implanted in the thigh of mice.The sciatic nerve was electrically stimulated at 5 Hz. Local transplantable liver tumor (TLT) and fibrosarcoma (FSaII) tumor oxygen pressure (pO(2)) and perfusion measurements were carried out using electron paramagnetic resonance (EPR) oximetry and the OxyLite/OxyFlo technique. The radiosensitizing effect of the protocol was assessed by irradiating FSaII tumors with X-rays.Tumor pO(2) increased from approximately 3 mm Hg to approximately 8 mm Hg, and relative tumor blood flow was increased by 241% and 162% for TLT and FSaII tumor models, respectively. The effect on the tumor oxygenation was inhibited by a nitric oxide synthase (NOS) inhibitor, and an increase in the tumor nitric oxide (NO) content was observed using EPR spin-trapping. The tumor oxygen consumption rate was decreased after the stimulation protocol. In addition, the electrical stimulation of the host tissue increased regrowth delays by a factor of 1.65.This increase in tumor oxygenation is due to the temporary increase in tumor blood flow, but particularly to a decrease in the tumor oxygen consumption rate (inhibition of respiration) that is mediated by a local production of NO during the protocol. Those tumor hemodynamic changes resulted in a radiosensitizing effect.

Altered metabolic pathways in cancer such as exacerbated glycolytic flux and increased glutamine metabolism are promising targets for anti-cancer therapies. While commonly observed in glycolytic tumors, extracellular acidosis has never been considered as a potential modulator of anti-metabolic drug activity such as dichloroacetate (DCA). Using cancer cells from various origins selected for their ability to proliferate under acidic conditions, we found that DCA exerts greater inhibitory effects on the growth of these acid-adapted cells than in parental cells. Moreover, daily DCA administration to mice led to a significant decrease in tumor growth from acid-adapted cells but not from parental cells. 13C-tracer studies revealed that DCA induced a double metabolic shift, diminishing glycolysis and increasing intracellular glutamine in acid-adapted cells. As a consequence, DCA reduced the pentose phosphate pathway activity more extensively and increased apoptosis in acid-adapted cells. Finally, the combination of DCA with a glutaminase inhibitor significantly enhanced the cytotoxic effects of DCA. Overall, the interplay between acidosis and DCA exposure leads to metabolic reprogramming that considerably alters cellular fitness.

We study price formation in intraday electricity markets in the presence of intermittent renewable generation. We consider the setting where a major producer may interact strategically with a large number of small producers. Using stochastic control theory, we identify the optimal strategies of agents with market impact and exhibit the Nash equilibrium in a closed form in the asymptotic framework of mean field games with a major player.

This review presents the set of electricity price models proposed in the literature since the opening of power markets. We focus on price models applied to financial pricing and risk management. We classify these models according to their ability to represent the random behavior of prices and some of their characteristics. In particular, this classification helps users to choose among the most suitable models for their risk management problems.

In 2020, the estimated number of new leukemia cases was higher than 30,000 in girls between 0 and 19 years old. Due to cancer treatment, some of these patients may lose both endocrine and reproductive functions. Transplantation of cryopreserved ovarian tissue is not advised after cancer remission because it has a high risk of reintroducing malignant cells in the patient, potentially leading to leukemia recurrence. To safely transplant the ovarian tissue from these patients and restore their fertility, our goal was to develop a photodynamic therapy (PDT) strategy to eliminate leukemia ex vivo. To this end, we designed, optimized, and characterized OR141-loaded niosomes (ORN) to develop the most effective formulation for ex vivo purging ovarian fragments from chronic myelogenous leukemia cells. After establishing the best ORN formulation, the PDT efficiency of optimized ORN was determined for human ovarian stromal cells and acute myeloid leukemia cell line (HL60). Blank niosomes treatment on ovarian stromal cells causes no significant toxicity, showing that the composition of the nanoparticle is not toxic. On the other hand, the in vitro studies showed that while ovarian stromal cells were still viable (82.04 ± 2.79%) after the treatment by 0.5 µM ORN, the same treatment yielded 95.43 ± 3.89% toxicity and cell death in the cancer cells. In conclusion, our results showed that our novel PDT procedure could be a promising strategy to destroy leukemia cells in ovarian tissue fragments allowing safe transplantation in cancer survivors.

1. The tissue-protective effects of calcium channel blockers in hypertension are not well dissociated from their effect on systolic blood pressure (SBP). We have previously shown that lacidipine, a dihydropyridine-type calcium antagonist, reduced the cardiac hypertrophy and the cardiac endothelin-1 (ET-1) gene overexpression occurring in salt-loaded stroke-prone spontaneously hypertensive rats (SL-SHRSP), an effect occurring without systolic blood pressure (SBP) change. In the present study, we have examined whether this action was dose-related and if it could be associated with ET receptor changes. The action of lacidipine was also examined in control SHRSP and in Wistar Kyoto rats (WKY). 2. The daily dose of 0.3 mg kg-1 lacidipine which did not lower SBP but significantly prevented ventricle hypertrophy and cardiac preproET-1-mRNA expression in SL-SHRSP was inactive in control SHRSP. With the higher dose of lacidipine (1 mg kg-1 day-1), we observed a further reduction of cardiac hypertrophy and of ET-1 gene expression in SL-SHRSP and a significant effect on those parameters in control SHRSP but only a small reduction of SBP in both groups. 3. In WKY, salt loading did not induce change in SBP or increase of cardiac ET-1 gene expression and ventricle mass. In these normotensive rats, lacidipine (1 mg kg-1 day-1) did not modulate the basal preproET-1-mRNA expression and did not affect SBP or heart weight. 4. The maximum binding capacity (Bmax) and the dissociation constant (KD) of [125I]-ET-1 binding and the relative proportion of low- and high-affinity binding sites for ET-3 were not significantly affected by salt loading or lacidipine treatment in SHRSP. 5. These results show that lacidipine exerted a dose-related inhibition of ventricle hypertrophy and preproET-1-mRNA expression in SHRSP and indicate that this effect was unrelated to SBP changes. The dose-dependency of this inhibition suggests that salt-induced cardiac hypertrophy could be related to ET-1 gene overexpression. The results further show that ET receptor changes are not involved in the pathophysiological process studied here.

Peritoneal dialysis (PD) is an established renal replacement therapy in diabetic patients, but the influence of diabetes on the peritoneal membrane (PM) remains debated. We have used functional, biochemical and molecular studies in vivo and in vitro to substantiate the changes induced by diabetes and hyperglycemia in the PM.Peritoneal equilibration tests were performed 2, 4, and 6 weeks after induction of diabetes with streptozotocin (STZ) in rats. Morphological analyses, determination of nitric oxide synthase (NOS) activities, and expression studies for NOS isoforms and advanced glycation end products (AGE) were performed in parallel. Additional studies were conducted in diabetic rats treated with insulin, non-diabetic rats fed with urea, and cultured bovine aortic endothelial cells (BAEC).In comparison with controls, diabetic rats were characterized by: increased permeability for small solutes and decreased sodium sieving; capillary proliferation; increased endothelial NOS (eNOS) and AGE immunoreactivity; up-regulation of eNOS and down-regulation of neuronal NOS; and increased NOS activity in the PM. The changes, which culminated at week 6, were prevented by chronic insulin treatment in diabetic rats. In contrast to hyperglycemia, hyperosmolality alone did not induce functional or structural changes in the PM. Studies in BAEC showed that high glucose incubation led to increased activity and expression of eNOS, a prerequisite for vascular proliferation.These data demonstrate that chronic hyperglycemia is associated with functional and structural changes in the peritoneum that parallel with selective regulation of NOS isoforms and AGE deposits. The alterations are prevented by insulin treatment, which suggests that adequate control of diabetes can preserve PM integrity in diabetic patients prior to PD.

Objective: Induction of angiogenesis has been reported subsequent to eNOS overexpression or activation, the latter involving Hsp90 as a chaperone protein. Here, we investigated the potential of regional Hsp90 overexpression to induce therapeutic neovascularization in vivo in a chronic rabbit hindlimb ischemia model.

Purpose of review Although saturated fatty acid (FA) (SFA) and monounsaturated FA (MUFA) are synthesized in cancer cells from acetyl-CoA, polyunsaturated FAs (PUFAs) are necessarily obtained from diet. Depending on concentrations and metabolism, these different FAs may support tumor proliferation but also exert growth inhibitory effects. The mutual interplay between them also requires to integrate the FA oxidation component that may be concomitant with FA synthesis is cancer cells. Recent findings New molecular mechanisms driving FA synthesis, lipotoxicity and anti-inflammatory activity of eicosanoids in mouse and human cancers were recently elicited. To block or take advantage of the above represent attractive perspectives of treatments to fight cancer progression. Summary The various enzymatic reactions leading to SFA synthesis represent as many targets to prevent tumor growth. Ironically excess SFAs are per-se toxic for cancer cells and the introduction of a double bound to form MUFA is actually limiting lipotoxicity in cancer cells. Blocking stearoyl-CoA desaturase therefore represents another attractive modality. By contrast, dietary PUFAs may exert direct cytotoxic effects by promoting apoptosis or by generating anti-inflammatory eicosanoids. Altogether, these data point out the intricate relationship between SFA, MUFA and PUFA at the heart of the metabolism of proliferating cancer cells.

Crosstalk between cancer-associated fibroblasts (CAFs) and colorectal cancer cells promotes tumor growth and contributes to chemoresistance. In this study, we assessed the sensitivity of a primary CAF cell line, CT5.3hTERT, to standard-of-care and alternative cytotoxic treatments. Paclitaxel (PTX) and acriflavine (ACF) were identified as the most promising molecules to inhibit CAF development. To allow the translational use of both drugs, we developed lipid nanocapsule (LNC) formulations for PTX and ACF. Finally, we mixed CAFs and tumor cell lines in a cocultured spheroid, and the effect of both drugs was investigated by histological analyses. We demonstrated CAF inhibition by LNC-ACF and whole tumor inhibition by LNC-PTX. Altogether, we proposed a new strategy to reduce CAF populations in the colorectal microenvironment that should be tested in vivo.

Abstract Epidemiological studies have shown that obese subjects have an increased risk of developing triple‐negative breast cancer (TNBC) and an overall reduced survival. However, the relation between obesity and TNBC remains difficult to understand. We hypothesize that apelin, an adipokine whose levels are increased in obesity, could be a major factor contributing to both tumour growth and metastatization in TNBC obese patients. We observed that development of obesity under high‐fat diet in TNBC tumour‐bearing mice significantly increased tumour growth. By showing no effect of high‐fat diet in obesity‐resistant mice, we demonstrated the necessity to develop obesity‐related disorders to increase tumour growth. Apelin mRNA expression was also increased in the subcutaneous adipose tissue and tumours of obese mice. We further highlighted that the reproduction of obesity‐related levels of apelin in lean mice led to an increased TNBC growth and brain metastases formation. Finally, injections of the apelinergic antagonist F13A to obese mice significantly reduced TNBC growth, suggesting that apelinergic system interference could be an interesting therapeutic strategy in the context of obesity and TNBC.

Specific diets for cancer patients have the potential to offer an adjuvant modality to conventional anticancer therapy. If the concept of starving cancer cells from nutrients to inhibit tumor growth is quite simple, the translation into the clinics is not straightforward. Several diets have been described including the Calorie-restricted diet based on a reduction in carbohydrate intake and the Ketogenic diet wherein the low carbohydrate content is compensated by a high fat intake. As for other diets that deviate from normal composition only by one or two amino acids, these diets most often revealed a reduction in tumor growth in mice, in particular when associated with chemo- or radiotherapy. By contrast, in cancer patients, the interest of these diets is almost exclusively supported by case reports precluding any conclusions on their real capacity to influence disease outcome. In parallel, the field of tumor lipid metabolism has emerged in the last decade offering a better understanding of how fatty acids are captured, synthesized or stored as lipid droplets in cancers. Fatty acids participate to cancer cell survival in the hypoxic and acidic tumor microenvironment and also support proliferation and invasiveness. Interestingly, while such addiction for fatty acids may account for cancer progression associated with high fat diet, it could also represent an Achilles heel for tumors. In particular n-3 polyunsaturated fatty acids represent a class of lipids that can exert potent cytotoxic effects in tumors and therefore represent an attractive diet supplementation to improve cancer patient outcomes.

Targeting enzymes involved in tumor metabolism is a promising way to tackle cancer progression. The inhibition of carnitine palmitoyltransferase 1 (CPT1) by etomoxir (Eto) efficiently slows down the growth of various cancers. Unfortunately, the clinical use of this drug was abandoned because of hepatotoxic effects. We report the development of pH-sensitive peptide (pHLIP)-drug conjugate to deliver Eto selectively to cancer cells exposed to acidic microenvironmental conditions. A newly designed sequence for the pHLIP peptide, named pHLIPd, was compared with a previously published reference pHLIP peptide, named pHLIPr. We showed that the conjugate between pHLIPd and Eto has a better pH-dependent insertion and structuration than the pHLIPr-based conjugate inside POPC vesicles. We observed antiproliferative effects when applied on acid-adapted cancer cells, reaching a larger inhibitory activity than Eto alone. In conclusion, this study brings the first evidence that pHLIP-based conjugates with a CPT1 inhibitor has the potential to specifically target the tumor acidic compartment and exert anticancer effects while sparing healthy tissues.

Abstract Change points detection in time series is an important area of research in statistics, has a long history and has many applications. However, very often change point analysis is only focused on the changes in the mean value of some quantity in a process. In this work we consider time series with discrete point changes which may contain a finite number of changes of probability density functions (pdf). We focus on the case where the data in all segments are modeled by Gaussian probability density functions with different means, variances and correlation lengths. We put a prior law on the change point occurances (Poisson process) as well as on these different parameters (conjugate priors) and give the expression of the posterior probability distributions of these change points. The computations are done by using an appropriate Markov Chain Monte Carlo (MCMC) technique. The problem as we stated can also be considered as an unsupervised classification and/or segmentation of the time serie. This analogy gives us the possibility to propose alternative modeling and computation of change points. © 2007 Wiley Periodicals, Inc. Int J Imaging Syst Technol, 16, 215–221, 2006

We propose a new structural model that can compute the electricity spot and forward prices in two coupled markets with limited interconnection and multiple fuels. We choose a structural approach in order to represent some key characteristics of electricity spot prices such as their link to fuel prices, consumption level and production fleet. With this model, explicit formulas are also available for forward prices and other derivatives. We give some illustrative results of the behaviour of spot, forward and transmission rights prices.

Is it possible to purge leukemia cells from ovarian tissue (OT) fragments before transplantation?Our photodynamic therapy (PDT) approach has been shown to efficiently destroy leukemia cells from tumor-infiltration mimicking models (TIMs), indicating the feasibility of this technique to purge OT samples.Autotransplantation of cryopreserved OT is the most suitable option to preserve fertility for prepubertal girls and women who require immediate cancer treatment. Up until now, more than 200 live births have already been reported after OT cryopreservation and transplantation. Leukemia is the 12th most common cancer in Europe among prepubertal girls and women of reproductive age and in 2020, the estimated number of new leukemia cases was higher than 33 000 in girls between 0 and 19 years old. Unfortunately, once their health has been restored, autotransplantation of cryopreserved OT for leukemia patients is not advised due to the high risk of transferring malignant cells back to the patient leading to leukemia recurrence.To safely transplant the OT from leukemia patients and restore their fertility, our goal was to develop a PDT strategy to eliminate leukemia ex vivo. To this end, we designed OR141-loaded niosomes (ORN) to create the most effective formulation for ex vivo purging of acute myelogenous leukemia cells from OT fragments (n = 4). Moreover, to ensure that such treatments are not harmful to follicle survival and development so they can be deemed a potential fertility restoration alternative, the effect of the ORN-based PDT purging procedure on follicles was assessed after xenografting the photodynamic-treated OT in SCID mice (n = 5). The work was carried out between September 2020 and April 2022 at the Catholic University of Louvain.After establishing the best ORN formulation, our PDT approach was used to eradicate HL60 cells from ex vivo TIMs prepared by microinjection of a cancer cell suspension into OT fragments. The purging efficiency was analyzed by droplet digital polymerase chain reaction and immunohistochemical analyses. Additionally, we evaluated the effect of ORN-based PDT on follicle density, survival and development, and tissue quality in terms of fibrotic areas and vascularization after 7-day xenotransplantation to immunodeficient mice.The ex vivo purging of TIMs demonstrated that our PDT strategy could selectively eradicate the malignant cells from tissue fragments without affecting OT normal cells, as evidenced by PCR and immunohistochemical analysis. Regarding the effect of our PDT approach on follicle population and OT quality, our results after xenotransplantation revealed no significant difference between the follicle density of control (non-treated, grafted OT) and PDT-treated groups (2.38 ± 0.63 and 3.21 ± 1.94 morphologically normal follicles/mm2, respectively). In addition, our findings showed that the control and PDT-treated OT could be equally vascularized (7.65 ± 1.45% and 9.89 ± 2.21%, respectively). Similarly, the proportions of fibrotic area did not differ between the control (15.96 ± 5.94%) and PDT-treated groups (13.32 ± 3.05%).N/A.This study did not use OT fragments from leukemia patients, but TIMs created after injection of HL60 cells into OT from healthy patients. Therefore, while the results are promising, whether our PDT approach will be equally successful in eliminating malignant cells from leukemia patients remains to be assessed.Our results showed that the purging procedure causes no significant impairment effect on follicle development and tissue quality, suggesting that our novel PDT procedure could be a promising strategy to destroy leukemia cells in fragments of OT, allowing safe transplantation in cancer survivors.This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A.); Fondation Louvain (awarded to C.A.A.; a Ph.D. scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and a Ph.D. scholarship awarded to A.D. as part of a legacy from Mrs. Ilse Schirmer); and Foundation Against Cancer (grant number 2018-042 awarded to A.C.). The authors declare no competing interests.

The palladium-catalyzed uncaging of alloc-protected amines in living cells has been extensively studied mostly in the form of nanostructures or mesostructures. However, the scarcity of kinetic and mechanistic studies of discrete palladium complexes for deallylation reactions has hindered progress. Herein, we report the development of a series of discrete palladium complexes bearing acetanilide ligands, which exhibit a good balance between reactivity and stability in living cells. We investigated the catalytic activity and cytotoxicity of these complexes in SiHa cells and found that acetanilide[tri(2-furyl)phosphine]palladium(II) triflate showed promising results. Under physiological conditions, this palladium complex exhibited a second-order reaction rate of 30 M−1 s−1, and liquid chromatography-mass spectrometry (LC-MS) studies suggested the possibility of a tandem Heck/Tsuji-Trost mechanism. Our results demonstrate the potential of using these discrete palladium complexes for bioorthogonal chemistry studies in living cells.

We study the calibration of specific multi-factorial Heath-Jarrow-Morton models to power market prices, with a focus on the estimation of the optimal number of Gaussian factors. We describe a common statistical procedure based on likelihood maximisation and Akaike/Bayesian information criteria, in the case of a joint calibration on both spot and futures prices. We perform a detailed analysis on three national markets within Europe: Belgium, France, and Germany. The results show a lot of similarities among all the markets we consider, especially on the optimal number of factors and on the behaviour of the different factors.

Tumour progression drives profound alterations in host metabolism, such as adipose tissue depletion, an early event of cancer cachexia. As fatty acid consumption by cancer cells increases upon acidosis of the tumour microenvironment, we reasoned that fatty acids derived from distant adipose lipolysis may sustain tumour fatty acid craving, leading to the adipose tissue loss observed in cancer cachexia. To evaluate the pro-lipolytic capacities of acid-exposed cancer cells, primary mouse adipocytes from subcutaneous and visceral adipose tissue were exposed to pH-matched conditioned medium from human and murine acid-exposed cancer cells (pH 6.5), compared to naive cancer cells (pH 7.4). To further address the role of tumoral acidosis on adipose tissue loss, a pH-low insertion peptide was injected into tumour-bearing mice, and tumoral acidosis was neutralised with a sodium bicarbonate buffer. Prolipolytic mediators were identified by transcriptomic approaches and validated on murine and human adipocytes. Here, we reveal that acid-exposed cancer cells promote lipolysis from subcutaneous and visceral adipocyte and that dampening acidosis in vivo inhibits adipose tissue depletion. We further found a set of well-known prolipolytic factors enhanced upon acidosis adaptation and unravelled a role for β-glucuronidase as a promising new actor in adipocyte lipolysis. Tumoral acidosis promotes the mobilization of fatty acids derived from adipocytes via the release of soluble factors by cancer cells. Our work paves the way for dual therapeutic approaches aimed at tackling cachexia by targeting the tumour acidic compartment.

Electricity price forecasting (EPF) plays a major role for electricity companies as a fundamental entry for trading decisions or energy management operations. As electricity can not be stored, electricity prices are highly volatile which make EPF a particularly difficult task. This is all the more true when dramatic fortuitous events disrupt the markets. Trading and more generally energy management decisions require risk management tools which are based on probabilistic EPF (PEPF). In this challenging context, we argue in favor of the deployment of highly adaptive black-boxes strategies allowing to turn any forecasts into a robust adaptive predictive interval, such as conformal prediction and online aggregation, as a fundamental last layer of any operational pipeline. We propose to investigate a novel data set containing the French electricity spot prices during the turbulent 2020-2021 years, and build a new explanatory feature revealing high predictive power, namely the nuclear availability. Benchmarking state-of-the-art PEPF on this data set highlights the difficulty of choosing a given model, as they all behave very differently in practice, and none of them is reliable. However, we propose an adequate conformalisation, OSSCP-horizon, that improves the performances of PEPF methods, even in the most hazardous period of late 2021. Finally, we emphasize that combining it with online aggregation significantly outperforms any other approaches, and should be the preferred pipeline, as it provides trustworthy probabilistic forecasts.

The interaction of pinaverium bromide, a quaternary ammonium compound, with binding sites for (L‐type) calcium channel blockers was investigated in rat ileum smooth muscle. Pinaverium inhibited [ 3 H]‐(+)‐PN200–110 ([ 3 H]‐(+)‐isradipine) specific binding to tissue homogenates incompletely ( K i 0.38 μ m ; maximal inhibition 80%). In contrast, binding to single cell preparations (obtained by collagenase treatment) and to saponin‐treated homogenates was completely inhibited. These data are compatible with the view that, in untreated homogenates, 20% of [ 3 H]‐(+)‐isradipine binding sites are not accessible to pinaverium because it is associated with sealed inside‐out vesicles. Pinaverium bromide increased the apparent K D of [ 3 H]‐(+)‐isradipine binding to saponin‐treated homogenates but did not significantly affect the B max value. Moreover, the dissociation rate constant of [ 3 H]‐(+)‐isradipine binding was not changed by pinaverium. These data suggest that pinaverium interacts with the dihydropyridine binding site in a competitive manner. However, in contrast to uncharged dihydropyridine calcium antagonists, pinaverium inhibited, rather than stimulated, [ 3 H]‐diltiazem binding to rat brain membranes (at 30–37°C). Although B max values of [ 3 H]‐(+)‐isradipine were similar in homogenates prepared from tissue and cells (collagenase‐treated), the K D value was significantly higher in cell homogenates (166 vs 95 p m ). Similarly, the K i value of pinaverium was higher in cell preparations than in tissue homogenates (0.77 vs 0.38 μ m ). Thus, collagenase can significantly modify the dihydropyridine recognition site. The competitive interaction of pinaverium, a permanently charged drug, with [ 3 H]‐(+)‐isradipine bound to intact cells and its absence of interaction with [ 3 H]‐(+)‐isradipine bound to sealed inside‐out vesicles imply that the dihydropyridine receptor lies near the external surface of the plasma membrane.

Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze-thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKβ1-/- mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.

We develop a tractable equilibrium model for price formation in intraday electricity markets in the presence of intermittent renewable generation. Using stochastic control theory, we identify the optimal strategies of agents with market impact and exhibit the Nash equilibrium in closed form for a finite number of agents as well as in the asymptotic framework of mean field games. Our model reproduces the empirical features of intraday market prices, such as increasing price volatility at the approach of the delivery date and the correlation between price and renewable infeed forecasts, and relates these features with market characteristics like liquidity, number of agents, and imbalance penalty.

The isoform of nitric oxide synthase (eNOS or NOS3) originally described in endothelial cells is also expressed in a number of other cell types, including cardiac myocytes. eNOS is activated in both atrial and ventricular myocytes, including specialized pacemaker cells, by M2AChR agonists, among other stimuli. In cardiac myocytes, as in endothelial cells, eNOS is targeted to sarcolemmal caveolae, due to both co-translational myristoylation and later palmitoylation, and by the presence of a caveolin binding domain in eNOS which interacts with the caveolin scaffolding domain. In the absence of ligand, the M2AChR is not associated with caveolar microdomains, but translates into caveolae upon agonist (but not antagonist) binding. Finally, the role of M2AChR-induced eNOS activation in regulating I(Ca-L) via activation of guanylyl cyclase has been confirmed in ventricular myocytes of mice that lack functional eNOS (i.e., eNOS(null)).

A Fourier transform IR spectrometer and a mass spectrometer have been coupled to a pyrocarbon low pressure CVD reactor in order to investigate the gas phase during pyrolysis of propane in the range 600–1060°C. The variations of the amounts of intermediate species such as ethylene, acetylene, and benzene are followed by infrared absorption measurements as a function of temperature and residence time. Mass spectrometry allows additional minor species to be identified. The overall study permits a reaction pathway from propane to diacetylene, benzene, and heavier species to be proposed.

This paper deals with Gibbs samplers that include high dimensional conditional Gaussian distributions.It proposes an efficient algorithm that avoids the high dimensional Gaussian sampling and relies on a random excursion along a small set of directions.The algorithm is proved to converge, i.e. the drawn samples are asymptotically distributed according to the target distribution.Our main motivation is in inverse problems related to general linear observation models and their solution in a hierarchical Bayesian framework implemented through sampling algorithms.It finds direct applications in semiblind / unsupervised methods as well as in some non-Gaussian methods.The paper provides an illustration focused on the unsupervised estimation for super-resolution methods.

Endothelin-1 is a potent vasoactive peptide which may play a role in the regulation of vascular resistance through its autocrine/paracrine effects. We have investigated the influence of salt loading on the renal and cardiac production of endothelin-1 in stroke prone spontaneously hypertensive rats, a classical model of hypertension. The results show that the dietary salt intake did not change systolic blood pressure nor the renal expression of the preproendothelin-1 mRNA but increased cardiac expression of the endothelin-1 gene transcript and a concomitant ventricular hypertrophy.

In this paper we propose a stochastic algorithm applied to an electromagnetic inverse scattering problem. The objective is to characterise an unknown object from measurements of the scattered fields at different frequencies and for several illuminations. This inverse problem is known to be nonlinear and ill-posed. It then needs to be regularized by introducing prior information. The particular prior information we account for is that the object is composed of a known finite number of different materials distributed in compact regions. The algorithm is applied to the inversion of experimental data collected at the Institut Fresnel (Marseille) and has already provided satisfactory results in a 2D-TM configuration. Herein, the goal is to test the same kind of method in a 2D-TE configuration.

Abstract Purpose: Optimal head and neck squamous cell carcinoma (HNSCC) patient selection for anti–EGFR-based therapy remains an unmet need since only a minority of patients derive long-term benefit from cetuximab treatment. We assessed the ability of state-of-the-art noninvasive in vivo metabolic imaging to probe metabolic shift in cetuximab-sensitive and -resistant HNSCC patient-derived tumor xenografts (PDTXs). Experimental Design: Three models selected based on their known sensitivity to cetuximab in patients (cetuximab-sensitive or acquired-resistant HNC007 PDTXs, cetuximab-naïve UCLHN4 PDTXs, and cetuximab-resistant HNC010 PDTXs) were inoculated in athymic nude mice. Results: Cetuximab induced tumor size stabilization in mice for 4 weeks in cetuximab-sensitive and -naïve models treated with weekly injections (30 mg/kg) of cetuximab. Hyperpolarized 13C-pyruvate–13C-lactate exchange was significantly decreased in vivo in cetuximab-sensitive xenograft models 8 days after treatment initiation, whereas it was not modified in cetuximab-resistant xenografts. Ex vivo analysis of sensitive tumors resected at day 8 after treatment highlighted specific metabolic changes, likely to participate in the decrease in the lactate to pyruvate ratio in vivo. Diffusion MRI showed a decrease in tumor cellularity in the HNC007-sensitive tumors, but failed to show sensitivity to cetuximab in the UCLHN4 model. Conclusions: This study constitutes the first in vivo demonstration of cetuximab-induced metabolic changes in cetuximab-sensitive HNSCC PDTXs that were not present in resistant tumors. Using metabolic imaging, we were able to identify hyperpolarized 13C-pyruvate as a potential marker for response and resistance to the EGFR inhibitor in HNSCC.

Nitric oxide production by the Ca2+/calmodulin-dependent enzyme endothelial nitric oxide synthase primarily reflects changes in intracellular [Ca2+], increasing as Ca2+ rises and decreasing as Ca2+ falls. This simplistic bimodal mechanism of regulation has recently been refined by the finding that the binding of Ca2+/calmodulin to endothelial nitric oxide synthase involves the disruption of the association of endothelial nitric oxide synthase from the scaffolding protein caveolin and the subsequent translocation of the enzyme from plasmalemmal caveolae. Moreover, other endothelial nitric oxide synthase-associated proteins could account for a delayed Ca2+-independent activation of nitric oxide production, and may be involved with caveolin in the reversible trafficking of a large endothelial nitric oxide synthase-containing heterocomplex between the caveolae and cytosolic cell structures.

In order to build a numerical model for the growth of ternary (InGaAs, InGaP) or quaternary materials (InGaAsP), data on growth rate and composition distributions in a horizontal reactor have been collected. Samples were grown from trimethylindium, trimethylgallium, tert-butylarsine and tert-butylphosphine. Composition analyses were carried out by X-ray photoelectron spectroscopy. The results are discussed in relation to preliminary simulation works and a linear combination model based on experimental data for binary materials growth. The study enhances the understanding of growth mechanisms. Henceforth, modelling of InGaAsP growth seems possible but accurate simulation should take into account multicomponent diffusion and the enhancement of precursors' decomposition occurring while mixed.

Abstract Study question Is it possible to purge leukemia cells from ovarian tissue (OT) fragments before transplantation? Summary answer Our photodynamic therapy (PDT) approach showed to efficiently destroy leukemia cells from a tumor model, indicating the feasibility of this technique to purge OT samples. What is known already Autotransplantation of the cryopreserved OT is the most suitable option to preserve fertility for prepubertal girls and women that require immediate cancer treatment [1, 2]. Up to now, more than 200 live births have already been reported after OT cryopreservation and transplantation [3]. Leukemia is the 12th most common cancer in Europe among prepubertal girls and women of reproductive age [4]. Unfortunately, once their health has been restored, autotransplantation of cryopreserved OT for leukemia patients is not advised due to the high risk of transferring malignant cells back to the patient, leading to leukemia recurrence [3]. Study design, size, duration To safely transplant the OT from these patients and restore their fertility, our goal here was to develop a PDT strategy to eliminate leukemia ex vivo. To this end, we designed OR141-loaded niosomes (ORN) to create the most effective formulation for ex vivo purging OT fragments from myelogenous leukemia cells. Participants/materials, setting, methods After establishing the best ORN formulation, our PDT approach was used to eradicate HL60 from ex vivo tumor models prepared by microinjection of cancer cell suspension in ovarian fragments [5]. Additionally, we evaluated the effect of ORN-based PDT on follicle density, survival, and tissue quality was evaluated after 7-day xenotransplantation to SCID mice. Main results and the role of chance The ex vivo purging of tumor models demonstrated that the PDT strategy with ORN could selectively eradicate the malignant cells from tissue fragments without affecting ovarian tissue normal cells, as evidenced by PCR and immunohistochemical analysis. Regarding the effect of our PDT approach on follicle population and OT quality, our results after xenotransplantation revealed no significant difference between the follicle density of control (non-treated, grafted OT) and ORN-treated groups (2.38 ± 0.63 and 3.21 ± 1.94 morphologically normal follicles/mm2, respectively). In addition, the results showed that the control and ORN-treated ovarian tissue can be equally vascularized (7.65±1.45% and 9.89±2.21%, respectively). Moreover, the proportion of fibrotic area in control and ORN-treated groups were 15.96±5.94%, and 13.32±3.05%, which is not significantly different from the pre-graft fragments (14.22±6.65%). Limitations, reasons for caution This study did not use OT fragments from leukemia patients, but tumor models created after injection of HL60 cells in OT from healthy patients. Therefore, while the results are promising, it still remains to assess if our PDT approach will be equally successful to eliminate malignant cells from leukemia patients. Wider implications of the findings Our results showed that the purging procedure causes no significant impairing effect on follicles development and tissue quality, suggesting our novel PDT procedure could be a promising strategy to destroy leukemia cells in fragments of ovarian tissue allowing its safe transplantation in cancer survivors. Trial registration number not applicable

Cycling hypoxia (cyH), neo-angiogenesis, and tumor-associated macrophages are key features of the tumor microenvironment. In this study, we demonstrate that cyH potentiates the induction by unpolarized and M1-like macrophages of endothelial inflammatory phenotype and adhesiveness for monocytes and cancer cells. This process triggers a positive feedback loop sustaining tumor inflammation. This work opens the door for innovative therapeutic strategies to treat tumor inflammation and metastasis. In cancers, the interaction between macrophages and endothelial cells (ECs) regulates tumor inflammation and metastasis. These cells are both affected by cycling hypoxia (cyH), also called intermittent hypoxia, a feature of the tumor microenvironment. cyH is also known to favor tumor inflammation and metastasis. Nonetheless, the potential impact of cyH on the dialog between macrophages and ECs is still unknown. In this work, the effects of unpolarized, M1-like, and M2-like macrophages exposed to normoxia, chronic hypoxia (chH), and cyH on endothelial adhesion molecule expression, pro-inflammatory gene expression, and EC adhesiveness for monocytes and cancer cells were investigated. cyH increased the ability of unpolarized and M1-like macrophages to induce EC inflammation and to increase the expression of the EC endothelial adhesion molecule ICAM1, respectively. Unpolarized, M1-like, and M2-like macrophages were all able to promote EC adhesive properties toward cancer cells. Furthermore, the ability of macrophages (mostly M1-like) to shift EC phenotype toward one allowing cancer cell and monocyte adhesion onto ECs was potentiated by cyH. These effects were specific to cyH because they were not observed with chH. Together, these results show that cyH amplifies the effects of macrophages on ECs, which may promote tumor inflammation and metastasis.

Supplementary Methods from Lactate Influx through the Endothelial Cell Monocarboxylate Transporter MCT1 Supports an NF-κB/IL-8 Pathway that Drives Tumor Angiogenesis

In contrast to canonical ferroptosis inducers, highly peroxidable conjugated linolenic acids (CLnA) directly fuel the lipid peroxidation cascade upon their incorporation into membrane phospholipids. Little is known, however, about the cytotoxicity level of CLnAs to normal epithelial cells. Caco-2 cells, derived from colorectal adenocarcinoma, spontaneously differentiate into enterocyte-like cells over a period of 21 days of cell culturing, allowing for graduated phenotypic shift from proliferative, undifferentiated cells to a functional intestinal barrier. We exploited this property to assess the sensitivity of Caco-2 cells to CLnAs at different stages of differentiation. Our results show a significant decrease in CLnA-induced ferroptotic cell death over time. The acquired resistance aligned with decreases in cell proliferation and in the extent of lipid peroxidation, as well as with an increase in the expression of GPX4 upon differentiation. These results highlight that while CLnAs are highly toxic for proliferating cancer cells, differentiated epithelial cells are resistant to CLnA-induced ferroptosis. Therefore, this study gives credential to the therapeutic use of CLnAs as an anticancer strategy and offers a new model study to further investigate the safety of peroxidable fatty acids in differentiated cells.

A bstract : Endothelial cells constitutively express the NOS isoform eNOS, which generates NO in response to specific extracellular signals to regulate vascular smooth muscle tone, vascular permeability, and platelet adhesion, among other actions. In addition to coronary vascular and endocardial endothelium, both atrial and ventricular myocytes express eNOS, the activation of which is also dependent on specific intracellular and extracellular signals. eNOS is targeted in cardiac myocytes to caveolae in plasma membranes and, in the case of cardiac myocytes, possibly T‐tubular membranes as well. eNOS targeting to caveolae in cardiac myocytes requires co‐translational myristoylation and subsequent palmitoylation for efficient targeting of the enzyme to the specialized lipid microdomains characteristic of caveolae. Although eNOS also contains a caveolin binding motif, this is insufficient for correct targeting of eNOS to caveolae. Recent evidence obtained from ventricular myocytes of mice with targeted disruption of the eNOS gene indicates that the lack of functional eNOS interrupts muscarinic cholinergic control of I CA‐L in these cells. eNOS‐/‐mice are hypertensive and develop cardiac hypertrophy as they age, and these animals also exhibit an accelerated degree of vascular remodeling in response to injury. Reconstitution experiments confirm both the essential role of eNOS in coupling m2 AchR signaling to the control of I CA‐L and myocyte automaticity and the importance of eNOS subcellular localization within caveolae in mediating this signal transduction pathway. It appears that translocation into caveolae is essential for signaling. However, this is not the case with all receptors associated with caveolae.

We have deposited III-V alloys on 200 mm Si miscut wafers with an oxide pattern. The selective epitaxial growth (SEG) of GaAs in large windows defined by SiO2 lines on a thick strained-relaxed Ge buffer layer served as a test vehicle which allowed us to demonstrate the integration of a III-V material deposition process step in a Si manufacturing line using an industrial reactor. High quality GaAs layers with high wafer-scale thickness uniformity were achieved. In a subsequent step, SEG of InP was successfully performed on wafers with a 300 nm shallow trench isolation pattern. The seed layer morphology depended on the treatment of the Ge surface and on the growth temperature. Such an approach for the integration of III-V materials on Si substrates allowed us to obtain extended-defect-free epitaxial regions suitable for the fabrication of high-performance devices.

Endothelium-derived nitric oxide is a key determinant of blood pressure homeostasis and platelet aggregation, and is synthesized by the endothelial isoform of nitric oxide synthase. In the vascular wall, endothelial nitric oxide synthase is activated by diverse cell surface receptors and by an increase in blood flow. Although initially classified as a constitutive enzyme, many substances and conditions that regulate endothelial nitric oxide synthase gene expression are now described. With the recent discovery of endothelial nitric oxide synthase being compartmentalized in signal-transducing microdomains of the plasma membrane termed caveolae, the past 2 years have mostly witnessed important advances in understanding the control of the functional expression of the enzyme.

Stroke-prone spontaneously hypertensive rats receiving a high salt diet were orally treated by the calcium antagonist lacidipine, at a dose which did not reduce systolic blood pressure. We observed that lacidipine inhibited the salt-induced cardiac hypertrophy and the concomitant increase of mRNA transcripts for preproendothelin-1 in ventricles. These data show that elevated blood pressure cannot necessarily account for cardiac hypertrophy and indicate that the therapeutic action of lacidipine is not only related to its haemodynamic properties, but also to the inhibition of the gene expression of growth factors such as endothelin.

Gas phase analyses and kinetic studies were carried out in-situ during the CVD of pyrocarbon from a methane/carbon tetrachloride mixture at low pressure (2 kPa) and constant flow rate (50 sccm). At low temperatures (<800°C), it is assumed that pyrocarbon is formed from chlorinated intermediate species with an apparent activation energy of 180 kJ/mol. At higher temperatures, the apparent activation energy is 140 kJ/mol and unsaturated hydrocarbons are the most probable carbon precursors.

In order to develop a computer-assisted process optimization of In 1- x Ga x As y P 1- y metalorganic chemical vapor deposition (MOCVD), the kinetics of GaAs growth was studied as the first step. For the accumulation of reaction data of source materials, the decomposition rates of trimethylgallium (TMGa) and tertiarybutylarsine (TBAs) were studied using a flow tube reactor and a Fourier transform infrared spectrometer (FT-IR). Special attention was paid to the effect of TBAs concentration on the decomposition rates of TMGa. The GaAs growth rate profile in a commercial MOCVD reactor was analyzed through both experiment and simulation. The profile was dependent on the gas velocity and total pressure. This dependency was explained by a reaction model which was deduced from the experimental observations: TMGa decomposes to a gas-phase intermediate which subsequently forms the GaAs film. The fluid dynamic calculations combined with this reaction model led to growth rate distributions which agreed well with the experimental data. The analysis revealed that the GaAs growth rate is limited by the gas-phase reactions of TMGa as well as the mass-transport of the intermediates, and that precise measurement of the reaction between TMGa and TBAs is essential for an accurate simulation.

This paper addresses the issue of model calibration to electricity prices. The non-storability of electricity introduces new problems in terms of modeling and calibration, especially when the objective is to represent both spot prices and forward products, the latter showing a particular time interval: the delivery period. The two main approaches to model electricity prices are: (i) models on a fictitious forward curve from what we can deduce spot prices and forward products with any delivery period, and (ii) models on spot prices from what we can deduce any forward products. In this paper we study both approaches and we focus on the calibration issues. The first part of the paper studies different calibration methods for a classic Gaussian factorial model as described in Benth and Koekebakker (2008), Kiesel, Schidlmayr, and Börger (2009) and mostly based on Heath-Jarrow-Morton approach (Heath, Jarrow, and Morton, Econometrica, 1992). In this case different calibration methods can be proposed, based on spot and/or forward prices, but the main objective is to compare or validate these estimation procedures. We compare these procedures on the valuation of specific portfolios and we then stress the high impact of the calibration method. The second part concerns the calibration issues of a structural model proposed in Aïd, Campi, Langrené (2013). In particular we study the reconstruction performances of forward prices and we address the issue of model calibration in terms of determining the parameters to exactly fit the observable forward products. We propose a modification in the structural model to ensure its ability to be calibrated on all the observed forward products and we give some illustrations of calibration performances.

We develop a tractable equilibrium model for price formation in intraday electricity markets in the presence of intermittent renewable generation. Using stochastic control theory we identify the optimal strategies of agents with market impact and exhibit the Nash equilibrium in closed form for a finite number of agents as well as in the asymptotic framework of mean field games. Our model reproduces the empirical features of intraday market prices, such as increasing price volatility at the approach of the delivery date and the correlation between price and renewable infeed forecasts, and relates these features with market characteristics like liquidity, number of agents, and imbalance penalty.

Objective: The aim was to analyze the early postnatal changes in myocardial density, subsarcolemmal localization and isoform expression of dihydropyridine receptors in rat ventricle and the influence of thyroid status on these changes. Methods: Newborn rats were treated from postnatal day 2 with L-triiodothyronine (T3) or 6-n-propyl-2-thiouracil (PTU) and ventricles were collected on day 1, 7 and 14. Radioligand binding and cell fractionation (density gradient centrifugation) techniques were used to determine the tissue density of various receptors and their subcellular localization. To analyze dihydropyridine receptor α1 subunit isoform expression, cDNA fragments corresponding to a large portion of motif IV were amplified by reverse transcriptase-polymerase chain reaction and treated with appropriate restriction endonucleases to determine the frequency of splicing events at the level of motif IV. Results: The myocardial density of dihydropyridine receptors increased 3-fold from day 1 to day 14 in control rats, and this increase occurred predominantly in membrane entities equilibrating at high densities in sucrose gradient, that is, presumably, in junctional structures (dyadic couplings). This maturation was delayed after PTU-treatment, and somewhat accelerated by excess T3. The proportion of mRNA variants typical of foetal heart (IVS3A variant and ‘deleted’ variant, showing a 33-nucleotide deletion at the level of the extracellular loop between IVS3 and IVS4) decreased with age in control rats. This reduction was delayed after treatment with PTU but was not influenced by excess T3. Conclusion: Hypothyroidism impaired the early postnatal maturation of dihydropyridine receptors as regards both their concentration into junctional structures and the decrease in the relative expression of α1-subunit mRNA variants typical of foetal heart.

In order to enhance the understanding of surface processes involved in MOCVD, the desorption of As and P from the (0 0 1) surface of binary and ternary III–V semiconductors was studied by in situ ellipsometry. The variations of ellipsometric parameters as a function of time give the group V desorption rate constant and Arrhenius laws were determined for all the materials. The evolution of activation energies suggests that these are influenced by the surface bond strengths. These kinetic data are essential for the control of heterostructures growth and the construction of numerical model devoted to the growth of InGaAsP. The study also suggests some specific surface reconstructions, especially in the case of the GaAs As-rich surface which probably adopts a c (4×4)-like structure for T⩾625°C and PTBAs=2 Pa and would present an As excess over the c (4×4) structure for T⩽600°C.

An in situ kinetic study of the SiC-based filament CVD from DCMS/H2 and DCMS/C3H6/H2 mixtures has been carried out. The results evidenced a low temperature regime (T<1150 °C) controlled by surface chemical reactions and a high temperature domain (T>1150 °C) governed by the diffusion of reactive species to the substrate (mass transfer regime). The addition of propene to the DCMS/H2 mixture leads to the decrease of the deposition rate, the increase of the activation energy (within the surface reaction limited regime) and the increase of the C/Si (at.) ratio. The dilution of DCMS in H2 promotes the co-deposition of free silicon. Conversely, the addition of propene results in lower amounts of free silicon or even traces of free carbon for high deposition temperatures. The increase of the C/Si (at.) ratio observed in presence of propene might be related to the inhibition of the deposition of Si-rich species derived from DCMS, through reactions with propene or related unsaturated hydrocarbons in the gas phase.

HomeCirculation ResearchVol. 90, No. 4Gaining Respectability Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBGaining RespectabilityMembrane-Delimited, Caveolar-Restricted Activation of Ion Channels Olivier Feron and Ralph A. Kelly Olivier FeronOlivier Feron From the Unit of Pharmacology and Therapeutics (O.F.), Department of Medicine, University of Louvain Medical School, Brussels, Belgium; and Genzyme Corporation (R.A.K.), Cambridge, Mass. Search for more papers by this author and Ralph A. KellyRalph A. Kelly From the Unit of Pharmacology and Therapeutics (O.F.), Department of Medicine, University of Louvain Medical School, Brussels, Belgium; and Genzyme Corporation (R.A.K.), Cambridge, Mass. Search for more papers by this author Originally published8 Mar 2002https://doi.org/10.1161/01.RES.0000012911.90134.EFCirculation Research. 2002;90:369–370Voltage-gated sodium channels play a key role in the excitability of myocardial cells and in impulse propagation.1,2 An increase in Na+ current is, for example, responsible for the generation of the rapid upstroke of the action potential (phase 0). Mutations in the gene encoding this channel (SCN5A) have been linked to the pathogenesis of several cardiac channelopathies, including the long-QT and the Brugada syndromes,1,2 the discoveries of which have helped in the functional characterization of these channels. In addition, the signaling pathways that regulate Na+ channel activity, such as the sympathetic nervous system, have been the subject of numerous studies.3–6 Indeed, stimulation of β-adrenergic receptors can both enhance conduction in normal ventricular myocardium but also induce arrhythmic events in a number of cardiac disease states.1,2Several mechanisms that contribute to the regulation of Na+ channels by β-adrenergic stimulation have been documented (see Figure). All of them involve G proteins, although diffusible second messengers are not necessarily involved. For example, besides the coupling of Gαs to adenylate cyclase and downstream protein kinase A (PKA)-mediated phosphorylation events,3,4 the so-called “membrane-delimited” pathway does not involve diffusible cytosolic factors. The Gαs subunit can, indeed, directly modify Na+ channel activity.5,6 This type of regulation is not unique to Gαs, as the direct binding of the Gβγ protein subunit to several Ca2+, K+, and Cl− channels has been extensively documented (for review, see Dascal7). Download figureDownload PowerPointSchematic representation f the 2 major pathways regulating voltage-dependent Na+ channels (Na+Ch) in cardiac myocytes. The PKA-dependent pathway (right) involves the coupling of the stimulated β-adrenergic receptor (β-AR) to Gαs and the activation of adenylate cyclase (Ad.C.). The subsequent phosphorylation of the Na+ channel by activated cAMP-dependent protein kinase (PKA) leads to a shift in voltage dependence of channel availability to more negative potentials. Of note, a PKA-dependent process has also been shown to regulate Na+ channel trafficking.3 The membrane-delimited pathway (left) is independent of soluble second messengers and involves the direct interaction of the Na+ channel with the N-terminal end of the Gαs protein subunit; this binding produces an increase in INa without a shift in voltage-dependent current activation.Shibata and colleagues have shown previously that the application of peptides derived from the Gαs sequence in inside-out macropatches enhanced isoproterenol-evoked sodium current (INa), attributable to an apparent increase in the number of functional channels.5,6 In this issue of Circulation Research, Shibata and colleagues (Yarbrough et al8) report that the source of these new channels could be caveolae. Using antibodies directed against caveolin-3, the muscle-specific isoform of caveolin, these investigators were able to block the direct effect of isoproterenol on the Na+ channel (ie, the PKA-independent, Gαs membrane-delimited pathway). Importantly, this effect seems specific, given that antibodies directed against other caveolin isoforms did not affect isoproterenol activation of Na+ channel activity. Moreover, Yarbrough et al8 also documented, in cardiac myocytes, the apparent preferential enrichment into caveolar microdomains of Na+ channels and Gαs. Although these data led the authors to conclude that an increase in sympathetic nervous system activity could lead to the recruitment of Na+ channels out of caveolar microdomains to the sarcolemma, thereby increasing INa, several caveats regarding this hypothesis should be noted.Definitive evidence for the translocation of Na+ channels out of caveolae upon isoproterenol stimulation would require further subcellular fractionation and/or electron microscopic studies. In the absence of such experiments, there is no reason to think that the Gαs-activated Na+ channels necessarily would need to exit caveolar microdomains to be active. The use of the anti-caveolin-3 antibody, although specific for this caveolin isoform, may have had effects on other signaling proteins in myocyte caveolae that affected Na+ function. Moreover, it is unlikely that the antibody resulted in the disruption of mature caveolae. This specific antibody, which is directed against an epitope in the nonscaffolding N-terminal end of caveolin-3, probably acts via steric hindrance to prevent Gαs translocation to the Na+ channels into the caveolar microenvironment. Alternatively (or additionally), because β-adrenergic receptors have been found to be enriched in cardiac myocyte caveolae,9,10 accumulation of the antibody could have altered receptor coupling to Gαs (and its consecutive targeting to Na+ channel) upon agonist stimulation. In either case, the argument that blockade of Na+ channel translocation out of caveolae is required to explain the decrease in INa does not necessarily hold.Indeed, sarcolemmal caveolar microdomains could be an important site of Na+ channel activation. Several lines of evidence support this concept. First, a caveat: the term “caveolae” should be used with caution in the context of cardiac muscle. In contrast to skeletal muscle, where caveolin-3 is only observed in association with sarcolemmal caveolae, caveolin-3 in cardiac myocytes has been observed to be associated with both the plasma membrane and the T-tubular system11,12 (Prof R.G. Parton, written communication, January 2002). Interestingly, several groups have reported the location of cardiac Na+ channels in T-tubules to be in close proximity with the Na+-Ca2+ exchanger and L-type Ca2+ channels (which are in close contact with the sarcoplasmic reticulum [SR]) (for review, see Scriven et al13). The data of Yarbrough et al8 could therefore be interpreted as complementary evidence that Na+ channels are colocalized with other key regulators of excitation-contraction coupling within caveolin-enriched structures in T-tubules.There are also parallels between the presence of functional Na+ channels in caveolae and the targeting to these discrete microdomains of other plasmalemmal channels, including water, Ca2+, K+, and Cl− channels.14–16 For some of these channels, a direct link between their subcellular localization in caveolae and their function has been clearly established. Using a dominant-negative caveolin, Trouet et al14 demonstrated that the activation of volume-regulated anion channels by cell swelling was dependent on the maintenance of caveolin integrity within caveolae. In smooth muscle and cardiac cells, Löhn et al15 also have documented that caveolae control the formation of local SR Ca2+ release events, thereby playing a role in triggering Ca2+ sparks.Finally, it should be pointed out that Yarbrough and colleagues8 used a PKA inhibitor to prevent cAMP-dependent activation of Na+ channels, a pathway that represents normally at least 50% of the increase in INa induced by isoproterenol5,6 (see Figure). Interestingly, several of the actors of this parallel Na+ channel activation pathway have also been shown to be located within caveolae. β-Adrenergic receptors, adenylate cyclase, and PKA were found closely associated with caveolin in cardiac myocytes.9,10 If true, this is additional evidence to suggest that Na+ channels might not be required to translocate out of caveolae to be activated. This question could be addressed by exposing patch-clamped cardiac myocytes to caveolin-3 antibodies or (preferably) caveolin-derived peptides and/or caveolae-disrupting drugs and then by evaluating their effects on PKA-dependent activation of Na+ channels.In conclusion, a half century after their discovery, the role of caveolae—and caveolins—in the regulation of intracellular signal transduction cascades has begun to reach maturity. In the cardiovascular system, for example, the recent development of caveolin knockout mice (for review, see Razani and Lisanti17) has to date confirmed the importance of the caveolar location of eNOS for the regulation of this NO synthase (see Feron and Kelly18 for references). Nevertheless, additional studies that build on the observations made by Yarbrough et al8 will be required to explore the functional consequences of caveolar compartmentation on the generation of calcium sparks15 and neuronal and humoral regulation of excitation-contraction coupling.19 Thus, from the unenviable status of being a “last refuge of scoundrels,” the compartmentation of signal transduction pathways has begun to gain respectability, if not yet nobility, in the molecular pharmacology of the cardiovascular system.The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.FootnotesCorrespondence to Olivier Feron, University of Louvain Medical School, Unit of Pharmacology and Therapeutics, UCL-FATH 5349, 53, Avenue E. Mounier, B-1200 Brussels, Belgium. E-mail [email protected] References 1 Marbán E. Cardiac channelopathies. Nature. 2002: 415: 213–218.CrossrefMedlineGoogle Scholar2 Bezzina CR, Rook MB, Wilde AA. Cardiac sodium channel and inherited arrhythmia syndromes. Cardiovasc Res. 2001; 49: 257–271.CrossrefMedlineGoogle Scholar3 Zhou J, Yi J, Hu N, George AL Jr, Murray KT. Activation of protein kinase A modulates trafficking of the human cardiac sodium channel in Xenopus oocytes. Circ Res. 2000; 87: 33–38.CrossrefMedlineGoogle Scholar4 Murphy BJ, Rogers J, Perdichizzi AP, Colvin AA, Catterall WA. cAMP-dependent phosphorylation of two sites in the α subunit of the cardiac sodium channel. J Biol Chem. 1996; 271: 28837–28843.CrossrefMedlineGoogle Scholar5 Lu T, Lee HC, Kabat JA, Shibata EF. Modulation of rat cardiac sodium channel by the stimulatory G protein α subunit. J Physiol. 1999; 518(pt 2): 371–384.MedlineGoogle Scholar6 Matsuda JJ, Lee H, Shibata EF. Enhancement of rabbit cardiac sodium channels by β-adrenergic stimulation. Circ Res. 1992; 70: 199–207.CrossrefMedlineGoogle Scholar7 Dascal N. Ion-channel regulation by G proteins. Trends Endocrinol Metab. 2001; 12: 391–398.CrossrefMedlineGoogle Scholar8 Yarbrough TL, Lu T, Lee H-C, Shibata EF. Localization of cardiac sodium channels in caveolin-rich membrane domains: regulation of sodium current amplitude. Circ Res. 2002; 90: 443–449.CrossrefMedlineGoogle Scholar9 Rybin VO, Xu X, Lisanti MP, Steinberg SF. Differential targeting of β-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae: a mechanism to functionally regulate the cAMP signaling pathway. J Biol Chem. 2000; 275: 41447–41457.CrossrefMedlineGoogle Scholar10 Ostrom RS, Gregorian C, Drenan RM, Xiang Y, Regan JW, Insel PA. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase. J Biol Chem. 2001; 276: 42063–42069.CrossrefMedlineGoogle Scholar11 Parton RG, Way M, Zorzi N, Stang E. Caveolin-3 associates with developing T-tubules during muscle differentiation. 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Akinci G, Topaloglu H, Akinci B, Onay H, Karadeniz C, Ergul Y, Demir T, Ozcan E, Altay C, Atik T and Garg A (2016) Spectrum of clinical manifestations in two young Turkish patients with congenital generalized lipodystrophy type 4, European Journal of Medical Genetics, 10.1016/j.ejmg.2016.05.001, 59:6-7, (320-324), Online publication date: 1-Jun-2016. Hedley P, Kanters J, Dembic M, Jespersen T, Skibsbye L, Aidt F, Eschen O, Graff C, Behr E, Schlamowitz S, Corfield V, McKenna W and Christiansen M (2013) The Role of CAV3 in Long–QT Syndrome, Circulation: Cardiovascular Genetics, 6:5, (452-461), Online publication date: 1-Oct-2013. Vatta M (2013) Caveolae and Arrhythmogenesis Electrical Diseases of the Heart, 10.1007/978-1-4471-4881-4_18, (299-315), . 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In this paper we consider the problem of joint segmentation of hyperspectral images in the Bayesian framework. The proposed approach is based on a Hidden Markov Modeling (HMM) of the images with common segmentation, or equivalently with common hidden classification label variables which is modeled by a Potts Markov Random Field. We introduce an appropriate Markov Chain Monte Carlo (MCMC) algorithm to implement the method and show some simulation results.

Abstract Acetate is reported as a regulator of fat mass but also as lipogenic source for cancer cells. Breast cancer is surrounded by adipose tissue and has been associated with obesity. However, whether acetate contributes to cancer cell metabolism as lipogenic substrate and/or by changing fat storage and eventually obesity‐induced breast cancer progression remains unknown. Therefore, we studied the contribution of acetate to breast cancer metabolism and progression. In vitro, we found that acetate is not a bioenergetic substrate under normoxia and did not result in a significant change of growth. However, by using lipidomic approaches, we discovered that acetate changes the lipid profiles of the cells under hypoxia. Moreover, while mice fed a high‐fat diet (HFD) developed bigger tumours than their lean counterparts, exogenous acetate supplementation leads to a complete abolishment of fat mass gain without reverting the HFD‐induced obesity‐driven tumour progression. In conclusion, although acetate protects against diet‐induced obesity, our data suggest that it is not affecting HFD‐driven tumour progression.

We develop a tractable equilibrium model for price formation in intraday electricity markets in the presence of intermittent renewable generation. Using stochastic control theory we identify the optimal strategies of agents with market impact and exhibit the Nash equilibrium in closed form for a finite number of agents as well as in the asymptotic framework of mean field games. Our model reproduces the empirical features of intraday market prices, such as increasing price volatility at the approach of the delivery date and the correlation between price and renewable infeed forecasts, and relates these features with market characteristics like liquidity, number of agents, and imbalance penalty.