Hugo R. de Jonge

Rectal organoids from subjects with cystic fibrosis can be used to assess responses to drugs that modulate CFTR.

cGMP-dependent protein kinases I and II conduct signals from widespread signaling systems. Whereas the type I kinase mediates numerous effects of natriuretic peptides and nitric oxide in cardiovascular cells, the type II kinase transduces signals from the Escherichia coli heat-stable enterotoxin, STa, and from the endogenous intestinal peptide, guanylin, stimulating Cl- conductance of the cystic fibrosis transmembrane conductance regulator (CFTR). Although the two kinases may be interchangeable for several functions, CFTR regulation specifically requires the type II kinase.

The epithelial anion channel CFTR interacts with multiple PDZ domain-containing proteins. Heterologous expression studies have demonstrated that the Na+/H+ exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO3- secretion in mouse models of Nherf1, Nherf2, and Pdzk1 loss of function. We found that Nherf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO3- secretory rates and blocked beta2-adrenergic receptor (beta2-AR) stimulation. Conversely, Nherf2-/- mice displayed augmented FSK-stimulated HCO3- secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO3- secretion in WT mice, this effect was lost in Nherf2-/- mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO3- secretion. In addition, laser microdissection and quantitative PCR revealed that the beta2-AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the beta2-AR and CFTR was reduced in the Nherf1-/- mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO3- secretion: while NHERF1 is an obligatory linker for beta2-AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.

Identification of Lgr5 as the intestinal stem cell marker as well as the growth factors necessary to replicate adult intestinal stem cell division has led to the establishment of the methods to generate “indefinite” ex vivo primary intestinal epithelial cultures, termed “mini-intestines.” Primary cultures developed from isolated intestinal crypts or stem cells (termed enteroids/colonoids) and from inducible pluripotent stem cells (termed intestinal organoids) are being applied to study human intestinal physiology and pathophysiology with great expectations for translational applications, including regenerative medicine. Here we discuss the physiologic properties of these cultures, their current use in understanding diarrhea-causing host-pathogen interactions, and potential future applications. Identification of Lgr5 as the intestinal stem cell marker as well as the growth factors necessary to replicate adult intestinal stem cell division has led to the establishment of the methods to generate “indefinite” ex vivo primary intestinal epithelial cultures, termed “mini-intestines.” Primary cultures developed from isolated intestinal crypts or stem cells (termed enteroids/colonoids) and from inducible pluripotent stem cells (termed intestinal organoids) are being applied to study human intestinal physiology and pathophysiology with great expectations for translational applications, including regenerative medicine. Here we discuss the physiologic properties of these cultures, their current use in understanding diarrhea-causing host-pathogen interactions, and potential future applications.

In vitro drug tests using patient-derived stem cell cultures offer opportunities to individually select efficacious treatments. Here, we provide a study that demonstrates that in vitro drug responses in rectal organoids from individual patients with cystic fibrosis (CF) correlate with changes in two in vivo therapeutic endpoints. We measured individual in vitro efficaciousness using a functional assay in rectum-derived organoids based on forskolin-induced swelling and studied the correlation with in vivo effects. The in vitro organoid responses correlated with both change in pulmonary response and change in sweat chloride concentration. Receiver operating characteristic curves indicated good-to-excellent accuracy of the organoid-based test for defining clinical responses. This study indicates that an in vitro assay using stem cell cultures can prospectively select efficacious treatments for patients and suggests that biobanked stem cell resources can be used to tailor individual treatments in a cost-effective and patient-friendly manner.

Using the human Intestine 407 cell line as a model, we investigated a possible role for tyrosine kinase(s) in regulating the ion efflux pathways induced by hyposmotic stimulation (regulatory volume decrease, RVD). Pretreatment of 125I(-)-and 86Rb(+)-loaded cells with the phosphotyrosine phosphatase inhibitor sodium orthovanadate (200 microM) potentiated isotope efflux triggered by mild hypotonicity (10-20%) but did not further increase the efflux in response to more vigorous osmotic stimulation (30% hypotonicity). The tyrosine kinase inhibitors herbimycin A and genistein largely reduced the osmoshock-induced efflux in both control and vanadate-pretreated cells, while not affecting calcium-activated 86Rb+ efflux. Potentiation of the RVD response by vanadate was confirmed by direct measurements of hypotonicity-induced changes in cell volume. Hypotonic shock alone triggered a rapid and transient increase in tyrosine phosphorylation of several proteins as well as phosphorylation of mitogen-activated protein kinase. Furthermore, the potentiating effects of vanadate on hypotonicity-induced ion efflux and mitogen-activated protein (MAP) kinase phosphorylation were mimicked by epidermal growth factor. Neither vanadate nor epidermal growth factor provoked a RVD-like ionic response under isotonic conditions. These results indicate that tyrosine phosphorylation is an essential step in the RVD response and suggest a novel role of growth factors in the cellular defense against osmotic stress.

Genetically modified mice have been studied for more than fifteen years as models of cystic fibrosis (CF). The large amount of experimental data generated illuminates the complex multi-organ pathology of CF and raises new questions relevant to human disease. CF mice have also been used to test experimental therapies prior to clinical trials. This review recapitulates the major phenotypic traits of CF mice and highlights important new findings including aberrant alveolar macrophages, bone and cartilage abnormalities and abnormal bioactive lipid metabolism. Novel data are presented on the intestinal and nasal physiology of F508del-CFTR CF mice backcrossed onto different genetic backgrounds. Caveats, and sources of variability including age, gender and animal husbandry, are discussed. Interspecies differences limit comparison of lung pathology in CF mice to the human disease. The recent development of genetically modified pigs and ferrets heralds the application of more advanced animal models to CF research and drug development.

A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl- secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl- channel. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells.

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.

The folding process for newly synthesized, multispanning membrane proteins in the endoplasmic reticulum (ER) is largely unknown. Here, we describe early folding events of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC-transporter family. In vitro translation of CFTR in the presence of semipermeabilized cells allowed us to investigate this protein during nascent chain elongation. We found that CFTR folds mostly during synthesis as determined by protease susceptibility. C-terminally truncated constructs showed that individual CFTR domains formed well-defined structures independent of C-terminal parts. We conclude that the multidomain protein CFTR folds mostly cotranslationally, domain by domain.

Lubiprostone alleviates constipation by stimulating intestinal fluid secretion, purportedly through activation of ClC-2-type Cl(-) channels. Intestinal obstruction is also a recurrent cause of distress in cystic fibrosis (CF) patients, caused by loss of CF transmembrane conductance regulator (CFTR) Cl(-) channel activity. Because ClC-2 recruitment might be beneficial to CF patients, we investigated lubiprostone's mode of action.Cl(-) transport was measured in an Ussing chamber, in 3 model systems: (1) T84 colonocytes, (2) intestinal epithelium of wild-type and CF mice, and (3) intestinal epithelium of CF patients and controls.In T84 monolayers, lubiprostone induced a robust secretory response. Selective permeabilization of the basolateral plasma membrane revealed that lubiprostone activated an apical Cl(-) conductance. The lubiprostone response was attenuated by H89, an inhibitor of the cAMP-dependent protein kinase, and lubiprostone precluded responsiveness to the cAMP agonist forskolin. CFTR blockage by CFTRinh172, but not ClC-2 blockage by CdCl(2), inhibited the lubiprostone response. Lubiprostone induced a CdCl(2)-insensitive secretory response in mouse intestine, but failed to induce intestinal Cl(-) secretion in Cftr-null mice. Correspondingly, lubiprostone induced a secretory response in human intestinal epithelium, but not in tissue of CF patients. The EP(4)-type prostanoid receptor antagonist L-161,982 blocked the lubiprostone response in all 3 models studied. In T84 cells, lubiprostone induced a rise in cAMP levels that was sensitive to EP(4)-receptor blockage.Lubiprostone enhances intestinal Cl(-) and fluid secretion via prostanoid receptor signaling, triggering activation of CFTR. Therefore, it is of limited use for treatment of CF-related intestinal disease.

In the majority of cases, there is no difficulty in diagnosing Cystic Fibrosis (CF). However, there may be wide variation in signs and symptoms between individuals which encourage the scientific community to constantly improve the diagnostic tests available and develop better methods to come to a final diagnosis in patients with milder phenotypes. This paper is the result of discussions held at meetings of the European Cystic Fibrosis Society Diagnostic Network supported by EuroCareCF. CFTR bioassays in the nasal epithelium (nasal potential difference measurements) and the rectal mucosa (intestinal current measurements) are discussed in detail including efforts to standardize the techniques across Europe. New approaches to evaluate the sweat gland, future of genetic testing and methods on the horizon like CFTR expression in human leucocytes and erythrocytes are discussed briefly.

Hepatobiliary complications are a leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Knowledge of the underlying pathological aspects and optimal clinical management is, however, sorely lacking.We provide a summary of the lectures given by international speakers at the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) monothematic conference on cystic fibrosis-related liver disease (CFLD) held in Paris in January 2016, to discuss the status of our current knowledge of liver disease in CF patients, to define the critical areas that need to be addressed, and to resolve actions to elucidate relevant mechanisms of disease to optimise future therapeutic options.The need for a universal consensus on the definition of CFLD to clarify disease stage and to identify relevant biomarkers to assess disease severity was highlighted. A deeper understanding of the pathophysiology and prognostic factors for the long-term evolution of CFLD is fundamental to move forward and has a strong bearing on identifying potential treatments. Novel experimental models and new treatment options under investigation are discussed and offer hope for the near future of CFLD.

The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse transcriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMP-dependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this clone, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKII isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK II localization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK II poly(A) RNA form was 4.8 kb, with minor forms of 6.2 and 3.1 kb. The cGK II RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.

To investigate the impact of chloride (Cl(-)) permeability, mediated by residual activity of the cystic fibrosis transmembrane conductance regulator (CFTR) or by other Cl(-) channels, on the manifestations of cystic fibrosis (CF), we determined Cl(-) transport properties of the respiratory and intestinal tracts in Delta F508 homozygous twins and siblings. In the majority of patients, cAMP and/or Ca(2+)-regulated Cl(-) conductance was detected in the airways and intestine. Our finding of cAMP-mediated Cl(-) conductance suggests that, in vivo, at least some Delta F508 CFTR can reach the plasma membrane and affect Cl(-) permeability. In respiratory tissue, the expression of basal CFTR-mediated Cl(-) conductance, demonstrated by 30% of Delta F508 homozygotes, was identified as a positive predictor of milder CF disease. In intestinal tissue, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive (DIDS-insensitive) Cl(-) secretion, which is indicative of functional CFTR channels, correlated with a milder phenotype, whereas DIDS-sensitive Cl(-) secretion was observed mainly in more severely affected patients. The more concordant Cl(-) secretory patterns within monozygous twins compared with dizygous pairs imply that genes other than CFTR significantly influence the manifestation of the basic defect.

The most prevalent mutation ( ∆ F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR).We have analyzed the properties of a ⌬ F508 CFTR mouse model, which we described recently.We show that the mRNA levels of mutant CFTR are normal in all tissues examined.Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units.Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal.Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice.In cultured gallbladder epithelial cells from ⌬ F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency.However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature.The biophysical characteristics of the mutant channel were not significantly different from normal.In homozygous ⌬ F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments.Our data show that like its human homologue, mouse ⌬ F508-CFTR is a temperature sensitive processing mutant.The ⌬ F508 mouse is therefore a valid in vivo model of human ⌬ F508-CFTR.It may help us to elucidate the processing pathways of complex membrane proteins.Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.( J. Clin.Invest.

Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type Iα cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl⁻ channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl⁻ channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl⁻ channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATPγS (adenosine 5′-<i>O</i>-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKIα, is a potential activator of chloride transport in CFTR-expressing cell types.

The aim of this study was to determine the role of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (cGK) type II in intestinal fluid homeostasis under basal conditions and following exposure to cGMP-linked secretagogues, e.g., Escherichia coli heat-stable enterotoxin (STa) and guanylin.Fluid and ion transport was determined in different segments of the intestine of wild-type and cGK II-deficient mice by ligated loop assays in vivo, and by short-circuit current and isotope flux measurements in vitro.Small intestinal fluid absorption in vivo was enhanced in cGK II-deficient mice under basal conditions and in the presence of STa. Furthermore, STa, guanylin, and 8-pCPT-cGMP stimulation of electrogenic anion secretion and inhibition of Na(+) absorption in vitro were markedly reduced in the small intestine from cGK II -/- mice but not in proximal colon. The type III phosphodiesterase inhibitor amrinone mimicked STa action in cGK II -/- mice, and also stimulated ion secretion in humans.This study shows that the cGMP/cGK II pathway regulates fluid homeostasis in the small intestine under basal conditions and mediates STa effects by both increasing anion secretion and inhibiting Na(+) absorption. It also demonstrates the presence of a cGK II-independent pathway for STa/cGMP-provoked secretion predominantly in the colon, which possibly involves a cGMP-inhibitable phosphodiesterase and/or activation of the cAMP-dependent protein kinase pathway.

Previous Ussing chamber measurements of secretagogue-provoked changes in short circuit current in rectal suction biopsies of cystic fibrosis (CF) patients showed that in a minority of patients chloride secretion in response to cholinergic agonists is reduced but not completely absent. To assess a possible relationship between this phenomenon and both the genotype and the phenotype, we performed Ussing chamber experiments on rectal suction biopsies of 51 CF patients. The CF mutation was identified in 89 out of 102 CF alleles. No apparent chloride secretion was found in 30 CF patients (group I). Low residual chloride secretion was found in 11 CF patients (group II), while a relatively high residual secretion appeared in 10 CF patients (group III). Pancreatic function was preserved more frequently in CF patients displaying residual secretion: 0% in group I, 27% in group II, and 60% in group III (P < 0.001). The age at diagnosis (mean +/- SEM) in group III (18.4 +/- 6.6) was significantly different from group I (1.2 +/- 0.4, P < 0.01) and group II (3.5 +/- 1.4, P = 0.05). Residual chloride secretion was found in some of the 28 dF508 homozygous patients (three in group II, and one in group III), disclosing that other factors than the CF gene defect itself affect the transepithelial chloride transport. The age at diagnosis correlates significantly with the magnitude of the secretory response, even within the dF508 homozygous patients (r = 0.4, P < 0.05). We conclude that residual chloride secretion in CF is the pathophysiological basis of preserved pancreatic function and delayed presentation of the disease, which is not exclusively determined by the CF genotype.

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia.

Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2+]i was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2+ and endothelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities in transepithelial electrolyte transport in cystic fibrosis rectum were analyzed by short-circuit current measurements on 11 control subjects and 11 subjects with cystic fibrosis in a modified Ussing chamber. As judged by the amiloride-sensitive component of the short-circuit current, electrogenic sodium absorption appeared unmodified in cystic fibrosis. In contrast, the short-circuit current response to specific stimuli of both cyclic adenosine monophosphate (cAMP)- and calcium-mediated chloride secretion was drastically altered in all of the cystic fibrosis biopsy specimens examined. Stimulation of the cAMP pathway by 8-bromo cAMP or forskolin resulted in a sustained increase in short-circuit current in control tissues (+ 2.51 +/- 0.63 microA/cm2) but in a slight change in the opposite direction in cystic fibrosis (-0.56 +/- 0.49 microA/cm2; P less than 0.05). Carbachol, a calcium-linked secretagogue, provoked a transient increase in short-circuit current in all of the control tissues (peak response, + 26.69 +/- 3.63 microA/cm2) but a transient change in the opposite direction in 6 of 11 cystic fibrosis tissues (-12.46 +/- 4.64 microA/cm2; P less than 0.05). In 2 of 11 patients with cystic fibrosis, however, a significant but subnormal and transient increase in short-circuit current was observed (+ 2.62 +/- 0.04 microA/cm2; P less than 0.05), whereas in 3 of 11 patients with cystic fibrosis a transient change in the opposite direction (-9.83 +/- 2.20 microA/cm2; P less than 0.05) was followed by a small and transient increase (+ 2.89 +/- 0.83 microA/cm2; P less than 0.05). Using the calcium-mediated secretory response therefore, patients with cystic fibrosis could be divided into two categories: a major population showing defective anion secretion but active cation secretion and a subclass (including three siblings) showing residual but subnormal anion secretion. The easy accessibility of rectal samples and the inversed direction of the cAMP- or calcium-provoked short-circuit current is of considerable advantage in the diagnosis of cystic fibrosis.

Background & AimsDiarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na+/H+ exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na+/H+ exchanger activity and Na+-dependent fluid absorption in the intestine.MethodsWe analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na+/H+ exchanger regulatory factor 2 (NHERF2; Nherf2−/−) or LPA2 (Lpa2−/−). Roles of LPA5 and NHERF2 were determined by analysis of heterologous expression.ResultsUnder basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA5 was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA5 signaling required coexpression of NHERF2, which interacted with LPA5. LPA-mediated intestinal fluid absorption was impaired in Nherf2−/− mice, demonstrating the requirement for NHERF2 in LPA5 activity. However, fluid absorption was unaltered in Lpa2−/− mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells.ConclusionsLPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA5. Regulation by LPA5 depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases. Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na+/H+ exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na+/H+ exchanger activity and Na+-dependent fluid absorption in the intestine. We analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na+/H+ exchanger regulatory factor 2 (NHERF2; Nherf2−/−) or LPA2 (Lpa2−/−). Roles of LPA5 and NHERF2 were determined by analysis of heterologous expression. Under basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA5 was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA5 signaling required coexpression of NHERF2, which interacted with LPA5. LPA-mediated intestinal fluid absorption was impaired in Nherf2−/− mice, demonstrating the requirement for NHERF2 in LPA5 activity. However, fluid absorption was unaltered in Lpa2−/− mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells. LPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA5. Regulation by LPA5 depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases.

Abstract Highly purified lysosomal membrane vesicles, obtained from rat liver lysosomes, were used to study characteristics of NeuAc transport across the lysosomal membrane. Uptake of [14C]NeuAc was found to be strongly influenced by a pH gradient across the membrane. When a proton gradient (pHin greater than pHout) was generated by impermeable buffers, NeuAc uptake above equilibrium level (overshoot) was observed. The influence of membrane diffusion potentials was ruled out by experiments where K+ and valinomycin were present. The overshoot appeared to be specifically produced by protons, since gradients of other cations (Na+ and K+) did not give stimulation. Proton-driven uptake was saturable (Kt = 0.24 mM) and mediated by a single system, as shown by linearity of the Scatchard plot. Stimulation of transport was also obtained by preincubation of vesicles with MgATP and the effect was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Monocarboxylic sugars like glycuronic acids were competitive inhibitors of sialic acid transport. Transstimulation of [14C] NeuAc uptake was observed when vesicles were preloaded either with unlabeled NeuAc or with glucuronic acid. The data demonstrate that lysosomal membrane vesicles from rat liver are a suitable system for kinetic studies of solute transport events. The presence of a proton-driven carrier in the lysosomal membrane specific for sialic acid and other acidic sugars, including glucuronic acid, is shown. The possible physiological significance of these findings for the human lysosomal carrier and the patients with a sialic acid transport defect is discussed.

The altered permeability characteristics of erythrocytes infected with malaria parasites have been a source of interest for over 30 years. Recent electrophysiological studies have provided strong evidence that these changes reflect transmembrane transport through ion channels in the host erythrocyte plasma membrane. However, conflicting results and differing interpretations of the data have led to confusion in this field. In an effort to unravel these issues, the groups involved recently came together for a week of discussion and experimentation. In this article, the various models for altered transport are reviewed, together with the areas of consensus in the field and those that require a better understanding.

Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has been documented in patients with CF. This study investigated the expression, subcellular distribution, and processing of CFTR in the kidney; used various mouse models to assess the role of CFTR in proximal tubule (PT) endocytosis; and tested the relevance of these findings in patients with CF. The level of CFTR mRNA in mouse kidney approached that found in lung. CFTR was located in the apical area of PT cells, with a maximal intensity in the straight part (S3) of the PT. Fractionation showed that CFTR co-distributed with the chloride/proton exchanger ClC-5 in PT endosomes. Cftr(-/-) mice showed impaired (125)I-beta(2)-microglobulin uptake, together with a decreased amount of the multiligand receptor cubilin in the S3 segment and a significant loss of cubilin and its low molecular weight (LMW) ligands into the urine. Defective receptor-mediated endocytosis was found less consistently in Cftr(DeltaF/DeltaF) mice, characterized by a large phenotypic heterogeneity and moderate versus mice that lacked ClC-5. A significant LMW proteinuria (and particularly transferrinuria) also was documented in a cohort of patients with CF but not in patients with asthma and chronic lung inflammation. In conclusion, CFTR inactivation leads to a moderate defect in receptor-mediated PT endocytosis, associated with a cubilin defect and a significant LMW proteinuria in mouse and human. The magnitude of the endocytosis defect that is caused by CFTR versus ClC-5 loss likely reflects functional heterogeneity along the PT.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.

Oral rehydration solutions reduce diarrhea-associated mortality. Stimulated sodium absorption by these solutions is mediated by the Na(+)/H(+) hydrogen exchanger NHE3 and is increased by Na(+)-glucose co-transport in vitro, but the mechanisms of this up-regulated process are only partially understood.Intracellular pH was measured in jejunal enterocytes of wild-type mice and mice with disrupted Na+/H+ exchange regulatory co-factor 2 (NHERF2-/- mice) by multiphoton microscopy. Diarrhea was induced by cholera toxin. Caco-2BBe cells that express NHE3 and the sodium/glucose cotransporter 1 (SGLT1) were studied by fluorometry, before and after siRNA-mediated knockdown of NHERF1 or NHERF2. NHE3 distribution was assessed by cell-surface biotinylation and confocal microscopy. Brush-border mobility was determined by fluorescence recovery after photobleaching and confocal microscopy.The nonmetabolized SGLT1 substrate α-methyl-D-Glu (α-MD-G) activated jejunal NHE3; this process required Akt and NHERF2. α-MD-G normalized NHE3 activity after cholera toxin-induced diarrhea. α-MD-G-stimulated jejunal NHE3 activity was defective in NHERF2-/- mice and cells with NHERF2 knockdown, but occurred normally with NHERF1 knockdown; was associated with increased NHE3 surface expression in Caco-2 cells, which also was NHERF2-dependent; was associated with dissociation of NHE3 from NHERF2 and an increase in the NHE3 mobile fraction from the brush border; and was accompanied by a NHERF2 ezrin-radixin-moesin-binding domain-dependent increase in co-precipitation of ezrin with NHE3.SGLT1-mediated Na-glucose co-transport stimulates NHE3 activity in vivo by an Akt- and NHERF2-dependent signaling pathway. It is associated with increased brush-border NHE3 and association between ezrin and NHE3. Activation of NHE3 corrects cholera toxin-induced defects in Na absorption and might contribute to the efficacy of oral rehydration solutions.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated anion channel expressed in epithelial cells. The most common mutation Delta Phe508 leads to protein misfolding, retention by the endoplasmic reticulum, and degradation. One promising therapeutic approach is to identify drugs that have been developed for other indications but that also correct the CFTR trafficking defect, thereby exploiting their known safety and bioavailability in humans and reducing the time required for clinical development. We have screened approved, marketed, and off-patent drugs with known safety and bioavailability using a Delta Phe508-CFTR trafficking assay. Among the confirmed hits was glafenine, an anthranilic acid derivative with analgesic properties. Its ability to correct the misprocessing of CFTR was confirmed by in vitro and in vivo studies using a concentration that is achieved clinically in plasma (10 microM). Glafenine increased the surface expression of Delta Phe508-CFTR in baby hamster kidney (BHK) cells to approximately 40% of that observed for wild-type CFTR, comparable with the known CFTR corrector 4-cyclohexyloxy-2-{1-[4-(4-methoxybenzensulfonyl)-piperazin-1-yl]-ethyl}-quinazoline (VRT-325). Partial correction was confirmed by the appearance of mature CFTR in Western blots and by two assays of halide permeability in unpolarized BHK and human embryonic kidney cells. Incubating polarized CFBE41o(-) monolayers and intestines isolated from Delta Phe508-CFTR mice (treated ex vivo) with glafenine increased the short-circuit current (I(sc)) response to forskolin + genistein, and this effect was abolished by 10 microM CFTR(inh)172. In vivo treatment with glafenine also partially restored total salivary secretion. We conclude that the discovery of glafenine as a CFTR corrector validates the approach of investigating existing drugs for the treatment of CF, although localized delivery or further medicinal chemistry may be needed to reduce side effects.

FEBS LettersVolume 53, Issue 2 p. 237-242 Full-length articleFree Access The localization of guanylate cyclase in rat small intestinal epithelium H.R. De Jonge, H.R. De Jonge Department of Biochemistry I, Faculty of Medicine, Erasmus University Rotterdam, P.O. Box 1738, Rotterdam, The NetherlandsSearch for more papers by this author H.R. De Jonge, H.R. De Jonge Department of Biochemistry I, Faculty of Medicine, Erasmus University Rotterdam, P.O. Box 1738, Rotterdam, The NetherlandsSearch for more papers by this author First published: May 01, 1975 https://doi.org/10.1016/0014-5793(75)80028-7Citations: 66AboutReferencesRelatedInformationPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 E. Ishikawa, S. Ishikawa, J.W. Davis, E.W. Sutherland, J. Biol. Chem., 244, (1969), 6371– 6376. 2 H. Kimura, F. Murad, J. Biol. Chem., 249, (1974), 6910– 6916. 3 D.K. Parkman, H. Ebel, D.R. Dibona, G.W.G. Sharp, J. Clin. Invest., 51, (1972), 2292– 2298. 4 R.J. Grand, F.M. Torti, S. Jaksina, J. Clin. Invest., 52, (1973), 2053– 2059. 5 Jonge H.R. de, Biochim. Biophys. Acta, 381, (1975), 128– 143. 6 H.L. Webster, D.D. Harrison, Exp. Cell Res., 56, (1969), 245– 253. 7 D.K. Podolsky, M.M. Weiser, J. Cell Biol., 58, (1973), 497– 500. 8 G.G. Forstner, S.M. Sabesin, K.J. Isselbacher, Biochem. J., 106, (1968), 381– 390. 9 A.P. Douglas, R. Kerley, K.J. Isselbacher, Biochem. J., 128, (1972), 1329– 1338. 10 de Jonge, H. R., manuscript in preparation. 11 W.G. Iemhoff, den Berg J.W.O. van, Pijper A.M. de, W.C. Hülsmann, Biochim. Biophys. Acta, 215, (1970), 229– 241. 12 C. Higgins, J. Lapides, J. Biol. Chem., 170, (1947), 467– 482. 13 A. Dahlqvist, Anal. Biochem., 7, (1964), 18– 25. 14 Jonge H.R. de, W.C. Hülsmann, Eur. J. Biochem., 32, (1972), 356– 364. 15 G. Ferrard, I. Sall, P. Metais, Enzyme, 19, (1975), 38– 47. 16 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 275. 17 A. Eichholz, Biochim. Biophys. Acta, 135, (1967), 475– 482. 18 S.L. Clark, Amer. J. Anat., 109, (1961), 57– 83. 19 M. Fujita, H. Ohta, K. Kawai, H. Matsui, M. Nakao, Biochim. Biophys. Acta, 274, (1972), 336– 347. 20 M.L. Clark, H.C. Lanz, J.R. Senior, Biochim. Biophys. Acta, 183, (1969), 233– 235. 21 O.V. Deimling, H. Madreiter, Histochemie, 29, (1972), 83– 96. 22 A.N. Charney, R.E. Gots, R.A. Giannella, Biochim. Biophys. Acta, 367, (1974), 265– 270. 23 G. Illiano, G.P.E. Tell, M.I. Siegel, P. Cuatrecasas, Proc. Natl. Acad. Sci. U.S.A., 70, (1973), 2443– 2447. 24 Both N.J. de, Dongen J.M. van, Hofwegen B. van, J. Keulemans, W.J. Visser, H. Galjaard, Dev. Biol., 38, (1974), 119– 137. 25 N.D. Goldberg, R.F. O'Dea, M.K. Haddox, P. Greengard G.A. Robison Advances in Cyclic Nucleotide Research 3, (1973), Raven Press New York 155– 223. 26 M. Field, Amer. J. Physiol., 221, (1971), 992– 997. 27 M. Field, Gastroenterology, 66, (1974), 1063– 1084. 28 E. Neville, E.S. Holdsworth, FEBS Lett., 2, (1969), 313– 316. 29 J.L. Kinzie, J.A. Ferrendelli, D.H. Alpers, J. Biol. Chem., 248, (1973), 7018– 7024. Citing Literature Volume53, Issue2May 01, 1975Pages 237-242 ReferencesRelatedInformation Metrics All-time Citations: 66 Details FEBS Letters 53 (1975) 1873-3468 © 2015 Federation of European Biochemical Societies Keywords Cyclic GMP cyclic 3′, 5′-guanosine monophosphate Cyclic AMP cyclic 3′, 5′-adenosine monophosphate GTP guanosine 5′-triphosphate Publication History Issue Online: 19 October 2001 Version of Record online: 19 October 2001 Manuscript received: 15 March 1975

Forskolin-induced swelling (FIS) of intestinal organoids from individuals with cystic fibrosis (CF) measures function of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein mutated in CF.We investigated whether FIS corresponds with clinical outcome parameters and biomarkers of CFTR function in 34 infants diagnosed with CF. Relationships with FIS were studied for indicators of pulmonary and gastrointestinal disease.Children with low FIS had higher levels of immunoreactive trypsinogen (p=0.030) and pancreatitis-associated protein (p=0.039), more often had pancreatic insufficiency (p<0.001), had more abnormalities on chest computed tomography (p=0.049), and had lower z-scores for maximal expiratory flow at functional residual capacity (p=0.033) when compared to children with high FIS values. FIS significantly correlated with sweat chloride concentration (SCC) and intestinal current measurement (ICM) (r= -0.82 and r=0.70, respectively; both p<0.001). Individual assessment of SCC, ICM and FIS suggested that FIS can help to classify individual disease severity.Thus, stratification by FIS identified subgroups that differed in pulmonary and gastrointestinal outcome parameters. FIS of intestinal organoids correlated well with established CFTR-dependent biomarkers such as SCC and ICM, and performed adequately at group and individual level in this proof-of-concept study.

Cystic fibrosis (CF), the most common, life-threatening monogenetic disease in Caucasians, is caused by mutations in the CFTR gene, encoding a cAMP- and cGMP-regulated epithelial chloride channel. Symptomatic therapies treating end-organ manifestations have increased the life expectancy of CF patients toward a mean of 40 years. The recent development of CFTR-targeted drugs that emerged from high-throughput screening and are capable of correcting the basic defect promises to transform the therapeutic landscape from a trial-and-error prescription to personalized medicine. This stratified approach is tailored to a specific functional class of mutations in CFTR, but can be refined further to an individual level by exploiting recent advances in ex vivo drug testing methods. These tests range from CFTR functional measurements in rectal biopsies donated by a CF patient to the use of patient-derived intestinal or pulmonary organoids. Such organoids may serve as an inexhaustible source of epithelial cells that can be stored in biobanks and allow medium- to high-throughput screening of CFTR activators, correctors and potentiators on the basis of a simple microscopic assay monitoring organoid swelling. Thus the recent breakthrough in stem cell biology allowing the culturing of mini-organs from individual patients is not only relevant for future stem cell therapy, but may also allow the preclinical testing of new drugs or combinations that are optimally suited for an individual patient.

Rationale: Gene therapy holds promise for a curative mutationindependent treatment applicable to all patients with cystic fibrosis (CF).The various viral vector-based clinical trials conducted in the past have demonstrated safety and tolerance of different vectors, but none have led to a clear and persistent clinical benefit.Recent clinical breakthroughs in recombinant adeno-associated viral vector (rAAV)based gene therapy encouraged us to reexplore an rAAV approach for CF.Objectives: We evaluated the preclinical potential of rAAV gene therapy for CF to restore chloride and fluid secretion in two complementary models: intestinal organoids derived from subjects with CF and a CF mouse model, an important milestone toward the development of a clinical rAAV candidate for CF gene therapy.Methods: We engineered an rAAV vector containing a truncated CF transmembrane conductance regulator (CFTRDR) combined with a short promoter (CMV173) to ensure optimal gene expression.A rescue in chloride and fluid secretion after rAAV-CFTRDR treatment was assessed by forskolin-induced swelling in CF transmembrane conductance regulator (CFTR)-deficient organoids and by nasal potential differences in DF508 mice.Measurements and Main Results: rAAV-CFTRDR transduction of human CFTR-deficient organoids resulted in forskolin-induced swelling, indicating a restoration of CFTR function.Nasal potential differences demonstrated a clear response to low chloride and forskolin perfusion in most rAAV-CFTRDR-treated CF mice.Conclusions: Our study provides robust evidence that rAAVmediated gene transfer of a truncated CFTR functionally rescues the CF phenotype across the nasal mucosa of CF mice and in patientderived organoids.These results underscore the clinical potential of rAAV-CFTRDR in offering a cure for all patients with CF in the future.

Abstract The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis.

This study aimed to investigate the effect of uridine on obesity, fat accumulation in liver, and gut microbiota composition in high-fat diet-fed mice.

The sex steroid 17beta-estradiol (17beta-E2) has a broad range of actions, including effects on calcium and bone metabolism. This study with 3-month-old Brown Norway rats was designed to investigate the role of 17beta-E2 in the regulation of calcium homeostasis. Rats were divided in four groups, sham-operated, ovariectomized (OVX), and OVX supplemented with either a 0.025-mg or 0.05-mg 17beta-E2 pellet implanted subcutaneously. After 4 weeks, in none of the groups was serum calcium, phosphate, or parathyroid hormone altered compared with the sham group, while only in the OVX rats was a significant reduction in urinary calcium found. Bone mineral density and osteocalcin were modified, as can be expected after OVX and 17beta-E2 supplementation. OVX resulted in a nonsignificant increase in serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Supplementation with either one of the 17beta-E2 dosages resulted in an 80% reduction of 1,25(OH)2D3 and only a 20% reduction in 25-hydroxyvitamin D3 levels. OVX, as well as supplementation with 17beta-E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3 levels were also significantly decreased in the 17beta-E2-supplemented group compared with the sham and OVX groups. Next, the consequences for intestinal calcium absorption were analyzed by the in situ intestinal loop technique. Although the 1,25(OH)2D3 serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3 levels (18. 1 +/- 2.1 and 16.4 +/- 2.2 pmol/l compared with 143.5 +/- 29 pmol/l for the OVX group), the OVX-induced decrease in calcium absorption could partially be restored by supplementation with either 0.025 mg or 0.05 mg of 17beta-E2. None of the treatments resulted in a significant change in calcium handling in the jejunum, although the trends were similar as those observed in the duodenum. 17beta-E2 did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17beta-E2 is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17beta-E2 exerts this effect independent of 1,25(OH)2D3. In general, 17beta-E2 not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption. (J Bone Miner Res 1999;14:57-64)

Previous studies have revealed an adenosine 3',5'-cyclic monophosphate (cAMP)-independent activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by the tyrosine kinase inhibitor genistein. To further explore its mechanism of action, we have reconstituted genistein activation of CFTR in excised inside-out membrane patches. In the presence or absence of ATP, genistein appeared unable to open silent CFTR Cl- channels. However, on CFTR prephosphorylation by cAMP-dependent protein kinase (cAK), genistein enhanced CFTR activity by twofold, resulting from a prolonged burst duration. Genistein could also hyperactivate partially phosphorylated CFTR in the absence of cAK and therefore is different from 5'-adenylylimidodiphosphate, which required fully phosphorylated CFTR. Phosphatase-resistant thiophosphorylation likewise primed the CFTR Cl- channel for hyperactivation by genistein in the absence of cAK. Replacement of ATP by GTP as a hydrolyzable nucleotide triphosphate for CFTR did not impair the ability of genistein to activate thiophosphorylated CFTR, despite the fact that GTP is a poor substrate for tyrosine kinases. These findings argue against a role of protein phosphatases or tyrosine kinases but suggest a more direct interaction of genistein with CFTR, possibly at the level of the second nucleotide-binding domain.

BACKGROUND & AIMS: Escherichia coli heat-stable enterotoxins (STa) provoke electrogenic Cl- secretion in the intestine through a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent signal transduction pathway. The cGMP receptor involved in the activation of the Cl- channel is not known with certainty but may comprise either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (cAK) or cGMP-dependent protein kinase (cGK) type II. The aim of this study was to discriminate between these possibilities using specific kinase inhibitors.METHODS: Intestinal electrogenic Cl- secretion was determined by measuring short-circuit current (Isc) in a Ussing chamber.RESULTS: The general protein kinase inhibitors staurosporine and H-8 inhibited rat cGK II activity in vitro with 50% inhibitory concentration values of 4 nmol/L and 3 mumol/L, respectively, which are lower than those reported for cAK. Both staurosporine and H-8, when added to rat proximal colon at concentrations that did not affect the Isc response to 8-bromo-cAMPS, inhibited the STa- and 8-bromo-cGMP- provoked Isc response for more than 80%. Furthermore, the relative specific cGK inhibitor Rp isomer of 8-(chlorophenylthio)-cGMP, but not the cAK inhibitor RP isomer of (Rp) 8-bromo-cAMPS, inhibited the Isc response to submaximal levels of STa in rat proximal colon.CONCLUSIONS: These data provide further evidence for an important role of cGK II in STa-mediated Cl- secretion in native rat intestinal epithelium.(Gastroenterology 1997 Feb;112(2):437-43)

Electroneutral NaCl absorption mediated by Na⁺/H⁺ exchanger 3 (NHE3) is important in intestinal and renal functions related to water/Na⁺ homeostasis. cGMP inhibits NHE3 in intact epithelia. However, unexpectedly it failed to inhibit NHE3 stably transfected in PS120 cells, even upon co-expression of cGMP-dependent protein kinase type II (cGKII). Additional co-expression of NHERF2, the tandem PDZ domain adapter protein involved in cAMP inhibition of NHE3, restored cGMP as well as cAMP inhibition, whereas NHERF1 solely restored cAMP inhibition. <i>In vitro</i> conditions were identified in which NHERF2 but not NHERF1 bound cGKII. The NHERF2 PDZ2 C terminus, which binds NHE3, also bound cGKII. A non-myristoylated mutant of cGKII did not support cGMP inhibition of NHE3. Although cGKI also bound NHERF2 <i>in vitro</i>, it did not evoke inhibition of NHE3 unless a myristoylation site was added. These results show that NHERF2, acting as a novel protein kinase G-anchoring protein, is required for cGMP inhibition of NHE3 and that cGKII must be bound both to the plasma membrane by its myristoyl anchor and to NHERF2 to inhibit NHE3.

Guanylyl cyclase C (GC-C) is a newly discovered receptor found in the intestine, and possibly in other epithelia, that binds bacterial heat-stable enterotoxins (STa). The receptor has now been stably expressed in human embryonic 293 cells, which do not normally contain the receptor. Cyclic GMP concentrations are elevated 40-fold in response to 1 microM STa, and membranes obtained from the overproducing cells contain GC-C activity that can be stimulated about 9-fold by STa alone and an additional 1.4- to 2-fold by a combination of ATP and STa. The ATP effect does not appear to be due to enzyme activation, but instead to protection of GC-C against inactivation. Antibody raised against the carboxyl-terminal sequence of GC-C identified two major proteins (M(r) 140,000 and 160,000) in 293 cells expressing GC-C. Both proteins bound to wheat germ lectin-Sepharose, and N-glycosidase F treatment converted both forms to a single M(r) 120,000 protein, the size predicted from amino acid composition. The addition of high concentrations of tunicamycin to 293-GC-C cells also reduced the M(r) to 120,000, indicating that GC-C is an N-linked glycoprotein. When rat intestinal membranes or 293-GC-C cells were cross-linked with 125I-labeled STa, the major 125I-labeled protein complexes had M(r) ranging between 45,000 and 80,000. On immunoblots of rat intestinal membranes treated with a reducing agent, 3 major proteins of M(r) 65,000, 85,000, and 140,000 were specifically recognized by a GC-C antibody. However, under nonreducing conditions one major GC-C related protein appeared at a higher position (M(r) 230,000). Its mobility was reduced in the presence of STa, similar to rCG-C expressed in 293 cells. These data indicate that at least part of the lower M(r) STa-binding proteins found in intestinal extracts represent proteolytic products of GC-C.

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [³H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 ± 5 to 35 ± 4%. Furthermore, a nonmyristoylated cGK II Gly² → Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the <i>K</i>_a for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.

Hypo-osmotic swelling of human Intestine 407 cells leads to a significant increase of intracellular MAPKAP-kinase 2 activity and Hsp27 phosphorylation. Pre-treatment of the cells with the p38 MAP kinase inhibitor SB-203580 blocks this activation, indicating that the hypotonicity-induced activation of MAPKAP kinase 2 is, similarly to that described for hyperosmotic treatment, the result of an activated p38 MAP kinase cascade. The activation of MAPKAP kinase 2 proceeds with kinetics similar to that of one of the first physiological responses of hypo-osmotic treatment, the opening of compensatory Cl- channels. However, inhibition of the p38 MAP kinase cascade does not block the osmo-sensitive anion efflux and, vice versa, activation of p38 MAP kinase by cytokines and anisomycin does not increase the efflux. These results indicate that the p38 MAP kinase cascade is not directly involved in Cl- channel activation but instead may play a role in subsequent cellular repair processes.

An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the "new permeation pathway" for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in <i>Plasmodium falciparum</i>-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on <i>in vitro</i> parasite growth or new permeation pathway activity, supporting an argument against a close association between the <i>Plasmodium</i>-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF.

1. Some kinetic properties of adenylate cyclase in separately isolated upper villous and crypt cells from rat and guinea pig small intestine were compared. An apparent Km of 0.4 mM was found for both enzymes in the rat. The slight difference between the V-values measured in the fluoride-stimulated state (132 and 165 pmoles cyclic AMP formed per min per mg protein respectively) indicated an approximately equal enzyme content of both cell populations and argues strongly against a preferential localization in the brushborder region of the epithelial cell. 2. Prolonged contact of the small intestine with luminally administered choleragen led to an irreversible activation of adenylate cyclase in both villous and crypt compartments. The maximal stimulation of the upper villous enzyme (4–7 times) exceeded the maximal effect on the crypt enzyme by two-fold. 3. A lag phase of at least 30 min was found between the first luminal contact with the purified choleragen and a significant activation of the adenylate cyclase associated with isolated intestinal brushborders from the upper villous region. 4. By using a short exposure time (2 min) of the luminal surface to high amounts of choleragen, adenylate cyclase activity in the upper villus could be optimally stimulated in the absence of any alteration of crypt cell activity. 5. By comparing, in vivo, the effects of short and prolonged contact with choleratoxin on the unidirectional and net flux of ions and water in ileal and jejunal segments, it was concluded that both villous and crypt regions contribute to the secretion of water and electrolytes (sodium, chloride and bicarbonate ions) during cholera. The serosal to mucosal flux of sodium and chloride ions increased without a significant alteration of the opposite flux. These results imply that absorptive and secretory processes occur within the same epithelial compartment. 6. The view that the crypt epithelium fulfills a specific role during the choleragen-induced secretion of ions and water is incompatible with the results of the present study.

We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

Background & AimsHuman enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation.MethodsHuman duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s).ResultsDifferentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids.ConclusionsHuman enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion. Human enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation. Human duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s). Differentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids. Human enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion.

Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt's base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus-surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt-villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.

Background: The effect of presently available CFTR modulator combinations, such as elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA), on rare CFTR alleles is often unknown. Several assays have been developed, such as forskolin-induced swelling (FIS), to evaluate the rescue of such uncommon CFTR alleles both by established and novel modulators in patient-derived primary cell cultures (organoids). Presently, we assessed the CFTR-mediated electrical current across rectal organoid-derived epithelial monolayers. This technique, which allows separate measurement of CFTR-dependent chloride or bicarbonate transport, was used to assess the effect of ELX/TEZ/IVA on two rare CFTR variants. Methods: Intestinal organoid cultures were established from rectal biopsies of CF patients carrying the rare missense mutations E193K or R334W paired with F508del. The effect of the CFTR modulator combination ELX/TEZ/IVA on CFTR-mediated Cl− and HCO3− secretion was assessed in organoid-derived intestinal epithelial monolayers. Non-CF organoids were used for comparison. Clinical biomarkers (sweat chloride, FEV1) were monitored in patients receiving modulator therapy. Results: ELX/TEZ/IVA markedly enhanced CFTR-mediated bicarbonate and chloride transport across intestinal epithelium of both patients. Consistent with the rescue of CFTR function in cultured intestinal cells, ELX/TEZ/IVA therapy improved biomarkers of CFTR function in the R334W/F508del patient. Conclusions: Current measurements in organoid-derived intestinal monolayers can readily be used to monitor CFTR-dependent epithelial Cl− and HCO3− transport. This technique can be explored to assess the functional consequences of rare CFTR mutations and the efficacy of CFTR modulators. We propose that this functional CFTR assay may guide personalized medicine in patients with CF-like clinical manifestations as well as in those carrying rare CFTR mutations.

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl⁻ secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels was analyzed after expression of cGK II or cGK Iβ in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by ¹²⁵I⁻ efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Iβ or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Iβ enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, <i>e.g.</i> intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Iβ nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

Cholinergic stimulation of chloride secretion is impaired in the intestines of patients with cystic fibrosis (CF). However, intestinal chloride secretion has been observed in patients with mild CF mutations. The aim of this study was to investigate residual Cl(-) secretion in the intestine of DeltaF508 homozygous CF patients, and examine the contribution of cystic fibrosis transmembrane conductance regulator (CFTR) and alternative Cl(-) conductances. Twins and siblings with identical CFTR genotypes were investigated to determine the impact of factors other than CFTR on chloride secretion.Chloride secretion in rectal tissue was investigated by applying Ca(2+) and adenosine 3',5'-cyclic monophosphate (cAMP)-linked agonists before and after the inhibition of alternative Cl(-) conductances with 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS).cAMP-mediated Cl(-) secretion was observed in 73% of patients, and 20% showed DIDS-sensitive Ca(2+)-activated Cl(-) secretion. This DIDS-sensitive alternative chloride conductance was seen only in CF patients who also responded to cAMP agonists. Chloride secretion was more concordant within monozygous twins than within dizygous pairs.These results suggest the presence of CFTR-mediated Cl(-) secretion in a subgroup of patients, implying that a portion of deltaF508 CFTR can be processed in vivo and function as a chloride channel in the apical membrane of intestinal cells. Moreover, a considerable number of deltaF508 homozygous patients express chloride conductances other than CFTR in their intestinal epithelia.

Intestinal current measurements (ICM) on rectal suction biopsies are a tool for the ex vivo diagnosis of classical and atypical cystic fibrosis (CF). We present the basic ICM protocol, typical tracings and their interpretation. The ICM technique allows the registration of CF-induced changes in electrogenic transepithelial ion transport (Cl-, HCO3-, K+) in a Cl- secretory epithelium, and on the basis of pharmacological criteria, is able to discriminate between CFTR-mediated Cl- secretion and secretion through alternative anion channels. ICM is particularly useful for the classification of individuals with CF-like clinical features with equivocal sweat test values and/or no or one identifiable CFTR mutation.

Animal models of cystic fibrosis, in particular several different mutant mouse strains obtained by homologous recombination, have contributed considerably to our understanding of CF pathology. In this review, we describe and compare the main phenotypic features of these models. Recent and possible future developments in this field are discussed.

The multi-PDZ domain containing protein Na⁺/H⁺ Exchanger Regulatory Factor 1 (NHERF1) binds to Na⁺/H⁺ exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1^-/- mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1^-/- ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1^-/- mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2′O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1^-/- mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.

Osmotic swelling of Intestine 407 cells leads to an immediate increase in cell surface membrane area as determined using the fluorescent membrane dye FM 1–43. In addition, as measured by tetramethylrhodamine isothiocyanate (TRITC)-dextran uptake, a robust (>100-fold) increase in the rate of endocytosis was observed, starting after a discrete lag time of 2–3 min and lasting for ∼10–15 min. The hypotonicity-induced increase in membrane surface area, like the cell swelling-induced release of ATP (Van der Wijk, T., De Jonge, H. R., and Tilly, B. C. (1999) <i>Biochem. J.</i> 343, 579–586), was diminished after 1,2-bis(2-aminophenoxy)ethane-<i>N</i>,<i>N</i>,<i>N</i>′,<i>N</i>′-tetraacetic acid-acetoxymethyl ester loading or cytochalasin B treatment. Uptake of TRITC-dextrans, however, was not affected. Treatment of the cells with the vesicle-soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor-specific protease <i>Clostridium botulinum</i> toxin F not only nearly eliminated the hypotonicity-induced increase in membrane surface area but also strongly diminished the release of ATP, indicating the involvement of regulated exocytosis. Both the ATP hydrolase apyrase and the MEK inhibitor PD098059 diminished the osmotic swelling-induced increase in membrane surface area as well as the subsequent uptake of TRITC-dextrans. Taken together, the results indicate that extracellular ATP is required for the hypotonicity-induced vesicle recycling and suggest that a positive feedback loop, involving purinergic activation of the Erk-1/2 pathway, may contribute to the release of ATP from hypo-osmotically stimulated cells.

FEBS LettersVolume 127, Issue 1 p. 139-143 Full-length articleFree Access On the role of cyclic AMP and Ca2+—calmodulin-dependent phosphorylation in the control of (Ca2+ + Mg2+)-ATPase of cardiac sarcolemma Jos M.J. Lamers, Jos M.J. Lamers Department of Biochemistry I, Medical Faculty, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The NetherlandsSearch for more papers by this authorHanny T. Stinis, Hanny T. Stinis Department of Biochemistry I, Medical Faculty, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The NetherlandsSearch for more papers by this authorHugo R. de Jonge, Hugo R. de Jonge Department of Biochemistry I, Medical Faculty, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The NetherlandsSearch for more papers by this author Jos M.J. Lamers, Jos M.J. Lamers Department of Biochemistry I, Medical Faculty, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The NetherlandsSearch for more papers by this authorHanny T. Stinis, Hanny T. Stinis Department of Biochemistry I, Medical Faculty, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The NetherlandsSearch for more papers by this authorHugo R. de Jonge, Hugo R. de Jonge Department of Biochemistry I, Medical Faculty, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The NetherlandsSearch for more papers by this author First published: May 05, 1981 https://doi.org/10.1016/0014-5793(81)80360-2Citations: 47AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 H. Will, B. Schirpke, A. Wollenberger, Biol. Med. Germ., 35, (1976), 529– 542. 2 P.V. Sulakhe, N.L. Leung, P.J. St Louis, Can. J. Biochem., 54, (1976), 438– 445. 3 C. Hui, M. Drummond, G.I. Drummond, Arch. Biochem. Biophys., 173, (1976), 415– 427. 4 J.M.J. Lamers, J.T. Stinis, Biochim. Biophys. Acta, 624, (1980), 443– 459. 5 L.R. Jones, H.R. Besch, M.M. Fleming, M.M. Mc Connaughey, A.M. Watanabe, J. Biol. Chem., 254, (1979), 530– 539. 6 L.R. Jones, S.W. Maddock, H.R. Besch, J. Biol. Chem., 255, (1980), 9971– 9980. 7 P.J. St Louis, P.V. Sulakhe, Arch. Biochem. Biophys., 198, (1979), 227– 240. 8 C.J. Le Peuch, D.A.M. Le Peuch, J.G. Demaille, Biochemistry, 19, (1980), 3368– 3373. 9 W.Y. Cheung, Science, 207, (1980), 19– 27. 10 D.J. Wolff, C.O. Brostrom, Adv. Cyclic Nucl. Res., 11, (1979), 27– 88. 11 H. Schulman, P. Greengard, Proc. Natl. Acad. Sci. USA, 75, (1978), 5432– 5436. 12 C.J. Le Peuch, J. Haiech, J.G. Demaille, Biochemistry, 18, (1979), 5150– 5157. 13 S. Katz, M.A. Remtulla, Biochem. Biophys. Res. Commun., 83, (1978), 1373– 1379. 14 E.G. Kranias, F. Mandel, T. Wang, A. Schwartz, Biochemistry, 19, (1980), 5434– 5439. 15 A.M. Katz, Adv. Cyclic Nucl. Res., 11, (1979), 303– 343. 16 M. Tada, M.A. Kirchberger, D.I. Repke, A.M. Katz, J. Biol. Chem., 249, (1974), 6174– 6180. 17 F. Wuytack, G. De Schutter, R. Casteels, Biochem. J., 190, (1980), 827– 831. 18 J.R. Dedman, J.D. Potter, R.L. Jackson, A.R. Means, J. Biol. Chem., 252, (1977), 8415– 8422. 19 C.W. Davis, J.W. Daly, J. Biol. Chem., 253, (1978), 8683– 8686. 20 J.M.J. Lamers, J.T. Stinis, Biochim. Biophys. Acta, 640, (1981), 521– 534. 21 W.R. Trumble, J.L. Sutko, J.P. Reeves, Life Sci., 27, (1980), 207– 214. 22 W. Ruitenbeek, J. Neurol. Sci., 41, (1979), 71– 80. 23 H.A. Pershadsingh, M.L. Mc Daniel, M. Landt, C.G. Bry, P.E. Lacy, J.M. Mc Donald, Nature, 288, (1980), 492– 495. 24 A. Fabiato, F. Fabiato, J. Physiol. Paris, 75, (1979), 463– 505. 25 J.M.J. Lamers, J.T. Stinis, E. Chazov Advances in Myocardiology 3, (1981), Plenum New York in press 26 N.C. Morcos, G.I. Drummond, Biochim. Biophys. Acta, 598, (1980), 27– 39. 27 P.F. Blackmore, M.F. El-Refai, J.-P. Dehaye, W.G. Strickland, B.P. Hughes, J.H. Exton, FEBS Lett., 123, (1981), 245– 248. 28 S. Muallem, S.J.D. Karlish, Biochim. Biophys. Acta, 597, (1980), 631– 636. 29 S. Shenolikar, P.T.W. Cohen, P. Cohen, A.C. Nairin, S.V. Perry, Eur. J. Biochem., 100, (1979), 329– 337. Citing Literature Volume127, Issue1May 05, 1981Pages 139-143 ReferencesRelatedInformation

Researchers need an available and easy-to-use model of the human intestine to better understand human intestinal physiology and pathophysiology of diseases, and to offer an enhanced platform for developing drug therapy. Our work employs human enteroids derived from each of the major intestinal sections to advance understanding of several diarrheal diseases, including those caused by cholera, rotavirus and enterohemorrhagic Escherichia coli. An enteroid bank is being established to facilitate comparison of segmental, developmental, and regulatory differences in transport proteins that can influence therapy efficacy. Basic characterization of major ion transport protein expression, localization and function in the human enteroid model sets the stage to study the effects of enteric infection at the transport level, as well as to monitor potential responses to pharmacological intervention.

The potentiator VX-770 (ivacaftor/KALYDECO™) targets defective gating of CFTR and has been approved for treatment of cystic fibrosis (CF) subjects carrying G551D, S1251N or one of 8 other mutations. Still, the current potentiator treatment does not normalize CFTR-dependent biomarkers, indicating the need for development of more effective potentiator strategies. We have recently pioneered a functional CFTR assay in primary rectal organoids and used this model to characterize interactions between VX-770, genistein and curcumin, the latter 2 being natural food components with established CFTR potentiation capacities. Results indicated that all possible combinations of VX-770, genistein and curcumin synergistically repaired CFTR-dependent forskolin-induced swelling of organoids with CFTR-S1251N or CFTR-G551D, even under suboptimal CFTR activation and compounds concentrations, conditions that may predominate in vivo. Genistein and curcumin also enhanced forskolin-induced swelling of F508del homozygous organoids that were treated with VX-770 and the prototypical CFTR corrector VX-809. These results indicate that VX-770, genistein and curcumin in double or triple combinations can synergize in restoring CFTR-dependent fluid secretion in primary CF cells and support the use of multiple potentiators for treatment of CF.

Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3′-azido-3′-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea. <b>Background:</b> Diarrhea is an adverse side effect associated with many therapeutics. <b>Results:</b> Irinotecan induced hyperactive cystic fibrosis transmembrane conductance regulator (CFTR) function by inhibiting multidrug resistance protein 4 (MRP4) and formation of MRP4-CFTR macromolecular complexes. <b>Conclusion:</b> MRP4-CFTR-containing macromolecular complexes play an important role in drug-induced diarrhea. <b>Significance:</b> These studies help define molecular mechanisms of drug-induced diarrhea.

Cystic fibrosis is caused by mutations in the CFTR gene, which are subdivided into six classes. Mutants of classes III and IV reach the cell surface but have limited function. Most class-III and class-IV mutants respond well to the recently approved potentiator VX-770, which opens the channel. We here revisited function and folding of some class-IV mutants and discovered that R347P is the only one that leads to major defects in folding. By this criterion and by its functional response to corrector drug VX-809, R347P qualifies also as a class-II mutation. Other class-IV mutants folded like wild-type CFTR and responded similarly to VX-809, demonstrating how function and folding are connected. Studies on both types of defects complement each other in understanding how compounds improve mutant CFTR function. This provides an attractive unbiased approach for characterizing mode of action of novel therapeutic compounds and helps address which drugs are efficacious for each cystic fibrosis disease variant.

Until recently, major advances in drug development have been hampered by a lack of proper cell and tissue models; but the introduction of organoid technology has revolutionized this field. At the level of the gastrointestinal tract, the so-called mini-gut comprises all major cell types of native intestine and recapitulates the composition and function of native intestinal epithelium. The mini-gut can be classified as an intestinal organoid (IO), derived from pluripotent stem cells, or as an enteroid, consisting only of epithelial cells and generated from adult stem cells. Both classifications have been used as models to develop drugs against cystic fibrosis, cancer and infectious disease, as well as for drug screening, personalized medicine and the development of new medical tools. In this review, we highlight and discuss the importance of mini-guts for drug development and point out their limitations and future prospects.

Abstract Biliary disorders can lead to life‐threatening disease and are also a challenging complication of liver transplantation. As there are limited treatment options, tissue engineered bile ducts could be employed to replace or repair damaged bile ducts. We explored how these constructs can be created by seeding hepatobiliary LGR5 + organoids onto tissue‐specific scaffold. For this, we decellularized discarded human extrahepatic bile ducts (EBD) that we recellularized with organoids of different origin, that is, liver biopsies, extrahepatic bile duct biopsies, and bile samples. Here, we demonstrate efficient decellularization of EBD tissue. Recellularization of the EBD extracellular matrix (ECM) with the organoids of extrahepatic origin (EBD tissue and bile derived organoids) showed more profound repopulation of the ductal ECM when compared with liver tissue (intrahepatic bile duct) derived organoids. The bile duct constructs that were repopulated with extrahepatic organoids expressed mature cholangiocyte‐markers and had increased electrical resistance, indicating restoration of the barrier function. Therefore, the organoids of extrahepatic sources are identified to be the optimal candidate for the development of personalized tissue engineered EBD constructs.

CFTR, the cystic fibrosis (CF) gene-encoded epithelial anion channel, has a prominent role in driving chloride, bicarbonate and fluid secretion in the ductal cells of the exocrine pancreas. Whereas severe mutations in CFTR cause fibrosis of the pancreas in utero, CFTR mutants with residual function, or CFTR variants with a normal chloride but defective bicarbonate permeability (CFTRBD), are associated with an enhanced risk of pancreatitis. Recent studies indicate that CFTR function is not only compromised in genetic but also in selected patients with an acquired form of pancreatitis induced by alcohol, bile salts or smoking. In this review, we summarize recent insights into the mechanism and regulation of CFTR-mediated and modulated bicarbonate secretion in the pancreatic duct, including the role of the osmotic stress/chloride sensor WNK1 and the scaffolding protein IRBIT, and current knowledge about the role of CFTR in genetic and acquired forms of pancreatitis. Furthermore, we discuss the perspectives for CFTR modulator therapy in the treatment of exocrine pancreatic insufficiency and pancreatitis and introduce pancreatic organoids as a promising model system to study CFTR function in the human pancreas, its role in the pathology of pancreatitis and its sensitivity to CFTR modulators on a personalized basis.

The cystic fibrosis transmembrane conductance regulator (CFTR), the ABC transporter encoded by the cystic fibrosis gene, is localized in the apical membrane of epithelial cells where it functions as a cyclic AMP-regulated chloride channel and as a regulator of other ion channels and transporters. Whereas a key role of cAMP-dependent phosphorylation in CFTR-channel gating has been firmly established, more recent studies have provided clear evidence for the existence of a second level of cAMP regulation, i.e. the exocytotic recruitment of CFFR to the plasma membrane and its endocytotic retrieval. Regulated trafficking of the CFTR Cl- channel has sofar been demonstrated only in a subset of CFTR-expressing cell types. However, with the introduction of more sensitive methods to measure CFTR cycling and submembrane localization, it might turn out to be a more general phenomenon that could contribute importantly to both the regulation of CFTR-mediated chloride transport itself and to the regulation of other transporters and CFTR-modulated cellular functions. This review aims to summarize the present state of knowledge regarding polarized and regulated CFTR trafficking and endosomal recycling in epithelial cells, to discuss present gaps in our understanding of these processes at the cellular and molecular level, and to consider its possible implications for cystic fibrosis.

Guanylyl cyclase (GC) C is a heat-stable enterotoxin (STa) receptor with a monomeric M(r) of approximately 140,000. We calculated from its hydrodynamic parameters that an active GC-C complex has a M(r) of 393,000, suggesting that GC-C is a trimer under native conditions. Both trimeric and dimeric GC-C complexes were detected by 125I-STa binding and SDS-polyacrylamide gel electrophoresis under non-reducing conditions. The GC activity and STa binding from intestinal brush border membranes comigrated in gel filtration and velocity sedimentation with recombinant GC-C. However, 125I-STa cross-linking demonstrated that STa receptors with molecular masses of 52 and 74 kDa are non-covalently attached to GC in the intestine. Radiation inactivation revealed different functional sizes for basal GC activity, STa-stimulated GC activity, and STa binding (59, 210-240, and 32-52 kDa, respectively). At low radiation doses, basal GC activity was stimulated, suggesting that GC-C is inhibited by a relatively large, probably internal structure. These results suggest that STa may activate GC-C by promoting monomer-monomer interaction (internal dimerization) within a homotrimeric GC-C complex, and that GC-C is proteolytically modified in the brush border membrane but retains its function.

In mammalian tissues two types of cGMP-dependent protein kinase (cGK) have been identified. In contrast to the dimeric cGK I, cGK II purified from pig intestine was shown previously to behave as a monomer. However, recombinant rat cGK II was found to have hydrodynamic parameters indicative of a homodimer. Chemical cross-linking studies showed that pig cGK II in intestinal membranes has a dimeric structure as well. However, after purification, cGK II was found to be partly proteolyzed into C-terminal monomeric fragments. Phosphorylation studies in rat intestinal brush borders revealed that the potency of cGMP analogs to stimulate or inhibit native cGK II in vitro (i.e.8-(4-chlorophenylthio)-cGMP > cGMP > β-phenyl-1,N 2-etheno-8-bromo-cGMP > β-phenyl-1,N 2-etheno-cGMP andR p-8-(4-chlorophenylthio)-cGMPs >R p-β-phenyl-1,N 2-etheno-8-bromo-cGMPs, respectively) correlated well with their potency to stimulate or inhibit cGK II-mediated Cl− secretion across intestinal epithelium but differed strikingly from their potency to affect cGK I activity. These data show that the N terminus of cGK II is involved in dimerization and that endogenous cGK II displays a distinct activation/inhibition profile with respect to cGMP analogs, which permits a pharmacological dissection between cGK II- and cGK I-mediated physiological processes. In mammalian tissues two types of cGMP-dependent protein kinase (cGK) have been identified. In contrast to the dimeric cGK I, cGK II purified from pig intestine was shown previously to behave as a monomer. However, recombinant rat cGK II was found to have hydrodynamic parameters indicative of a homodimer. Chemical cross-linking studies showed that pig cGK II in intestinal membranes has a dimeric structure as well. However, after purification, cGK II was found to be partly proteolyzed into C-terminal monomeric fragments. Phosphorylation studies in rat intestinal brush borders revealed that the potency of cGMP analogs to stimulate or inhibit native cGK II in vitro (i.e.8-(4-chlorophenylthio)-cGMP > cGMP > β-phenyl-1,N 2-etheno-8-bromo-cGMP > β-phenyl-1,N 2-etheno-cGMP andR p-8-(4-chlorophenylthio)-cGMPs >R p-β-phenyl-1,N 2-etheno-8-bromo-cGMPs, respectively) correlated well with their potency to stimulate or inhibit cGK II-mediated Cl− secretion across intestinal epithelium but differed strikingly from their potency to affect cGK I activity. These data show that the N terminus of cGK II is involved in dimerization and that endogenous cGK II displays a distinct activation/inhibition profile with respect to cGMP analogs, which permits a pharmacological dissection between cGK II- and cGK I-mediated physiological processes. Cyclic GMP-dependent protein kinases (cGKs) 1The abbreviations used are: cGK, cGMP-dependent protein kinase; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-cGMP; PET-cGMP, β-phenyl-1,N 2-etheno-cGMP; 8-Br-PET-cGMP, β-phenyl-1,N 2-etheno-8-bromo-cGMP;R p-8-pCPT-cGMPS, R pisomer of 8-(4-chlorophenylthio)-guanosine-3′-5′-cyclic monophosphoro-thioate; R p-8-Br-PET-cGMPS,R p isomer of β-phenyl-1,N 2-etheno-8-bromo-guanosine-3′-5′-cyclic monophosphorothioate; ISC, short-circuit current; PAGE, polyacrylamide gel electrophoresis. play an important role in the signaling of various cGMP-linked hormones and neurotransmitters including nitric oxide (NO), natriuretic peptides, and guanylin (1Butt E. Geiger J. Jarchau T. Lohmann S.M. Walter U. Neurochem. Res. 1993; 18: 27-42Google Scholar, 2Vaandrager A.B. De Jonge H.R. Mol. Cell. Biochem. 1996; 157: 23-30Google Scholar). In mammalian tissues two types of cGK have been identified. Type I cGK, consisting of α and β isoforms, is more generally expressed and acts as a key regulator of cardiovascular homeostasis (1Butt E. Geiger J. Jarchau T. Lohmann S.M. Walter U. Neurochem. Res. 1993; 18: 27-42Google Scholar, 2Vaandrager A.B. De Jonge H.R. Mol. Cell. Biochem. 1996; 157: 23-30Google Scholar). In contrast, type II cGK was described originally as an intestine-specific form (3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar). Molecular cloning demonstrated that cGK II is indeed a distinct gene product expressed predominantly in epithelial cells of the intestine (4Jarchau T. Häusler C. Markert T. Pöhler D. Vandekerckhove J. De Jonge H.R. Lohmann S.M. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9426-9430Google Scholar), although its mRNA was also detected in kidney, bone, and brain (4Jarchau T. Häusler C. Markert T. Pöhler D. Vandekerckhove J. De Jonge H.R. Lohmann S.M. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9426-9430Google Scholar, 5Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Google Scholar, 6El-Husseini A. Bladen C. Vincent S.R. J. Neurochem. 1995; 64: 2814-2817Google Scholar, 7Pfeifer A. Aszódi A. Seidler U. Ruth P. Hofmann F. Fässler R. Science. 1996; 274: 2082-2086Google Scholar). Its widespread distribution in various areas of the brain suggests an important role of cGK II in NO/cGMP signaling in the central nervous system (6El-Husseini A. Bladen C. Vincent S.R. J. Neurochem. 1995; 64: 2814-2817Google Scholar). In the intestine, cGMP is involved in the regulation of ion and water transport. It inhibits the uptake of NaCl and stimulates the secretion of Cl− by activating the cystic fibrosis trans-membrane conductance regulator, a Cl− channel that is mutated in CF patients (8Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (, 879–883): 800-806Google Scholar, 9Vaandrager A.B. De Jonge H.R. Adv. Pharmacol. 1994; 26: 253-283Google Scholar). Guanylin, a small peptide derived from a larger precursor protein released luminally by intestinal epithelial cells, may function as the physiological activator of the cGMP-mediated signaling pathway in intestine by activating guanylyl cyclase C (10Schulz S. Green C.K. Yuen P.S.T. Garbers D.L. Cell. 1990; 63: 941-948Google Scholar,11Currie M.G. Fok K.F. Kato J. Moore R.J. Hamra F.K. Duffin K.L. Smith C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 947-951Google Scholar). Heat-stable enterotoxins secreted by various pathogenic strains ofEscherichia coli mimic the action of guanylin and elicit a severe secretory diarrhea by hyperactivating guanylyl cyclase C (8Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (, 879–883): 800-806Google Scholar, 9Vaandrager A.B. De Jonge H.R. Adv. Pharmacol. 1994; 26: 253-283Google Scholar). Electrophysiological and immunolocalization studies have provided evidence for a key role for cGK II as a mediator of the cGMP-provoked intestinal Cl− secretion (12Markert T. Vaandrager A.B. Gambaryan S. Pöhler D. Häusler C. Walter U. De Jonge H.R. Jarchau T. Lohmann S.M. J. Clin. Invest. 1995; 96: 822-830Google Scholar, 13French P.J. Bijman J. Edixhoven M. Vaandrager A.B. Scholte B.J. Lohmann S.M. Nairn A.C. de Jonge H.R. J. Biol. Chem. 1995; 270: 26626-26631Google Scholar, 14Vaandrager A.B. Tilly B.C. Smolenski A. Schneider-Rasp Bot A.G.M. Edixhoven M. Scholte B.J. Jarchau T. Walter U. Lohmann S.M. Poller W.C. De Jonge H.R. J. Biol. Chem. 1997; 272: 4195-4200Google Scholar). The critical role of cGK II in STa/cGMP-induced diarrhea was recently confirmed by pharmacological and gene disruption techniques (7Pfeifer A. Aszódi A. Seidler U. Ruth P. Hofmann F. Fässler R. Science. 1996; 274: 2082-2086Google Scholar, 15Vaandrager A.B. Bot A.G.M. De Jonge H.R. Gastroenterology. 1997; 112: 437-443Google Scholar). Sequence comparison and biochemical analysis revealed a large degree of similarity in the structural organization of cGK I and II (2Vaandrager A.B. De Jonge H.R. Mol. Cell. Biochem. 1996; 157: 23-30Google Scholar, 3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar, 4Jarchau T. Häusler C. Markert T. Pöhler D. Vandekerckhove J. De Jonge H.R. Lohmann S.M. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9426-9430Google Scholar, 5Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Google Scholar). Both isotypes possess two cGMP binding domains on one polypeptide chain which is covalently coupled to a catalytic domain. Their N terminus contains an autoinhibitory region, one or more autophosphorylation sites, and a leucine zipper motif, and both proteins are devoid of hydrophobic trans-membrane domains. Despite these similarities, cGK II was shown to differ from soluble dimeric type I cGK in that it behaved as a membrane- and cytoskeleton-associated protein in intestinal brush borders and as a monomer following its solubilization and purification (3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar). However, recombinant mouse brain cGK II was reported to be soluble and dimeric after expression in mammalian and insect cells (5Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Google Scholar, 16Gamm D.M. Francis S.H. Angelotti T.P. Corbin J.D. Uhler M.D. J. Biol. Chem. 1995; 270: 27380-27388Google Scholar). In contrast, recombinant rat intestinal cGK II was found to be tightly bound to the membrane after expression in similar cell systems (4Jarchau T. Häusler C. Markert T. Pöhler D. Vandekerckhove J. De Jonge H.R. Lohmann S.M. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9426-9430Google Scholar, 17Pöhler D. Butt E. Meissner J. Müller S. Lohse M. Walter U. Lohmann S.M. Jarchau T. FEBS Lett. 1995; 374: 419-425Google Scholar,18Vaandrager A.B. Ehlert E.M.E. Jarchau T. Lohmann S.M. de Jonge H.R. J. Biol. Chem. 1996; 271: 7025-7029Google Scholar), but its oligomerization state was not established. Furthermore, the K a values for cGMP and cGMP analogs of recombinant rat intestine cGK II purified from Sf9 cells (17Pöhler D. Butt E. Meissner J. Müller S. Lohse M. Walter U. Lohmann S.M. Jarchau T. FEBS Lett. 1995; 374: 419-425Google Scholar) differed considerably from those of recombinant mouse brain cGK II, which contained an additional N-terminal histidine tag (16Gamm D.M. Francis S.H. Angelotti T.P. Corbin J.D. Uhler M.D. J. Biol. Chem. 1995; 270: 27380-27388Google Scholar). Since rat and mouse cGK II have very similar sequences, are endogenously both membrane-bound in intestine, and are activable by similar concentrations of cGMP after expression in COS-1 cells (5Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Google Scholar, 18Vaandrager A.B. Ehlert E.M.E. Jarchau T. Lohmann S.M. de Jonge H.R. J. Biol. Chem. 1996; 271: 7025-7029Google Scholar), the observed kinetic and structural differences among purified cGK II preparations may reflect artifacts of the expression system and/or purification rather than fundamental differences. To circumvent such potential artifacts, we characterized the oligomerization state of cGK II and its kinetic properties in an environment most relevant for its physiological functioning, i.e. intestinal brush border membranes. We also compared the stimulatory or inhibitory potency of various cGMP analogs toward cGK II in vitro, with their effects on Cl− secretion in rat intestinal epithelium, presumably a cGK II-mediated process (7Pfeifer A. Aszódi A. Seidler U. Ruth P. Hofmann F. Fässler R. Science. 1996; 274: 2082-2086Google Scholar, 9Vaandrager A.B. De Jonge H.R. Adv. Pharmacol. 1994; 26: 253-283Google Scholar, 12Markert T. Vaandrager A.B. Gambaryan S. Pöhler D. Häusler C. Walter U. De Jonge H.R. Jarchau T. Lohmann S.M. J. Clin. Invest. 1995; 96: 822-830Google Scholar, 13French P.J. Bijman J. Edixhoven M. Vaandrager A.B. Scholte B.J. Lohmann S.M. Nairn A.C. de Jonge H.R. J. Biol. Chem. 1995; 270: 26626-26631Google Scholar, 14Vaandrager A.B. Tilly B.C. Smolenski A. Schneider-Rasp Bot A.G.M. Edixhoven M. Scholte B.J. Jarchau T. Walter U. Lohmann S.M. Poller W.C. De Jonge H.R. J. Biol. Chem. 1997; 272: 4195-4200Google Scholar, 15Vaandrager A.B. Bot A.G.M. De Jonge H.R. Gastroenterology. 1997; 112: 437-443Google Scholar). We report here that the endogenous intestinal membrane-bound type II cGMP-dependent protein kinase exists as a dimer and displays a distinct activation profile with respect to cGMP analogs, which permits discrimination between cGK II and cGK I effects in physiological processes. Disuccinimidyl suberate was obtained from Pierce, and 3-isobutyl-1-methylxanthine was from Sigma. Polyclonal cGK II or cGK I antibodies, raised against recombinant cGK II or cGK Iβ expressed in E. coli, were prepared as described (12Markert T. Vaandrager A.B. Gambaryan S. Pöhler D. Häusler C. Walter U. De Jonge H.R. Jarchau T. Lohmann S.M. J. Clin. Invest. 1995; 96: 822-830Google Scholar). The cGK substrate peptide 2A3 (RRKVSKQE) and the Walsh inhibitor peptide (protein kinase A inhibitor-(5–24)-amide) were synthesized by D. Palm (University of Würzburg, Germany), and the cGMP analogs were from Biolog (Bremen). cGK I (primarily the Iα isoform; Ref. 13French P.J. Bijman J. Edixhoven M. Vaandrager A.B. Scholte B.J. Lohmann S.M. Nairn A.C. de Jonge H.R. J. Biol. Chem. 1995; 270: 26626-26631Google Scholar) was purified from bovine lung as described (19Walter U. Miller P. Wilson F. Menkes D. Greengard P. J. Biol. Chem. 1980; 255: 3757-3762Google Scholar). Confluent HEK-293 or NIH-3T3 cells, stably transfected with cGK II (18Vaandrager A.B. Ehlert E.M.E. Jarchau T. Lohmann S.M. de Jonge H.R. J. Biol. Chem. 1996; 271: 7025-7029Google Scholar), were washed twice with ice-cold phosphate-buffered saline, scraped with a rubber policeman in buffer A (150 mmNaCl, 10 mm NaPO4, pH 7.4, 1 mmEDTA, 100 μg/ml trypsin inhibitor, and 20 μg/ml leupeptin) and processed directly or frozen in liquid N2 and stored at −80 °C. The cells were homogenized by brief sonication (three bursts of 3 s, peak-to-peak amplitude 15–20 μm), and a crude membrane fraction was prepared by centrifugation for 15 min at 20,000 × g. Membranes from HEK-cGK II cells were resuspended in buffer A (2–3 mg of protein/ml), and membranes from 3T3-cGK II cells (which had lower basal protein kinase activity in cGMP activation assays) were resuspended to 1 mg of protein/ml in buffer B (20 mm Tris/HCl, pH 7.4, 5 mmβ-mercaptoethanol, 2 mm EDTA, 100 μg/ml trypsin inhibitor, and 20 μg/ml leupeptin). Non-vesiculated brush border caps were prepared from jejunum and ileum of male Wistar rats by vibration of everted intestine in hypotonic EDTA buffer and low speed centrifugation, essentially as described previously (20Vaandrager A.B. Bot A.G.M. De Vente J. De Jonge H.R. Gastroenterology. 1992; 102: 1161-1169Google Scholar), and were finally resuspended to 0.4 mg of protein/ml in buffer B. Brush border membrane vesicles were isolated from everted pig intestine by freeze-thawing, followed by Mg2+ precipitation according to Ref. 21Van Dommelen F.S. Hamer C.M. de Jonge H.R. Biochem. J. 1986; 236: 771-778Google Scholar. cGK II was solubilized from HEK-cGK II membranes or pig brush border membrane vesicles by addition of 0.5 m NaCl and 1% Triton X-100 and subsequently purified by affinity chromatography on 8-(2-aminoethyl)-amino-cAMP-Sepharose as described (3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar). Gel filtration was performed essentially as described (22Vaandrager A.B. van der Wiel E. Hom M.L. Luthjens L.H. de Jonge H.R. J. Biol. Chem. 1994; 269: 16409-16415Google Scholar) using Superose 6 HR 10/30 or Superdex 200 HR 10/30 analytical gel filtration columns (Pharmacia Biotech Inc.) equilibrated and subsequently eluted at 0.2 ml/min with 500 mm NaCl, 10 mm sodium phosphate buffer, pH 7.4, 10 mm EDTA, and 0.1% Triton X-100. Fractions of 400 μl were collected and analyzed by immunoblotting or protein kinase activity assays. For each experiment the column was calibrated using standards. Sucrose density centrifugation was performed with linear 7.5–25% sucrose gradients in the same buffer used for gel filtration (22Vaandrager A.B. van der Wiel E. Hom M.L. Luthjens L.H. de Jonge H.R. J. Biol. Chem. 1994; 269: 16409-16415Google Scholar). Apparent sedimentation coefficients (s w, 20) were calculated by plotting the distance from the top of the gradient against the position of the following standards: catalase (11.3 S), bovine serum albumin (4.9 S), and cytochrome c (1.9 S). The molecular mass (m) of cGK I and cGK II was calculated from: m = 6πηNas w, 20/(1 − υρ) where η = viscosity of medium, n = Avogadro's number, a = Stokes radius,s w, 20 = sedimentation coefficient, υ = partial specific volume, and ρ = density of the medium. We assumed υ for proteins to be 0.73 cm3/g (22Vaandrager A.B. van der Wiel E. Hom M.L. Luthjens L.H. de Jonge H.R. J. Biol. Chem. 1994; 269: 16409-16415Google Scholar). Purified pig cGK II was separated by SDS-PAGE and blotted to polyvinylidene difluoride ProblottR membrane. Pieces of membrane containing the 75- and 70-kDa forms of cGK II were cut out separately, and both protein fragments were N-terminally sequenced by automatic Edman degradation with a 473A protein sequencer (Applied Biosystems). For cross-linking, samples (2 mg of protein/ml) were incubated for 15 min at 0 °C in 10 mm phosphate buffer, pH 7.4, 100 mm NaCl, 10 mm EDTA with or without 0.6 mm disuccinimidyl suberate. The reaction was stopped by addition of SDS-PAGE sample buffer and cGK II was analyzed by immunoblotting. Immunoblotting was performed as described earlier (23Vaandrager A.B. Schulz S. De Jonge H.R. Garbers D.L. J. Biol. Chem. 1993; 268: 2174-2179Google Scholar). Immunoreactive proteins were detected after incubation with cGK II or cGK I antibody (1:3000) by the enhanced chemiluminescence method (Amersham Corp.) and quantitated by densitometric scanning (Bio-Rad, model 620). Protein kinase activity was determined by incubation of the samples (4–10 μg of membrane protein in case of cGK II or 30 ng of purified cGK I provided with 10 μg of bovine serum albumin) at 30 °C for different times in 40 μl of 20 mm Tris/HCl, pH 7.4, 10 mm MgCl2, 5 mm β-mercaptoethanol, 0.1 mm 3-isobutyl-1-methylxanthine, 25 mmsodium β-glycerophosphate, 200 nm protein kinase A inhibitor, 0.1 mg/ml cGK substrate peptide 2A3 (RRKVSKQE; Ref. 17Pöhler D. Butt E. Meissner J. Müller S. Lohse M. Walter U. Lohmann S.M. Jarchau T. FEBS Lett. 1995; 374: 419-425Google Scholar), 1 μCi of [γ-32P]ATP, and various concentrations of nonradioactive Mg-ATP and cGMP or cGMP analogs as indicated. The reaction was started by addition of 10-μl aliquots of the cGK II preparations to 30 μl of prewarmed incubation buffer and quenched by addition of 10 μl of 0.5 m EDTA. The samples were subsequently centrifuged for 10 min at 20,000 × g. The pellet fraction was resuspended in SDS-PAGE sample buffer for determination of the autophosphorylation of cGK II (3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar), and 15 μl of the supernatant was spotted in duplicate on sheets of P-81 chromatography paper (Whatman). After 4 washes with 1% phosphoric acid the amount of label incorporated was quantitated with the Molecular Imaging System GS-363 (Bio-Rad). The phosphotransferase activity of cGK II as measured in intestinal brush border and 3T3 cell membranes and the activity of cGK I were linear with the amount of enzyme up to the concentration used in the kinetic study and with time up to 4 min in the presence of relatively high ATP concentrations (≥300 μm). However, in the presence of low ATP, the cGK II kinase activity started to deviate from linearity after 2 min. This nonlinearity was independent of the concentration of cGMP or cGMP analogs and apparently resulted from ATP depletion caused by the action of endogenous ATPases (data not shown). The K m of cGK II for ATP was therefore determined from 2-min incubations, whereas the kinetic parameters for the cGMP analogs were routinely determined on the basis of 4-min incubations. The latter condition resulted in similar kinetic values but higher signal to noise ratios in comparison with 2-min incubations. A 1-cm long segment of rat cecum was removed under light diethyl ether anesthesia. The muscle layers were stripped off by blunt dissection, and the mucosa was mounted in an Ussing chamber (0.3-cm2area exposed) for measurements of short-circuit current (Isc) across the tissue as described (24Veeze H.J. Sinaasappel M. Bijman J. Bouquet J. De Jonge H.R. Gastroenterology. 1991; 101: 398-403Google Scholar). Dose-response curves were obtained by cumulative additions of agonists or antagonists, and subsequent measurements of the plateau phase of the ISC reached 10–20 min after each addition. The values for EC50, IC50, and apparent K a (defined as the concentration required for half-maximal activation) were determined from dose-response curves fitted by the program Slidewrite.K m and Hill coefficients were calculated with the program Enzfitter. K i values were determined from Dixon plots at half-maximal concentration of agonist (cGMP), assuming a single site. The lipophilicities of cGMP analogs were determined by gradient reversed phase chromatography essentially as described (25Braumann T. Jastorff B.J. J. Chromatogr. 1985; 350: 105-118Google Scholar) and comparable to common log p values. Immunoblots demonstrated that recombinant rat cGK II solubilized from HEK 293 membranes was eluted from gel filtration as a single peak with a Stokes radius of 6.6 which is significantly larger than that of purified cGK Iα (5.1) eluted under the same conditions (Fig. 1 A and TableI). Velocity sedimentation analysis likewise revealed single peaks for cGK II and Iα with sedimentation coefficients of 6.8 and 7.2 S, respectively (Table I). From these hydrodynamic parameters the molecular mass of cGK II was calculated as 190 kDa, more than twice the monomeric mass of 87 kDa (4Jarchau T. Häusler C. Markert T. Pöhler D. Vandekerckhove J. De Jonge H.R. Lohmann S.M. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9426-9430Google Scholar), indicating that cGK II most likely exists as a homodimer in solution. cGK II immunoprecipitation or purification by cAMP-Sepharose chromatography did not reveal any associated proteins that could falsify the mass determination (data not shown, Ref. 18Vaandrager A.B. Ehlert E.M.E. Jarchau T. Lohmann S.M. de Jonge H.R. J. Biol. Chem. 1996; 271: 7025-7029Google Scholar).Table IHydrodynamic parameters of recombinant and endogenous cGK IISampleStokes radiusApparents 20,wM r × 10−3 calculatednmSRecomb. cGK II (rat)6.6 ± 0.26.8 ± 0.3190Recomb. cGK II pure6.4 ± 0.27.0 ± 0.2190cGK II pure (pig intestine)86-kDa form6.3 ± 0.2ND75–70-kDa forms3.5 ± 0.1NDcGK I pure (bovine lung)5.1 ± 0.27.2 ± 0.4157The Stokes radius was determined by gel filtration; the sedimentation coefficient by sucrose density centrifugation, and the molecular weight was calculated from these parameters as described under “Experimental Procedures.” Recombinant cGK II was analyzed either directly after solubilization of membranes from HEK-293 cells stably transfected with rat cGK II (Recomb. cGK II) or after subsequent purification (recomb. cGK II pure). cGK II purified from pig intestine was found to consist of a 86-kDa full-length form and two fragments of approximately 75 and 70 kDa (see Fig. 1). Purified bovine lung cGK I (consisting mainly of the type 1α form) was added to the recombinant cGK II preparations as an internal standard (Stokes radius = 5.0–5.3 nm, apparents 20,w = 6.9–7.8 S and M r= 150,000–178,000; Refs. 3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar, 13French P.J. Bijman J. Edixhoven M. Vaandrager A.B. Scholte B.J. Lohmann S.M. Nairn A.C. de Jonge H.R. J. Biol. Chem. 1995; 270: 26626-26631Google Scholar, and 27Atkinson R.A. Saudek V. Huggins J.P. Pelton J.T. Biochemistry. 1991; 30: 9387-9395Google Scholar). Data represent means ± S.E. of three experiments, ND, not determined. Open table in a new tab The Stokes radius was determined by gel filtration; the sedimentation coefficient by sucrose density centrifugation, and the molecular weight was calculated from these parameters as described under “Experimental Procedures.” Recombinant cGK II was analyzed either directly after solubilization of membranes from HEK-293 cells stably transfected with rat cGK II (Recomb. cGK II) or after subsequent purification (recomb. cGK II pure). cGK II purified from pig intestine was found to consist of a 86-kDa full-length form and two fragments of approximately 75 and 70 kDa (see Fig. 1). Purified bovine lung cGK I (consisting mainly of the type 1α form) was added to the recombinant cGK II preparations as an internal standard (Stokes radius = 5.0–5.3 nm, apparents 20,w = 6.9–7.8 S and M r= 150,000–178,000; Refs. 3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar, 13French P.J. Bijman J. Edixhoven M. Vaandrager A.B. Scholte B.J. Lohmann S.M. Nairn A.C. de Jonge H.R. J. Biol. Chem. 1995; 270: 26626-26631Google Scholar, and 27Atkinson R.A. Saudek V. Huggins J.P. Pelton J.T. Biochemistry. 1991; 30: 9387-9395Google Scholar). Data represent means ± S.E. of three experiments, ND, not determined. In previous studies cGK II purified from pig intestine was found to behave as a monomer in gel filtration and sucrose density sedimentation analyses, although kinase activity, not immunoblotting as above, was used for cGK II detection (3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar). We therefore compared the hydrodynamic parameters of recombinant rat cGK II with those of native pig cGK II after purification. As shown in Table I, purification had no effect on the dimeric state of recombinant rat cGK II. However, purified pig cGK II was recovered in two major peaks after gel filtration as detected by immunoblotting (see Fig. 1 B). In the first peak, full-length cGK II (86 kDa) eluted at a position indicative of a dimer. Subsequently, 75- and 70-kDa cGK II fragments eluted at a position corresponding to a Stokes radius of 3.5 nm, similar to that found for monomeric cGK II previously (3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar). In accordance with the previous study, most of the kinase activity (>90%) was recovered in the second peak (data not shown). The proteins in the second peak were identified by protein sequencing as C-terminal, proteolytic fragments of cGK II. The N-terminal sequences VPLDV and PPEF obtained from the 75- and the 70-kDa form, respectively, matched those of a 75-kDa cGK II fragment beginning at Val101 and of a 70-kDa fragment starting at Pro139 (4Jarchau T. Häusler C. Markert T. Pöhler D. Vandekerckhove J. De Jonge H.R. Lohmann S.M. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9426-9430Google Scholar). To exclude the possibility that cGK II dimerization could be an artifact of the solubilization process, we compared the oligomeric structure of cGK II in membranes with that of solubilized cGK II by chemical cross-linking. As shown in Fig. 2, addition of the cross-linker disuccinimidyl suberate to pig brush border membrane vesicles or to membranes of HEK cells stably transfected with recombinant cGK II, either before or after solubilization, resulted in the appearance of a cGK II complex at the position of a dimer (170 kDa) in SDS-PAGE in all cases, indicating that cGK II is also dimeric in membranes. The additional cross-linked bands observed at 80- and 210-kDa positions may represent intrachain or multiple interchain cross-linked forms of cGK II. Non-vesiculated brush border caps freshly isolated from rat intestine were found to offer a suitable model for measuring cGK II activity in its native membrane environment. They are enriched in cGK II (0.5–1 μg of kinase/mg of protein; Refs. 3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar, 21Van Dommelen F.S. Hamer C.M. de Jonge H.R. Biochem. J. 1986; 236: 771-778Google Scholar), contain no detectable levels of cGK I (data not shown, cf. Refs. 3De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333Google Scholar, 9Vaandrager A.B. De Jonge H.R. Adv. Pharmacol. 1994; 26: 253-283Google Scholar, and 12Markert T. Vaandrager A.B. Gambaryan S. Pöhler D. Häusler C. Walter U. De Jonge H.R. Jarchau T. Lohmann S.M. J. Clin. Invest. 1995; 96: 822-830Google Scholar), and are fully accessible to ATP and exogenous peptide substrates (21Van Dommelen F.S. Hamer C.M. de Jonge H.R. Biochem. J. 1986; 236: 771-778Google Scholar). Furthermore, cGMP could stimulate the phosphorylation of exogenous substrate by brush border preparations to a similar extent (7-fold) as reported for purified recombinant cGK II (Fig.3; Refs. 16Gamm D.M. Francis S.H. Angelotti T.P. Corbin J.D. Uhler M.D. J. Biol. Chem. 1995; 270: 27380-27388Google Scholar and 17Pöhler D. Butt E. Meissner J. Müller S. Lohse M. Walter U. Lohmann S.M. Jarchau T. FEBS Lett. 1995; 374: 419-425Google Scholar). The apparent K m of membrane-bound cGK II for the substrate peptide 2A3 was found to be similar to that of cGK I (0.10 and 0.12 mm, respectively; data not shown). However, endogenous cGK II at saturating cGMP (10 μm) has a relatively high K m for ATP (0.40 mm) in comparison to cGK I (0.066 mm) (see Fig. 4 A). Moreover, ATP was observed to decrease the sensitivity of cGK II for cGMP. As shown in Fig.4 B, the apparent K a for cGMP shifted more than 10-fold, from 50 nm at 10 μm ATP to 560 nm at 1 mm ATP. A similar shift in the apparentK a for cGMP by ATP in the range of 10–100 μm was noticed in case of cGK I. In the following experiments we routinely determined cGK kinase activity in the presence of 300 μm ATP, since this condition ensured a relatively high signal-to-noise ratio and was comparable with that used in similar studies characterizing purified cGK II (16Gamm D.M. Francis S.H. Angelotti T.P. Corbin J.D. Uhler M.D. J. Biol. Chem. 1995; 270: 27380-27388Google Scholar, 17Pöhler D. Butt E. Meissner J. Müller S. Lohse M. Walter U. Lohmann S.M. Jarchau T. FEBS Lett. 1995; 374: 419-425Google Scholar). At 300 μm ATP using 2A3 as substrate, the apparentK a for cGMP of endogenous cGK II was found to be almost identical to that of purified bovine lung

We studied the activation and inactivation of recombinant guanylyl cyclase (GC) C stably expressed in human kidney 293 cells. Activation of GC-C by heat-stable enterotoxin (STa), Cd2+, hemin, or the detergent Triton X-100 was followed by a rapid inactivation of the enzyme. Adenine nucleotides were found to prevent the inactivation process in native membranes, as well as following enzyme solubilization and immunopurification. Inactivation of GC-C was not associated with dephosphorylation. However, the phosphorylation of GC-C was promoted by phorbol esters, known activators of protein kinase C. The activation of purified GC-C by STa required the presence of a nonspecific factor as exemplified by bovine serum albumin. When immunopurified GC-C, stabilized by ATP and bovine serum albumin, was analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, proteins with almost twice the molecular mass (220 and 245 kDa) of the monomer were observed. The mobility of these high M(r) GC-C forms was reduced by STa, suggesting that STa induces a conformation change in a preexisting GC-C dimer. These results indicate that ATP interacts directly with GC-C, stabilizing its active conformation and that the activation of GC-C may occur in the absence of other specific regulatory factors.

Abstract The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-thiophosphate (GTP gamma S) led to a large increase in Cl- conductance, as evidenced by an increased 125I- uptake and faster SPQ quenching. The enhancement was observed in the presence, but not in the absence of the K+ ionophore valinomycin, indicating that the increased Cl- permeability is not secondary to the opening of K+ channels. The effect of GTP gamma S was counteracted by guanosine 5'-O-(2-thiophosphate (GDP beta S) and appeared to be independent of cytosolic messengers, including ATP, cAMP, and Ca2+, suggesting that protein phosphorylation and/or phospholipase C activation is not involved. Patch clamp analysis of apical membrane patches of HT29-cl.19A colonocytes revealed a GTP gamma S-activated, inwardly rectifying, anion-selective channel with a unitary conductance of 20 +/- 4 pS. No spontaneous channel openings were observed in the absence of GTP gamma S, while the open time probability (Po) increases dramatically to 0.81 +/- 0.09 upon addition with GTP gamma S. Since the electrophysiological characteristics and regulatory properties of this channel are markedly different from those of the more widely studied cAMP/protein kinase A-operated channel, we propose the existence of a separate Cl(-)-selective ion channel in the apical border of intestinal epithelial cells. Our results suggest an alternative regulatory pathway in transepithelial salt transport and a possible site for anomalous channel regulation as observed in cystic fibrosis patients.

Purified basolateral membranes of rat duodenal epithelium were phosphorylated in the presence of [γ-32P]ATP and 1 μM free Ca2+ under conditions favorable for phospho-intermediate formation. Analysis of 32P-labeled membrane proteins on SDS-acrylamide gel electrophoresis revealed the presence of three Ca2+-sensitive phosphoproteins with molecular weights of 64 000 (protein I), 84 000 (II) and 115 000 (III). Both proteins I and II were identified as phospho-intermediate forms of alkaline phosphatase on the basis of the following criteria: these phosphoproteins were resistant to hydroxylamine and alkaline treatment, phosphate incorporation was stimulated by Zn2+ but totally inhibited by excess β-glycerophosphate during phosphorylation. A similar labeling efficiency was obtained with inorganic 32P instead of [γ-32P]ATP. Protein III comigrated on SDS gels with the phosphorylated intermediate of Ca2+-ATPase from rat heart sarcolemnal membranes and was identified as the intestinal form of Ca2+-ATPase in view of the following observations: binding of 32P to this protein was hydroxylamine sensitive and alkaline labile, which is in agreement with an acylphosphate character of an ATPase intermediate, phospho-intermediate formation was dependent on low free Ca2+ concentrations (1 μM) in the presence of 1 mM Mg2+, phosphorylation was strongly inhibited in the presence of phenothiazines (0.1 mM chlorpromazine or trifluoperazine). The amount of 32P bound to Ca2+-ATPase in basolateral membranes was only 6% of the amount incorporated into (Na+ + K+)-ATPase. The turnover number of duodenal Ca2+-ATPase estimated from this phosphorylation study is remarkably similar to the value for the red blood cell Ca2+-ATPase. Phosphorylation experiments with purified leaky brush border membranes under similar conditions did not reveal a phospho-intermediate from of Ca2+-ATPase as demonstrated in basolateral membranes. In contrast about 7-fold higher levels of 32P in phosphoprotein bands I and II were found. The 32P-labeling characteristics of these proteins were similar to those of alkaline phosphatase intermediates in the basolateral membrane. The results of this study are fully in support of an earlier study in which high affinity Ca2+-ATPase was found exclusively in the basolateral membrane of duodenal epithelium.

Conference Article| April 01 1984 The mechanism of action of Escherichia coli heat-stable toxin HUGO R. de JONGE HUGO R. de JONGE 1Department of Biochemistry I, Medical Faculty, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1984) 12 (2): 180–184. https://doi.org/10.1042/bst0120180 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation HUGO R. de JONGE; The mechanism of action of Escherichia coli heat-stable toxin. Biochem Soc Trans 1 April 1984; 12 (2): 180–184. doi: https://doi.org/10.1042/bst0120180 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CAMPdPKase, cyclic AMP-dependent protein kinase, CGMPdPKase, cyclic GMP-dependent protein kinase, GC, guanylate cyclase, ST, heat-stable enterotoxin This content is only available as a PDF. © 1984 Biochemical Society1984 Article PDF first page preview Close Modal You do not currently have access to this content.

Binding of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to the Na⁺/H⁺ exchanger 3 regulatory factor 1 (NHERF-1) and NHERF-2 scaffolding proteins has been shown to affect its localization and activation. We have for the first time studied the physiological role of these proteins in CFTR regulation in native tissue by determining CFTR-dependent chloride current in NHERF-1- and NHERF-2-deficient mice. The cAMP- and cGMP-activated chloride current and the basal chloride current in basolaterally permeabilized jejunum were reduced by ∼30% in NHERF-1-deficient mice but not in NHERF-2-deficient mice. The duodenal bicarbonate secretion was affected in a similar way, whereas no significant differences in CFTR activity were observed in ileum. CFTR abundance as determined by Western blotting was unaltered in jejunal epithelial cells and brush border membranes of NHERF-1 and NHERF-2 mutant mice. However, semi-quantitative detection of CFTR by confocal microscopy showed that the level of apically localized CFTR in jejunal crypts was reduced by ∼35% in NHERF-1-deficient and NHERF-1/2 double deficient mice but not in NHERF-2 null mice. Together our results indicate that NHERF-1 is required for full activation of CFTR in murine duodenal and jejunal mucosa and that NHERF-1 affects the local distribution of CFTR in or near the plasma membrane. These studies provide the first evidence in native intestinal epithelium that NHERF-1 but not NHERF-2 is involved in the formation of CFTR-containing functional complexes that serve to position CFTR in the crypt apical membrane and/or to optimize its function as a cAMP- and cGMP-regulated anion channel.

Cystic fibrosis, an autosomal recessive disease frequently diagnosed in the Caucasian population, is characterized by deficient Cl- transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A second major hall-mark of the disease is Na+ hyperabsorption by the airways, mediated by the epithelial Na+ channel (ENaC). In this study, we report that in human airway epithelial CF15 cells treated with the CFTR corrector miglustat (n-butyldeoxynojyrimicin), whole-cell patch-clamp experiments showed reduced amiloride-sensitive ENaC current in parallel with a rescue of defective CFTR Cl- channel activity activated by forskolin and genistein. Similar results were obtained with cells maintained in culture at 27 degrees C for 24 h before electrophysiology experiments. With monolayers of polarized CF15 cells, short-circuit current (Isc) measurements also show normalization of Na+ and Cl- currents. In excised nasal epithelium of cftr(F508del/F508del) mice, like with CF15 cells, we found normalization of amiloride-sensitive Isc. Moreover, oral administration of miglustat (6 days) decreased the amiloride-sensitive Isc in cftr(F508del/F508del) mice but had no effect on cftr-/- mice. Our results thus show that rescuing the trafficking-deficient F508del-CFTR by miglustat down-regulates Na+ absorption. A miglustat-based treatment of CF patients may thus have a beneficial effect both on Cl- and Na+ transports.

Rats, treated for 3 days or longer with high concentrations of oxytetracycline (OTC) suffered from a severe damage of structure and function of the small intestinal epithelium and showed an excessive accumulation of fat in the liver. In an attempt to explain these toxic effects, several possible modes of action of this drug were investigated. No direct correlation was found between the strong inhibitory effect on mitochondrial protein synthesis, leading to drastically lowered mitochondrial cytochrome levels in the rapidly dividing small intestinal epithelial cells, and a disturbance of mitochondrial energy production. After chloramphenicol treatment, producing a similar decrease of cytochrome contents, no epithelial cell damage was observed. At a stage of the OTC-treatment in which minimal cytochrome levels were reached but still no structural damage of the epithelium was observed, the energy-stores in the epithelial cells and the rate of oxidative phosphorylation in isolated intestinal mitochondria appeared to be unaffected. After 3–4 days, when morphological alterations of the villus were clearly observed histologically, the rate of proliferation of the crypt cell population was not appreciably affected, suggesting again a lack of influence on energy metabolism in the crypt cells. During short incubations of isolated villous cells, the cellular energy charge and the rate of aminoacid incorporation into protein was strongly decreased after anaerobiosis and addition of KCN, oligomycin or 2,4-DNP, conditions which impair the mitochondrial energetic function. However, incubation with 100–500 μg/ml of several tetracyclines, of which doxycycline showed the strongest effect, led to a significant inhibition of cytoplasmic protein synthesis without affecting the rate of glycolysis and respiration or the energy charge in the epithelial cells. These observations tend to support the view that the toxic effects of OTC are mainly based on its interaction with cytoplasmic protein synthesis under conditions of drug accumulation in blood and tissues. The lowering of energy charge in liver and intestine after prolonged treatment may be interpreted as secondary effects of this action, namely of fatty infiltration in the liver and structural damage of the villous epithelium respectively.

To test the hypothesis that Na + /H + exchanger (NHE) regulatory factor 2 (NHERF2) is necessary for multiple aspects of acute regulation of NHE3 in intact mouse small intestine, distal ileal NHE3 activity was determined using two-photon microscopy/SNARF-4F in a NHERF2-null mouse model. The NHERF2-null mouse ileum had shorter villi, deeper crypts, and decreased epithelial cell number. Basal rates of NHE3 activity were reduced in NHERF2-null mice, which was associated with a reduced percentage of NHE3 in the apical domain and an increase in intracellular NHE3 amount but no change in total level of NHE3 protein. cAMP, cGMP, and elevated Ca 2+ due to apical exposure to UTP all inhibited NHE3 activity in wild-type mouse ileum but not in NHERF2-null mice, while inhibition by hyperosmolarity occurred normally. The cAMP-increased phosphorylation of NHE3 at aa 552; levels of PKAIIα and cGMP-dependent protein kinase II (cGKII); and elevation of Ca 2+ were similar in wild-type and NHERF2-null mouse ileum. Luminal lysophosphatidic acid (LPA) stimulated NHE3 in wild-type but not in NHERF2-null ileum. In conclusion, 1) there are subtle structural abnormalities in the small intestine of NHERF2-null mouse which include fewer villus epithelial cells; 2) the decreased basal NHE3 activity and reduced brush border NHE3 amount in NHERF2-null mice show that NHERF2 is necessary for normal basal trafficking or retention of NHE3 in the apical domain; 3) hyperosmolar inhibition of NHE3 occurs similarly in wild-type and NHERF2-null ileum, demonstrating that some inhibitory mechanisms of NHE3 are not NHERF2 dependent; 4) cAMP inhibition of NHE3 is NHERF2 dependent at a step downstream of cAMP/PKAII phosphorylation of NHE3 at aa 552; 5) cGMP- and UTP-induced inhibition of NHE3 are NHERF2 dependent at steps beyond cGKII and the UTP-induced increase of intracellular Ca 2+ ; and 6) LPA stimulation of NHE3 is also NHERF2 dependent.

Key points Cystic fibrosis (CF) is a lethal disease characterized by low rates of epithelial Cl − and HCO 3 − secretion and obstruction of the airways and gastrointestinal and reproductive organs by sticky mucus. HCO 3 − secretion has recently been demonstrated to be necessary for mucus hydration. The most frequent CF mutation is F508del. This mutant protein is usually degraded in the proteasome. New therapeutic strategies have been developed which deliver F508del to the plasma membrane. Utilizing transgenic F508del mutant and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice, apical membrane expression of F508del protein was found to be associated with enhanced stimulation of intestinal HCO 3 − secretion. The predominant molecular mechanism for enhanced F508del HCO 3 − stimulation appeared to be the activation of a Cl − recycling pathway, with Cl − exit via membrane‐resident F508del protein and Cl − entry in exchange for HCO 3 − by apical Cl − /HCO 3 − exchange. In contrast, the predominant molecular mechanism for cAMP‐activated HCO 3 − secretion in WT intestine appears to be HCO 3 − exit via CFTR itself. Abstract This study investigated whether expression of the common cystic fibrosis transmembrane conductance regulator (CFTR) mutant F508del in the apical membrane of enterocytes confers increased bicarbonate secretory capacity on the intestinal epithelium of F508del mutant mice compared to that of CFTR knockout (KO) mice. CFTR KO mice, F508del mutant mice (F508del) and wild‐type (WT) littermates were bred on the FVB/N background. F508del isolated brush border membrane (BBM) contained approximately 5–10% fully glycosylated band C protein compared to WT BBM. Similarly, the forskolin (FSK)‐induced, CFTR‐dependent short‐circuit current (Δ I sc ) of F508del mucosa was approximately 5–10% of WT, whereas the HCO 3 − secretory response ( ) was almost half that of WT in both duodenum and mid‐colon studied in vitro and in vivo. While WT intestine retained full FSK‐induced in the absence of luminal Cl − , the markedly higher than Δ I sc in F508del intestine was dependent on the presence of luminal Cl − , and was blocked by CFTR inhibitors. The Ste20‐related proline–alanine‐rich kinases (SPAK/OSR1), which are downstream of the with‐no‐lysine (K) protein kinases (WNK), were rapidly phosphorylated by FSK in WT and F508del, but significantly more slowly in CFTR KO intestine. In conclusion, the data demonstrate that low levels of F508del membrane expression in the intestine of F508del mice significantly increased FSK‐induced HCO 3 − secretion mediated by Cl − /HCO 3 − exchange. However, in WT mucosa FSK elicited strong SPAK/OSR1 phosphorylation and Cl − ‐independent HCO 3 − efflux. This suggests that therapeutic strategies which deliver F508del to the apical membrane have the potential to significantly enhance epithelial HCO 3 − secretion.

We hypothesized that people with cystic fibrosis (CF) who express CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function. We used intestinal organoids to screen a GPCR-modulating compound library and identified β 2 -adrenergic receptor agonists as the most potent inducers of CFTR function. β 2 -Agonist-induced organoid swelling correlated with the CFTR genotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. The in vivo response to treatment with an oral or inhaled β 2 -agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35 mV, p&lt;0.05), suggesting that this treatment might be effective in vivo . Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administered ex vivo to organoids. This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR function in vivo and to select route of administration.

Disruption of the enterohepatic circulation of bile acids (BAs) is part of the gastrointestinal phenotype of cystic fibrosis (CF). Ivacaftor (VX-770), a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, improves pulmonary function in CF patients with class III gating mutations. We studied the effect of ivacaftor on the enterohepatic circulation by assessing markers of BA homeostasis and their changes in CF patients.In CF patients with an S1251N mutation (N = 16; age 9-35 years S125N study/NTR4873) or a G551D mutation (N = 101; age 10-24 years; GOAL study/ NCT01521338) we analyzed plasma fibroblast growth factor 19 (FGF19) and 7α-hydroxy-4-cholesten-3-one (C4) levels, surrogate markers for intestinal BA absorption and hepatic synthesis, respectively, before and after treatment with ivacaftor.At baseline, median FGF19 was lower (52% and 53%, P < .001) and median C4 higher (350% and 364%, P < .001), respectively, for the S1251 N and G551D mutation patient groups compared to healthy controls. Treatment with ivacaftor significantly increased FGF19 and reduced C4 levels towards normalization in both cohorts but this did not correlate with CFTR function in other organs, as measured by sweat chloride levels or pulmonary function.We demonstrate that patients with CFTR gating mutations display interruption of the enterohepatic circulation of BAs reflected by lower FGF19 and elevated C4 levels. Treatment with ivacaftor partially restored this disruption of BA homeostasis. The improvement did not correlate with established outcome measures of CF, suggesting involvement of modulating factors of CFTR correction in different organs.

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl − channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl − channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl − channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl − channels.

Although rotavirus infection is usually acute and self-limiting, it can cause chronic infection with severe diseases in immunocompromised patients, including organ transplantation recipients and cancer patients irrespective of pediatric or adult patients. Since no approved medication against rotavirus infection is available, this study screened a library of safe-in-man broad-spectrum antivirals. We identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of rotavirus infection. We confirmed this effect in 2D cell cultures and 3D cultured human intestinal organoids with both laboratory-adapted rotavirus strains and five clinical isolates. Supplementation of UTP or uridine largely abolished the anti-rotavirus activity of gemcitabine, suggesting its function through inhibition of pyrimidine biosynthesis pathway. Our results support repositioning of gemcitabine for treating rotavirus infection, especially for infected cancer patients.

In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport.Epithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique.ELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation.ELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues.

Abstract Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Defective CFTR leads to accumulation of dehydrated viscous mucus within the small intestine, luminal acidification and altered intestinal motility, resulting in blockage. These changes promote gut microbial dysbiosis, adversely influencing the normal proliferation and differentiation of intestinal epithelial cells. Using Illumina 16S rRNA gene sequencing and immunohistochemistry, we assessed changes in mucosa-attached microbiome and epithelial cell profile in the small intestine of CF mice and a CF patient compared to wild-type mice and non-CF humans. We found increased abundance of pro-inflammatory Escherichia and depletion of beneficial secondary bile-acid producing bacteria in the ileal mucosa-attached microbiome of CFTR-null mice. The ileal mucosa in a CF patient was dominated by a non- aeruginosa Pseudomonas species and lacked numerous beneficial anti-inflammatory and short-chain fatty acid-producing bacteria. In the ileum of both CF mice and a CF patient, the number of absorptive enterocytes, Paneth and glucagon-like peptide 1 and 2 secreting L-type enteroendocrine cells were decreased, whereas stem and goblet cell numbers were increased. These changes in mucosa-attached microbiome and epithelial cell profile suggest that microbiota-host interactions may contribute to intestinal CF disease development with implications for therapy.

Human intestine 407 cells respond to hypo-osmotic stress by the rapid release of ATP into the extracellular medium. A difference in the time course of activation as well as in the sensitivity to cytochalasin B treatment and BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester] loading suggests that ATP leaves the cell through a pathway distinct from volume-regulated anion channels. To evaluate a putative role for nucleotides as autocrinic/paracrinic factors in osmotic signalling, the effects of extracellular ATP on the regulation of volume-sensitive anion channels as well as on the hypotonicity-induced activation of extracellular signal-regulated protein kinases (Erk-1/2) were investigated. Micromolar concentrations of ATP were unable to elicit an isotope efflux from (125)I(-)-loaded cells by itself, but strongly potentiated the hypotonicity-provoked anion efflux through a Ca(2+)-dependent mechanism. The order of potency of nucleotides (ATP = UTP = ATP[S] > ADP = AMP >> adenosine = cAMP) indicated the involvement of P2Y(2) receptors. In contrast, millimolar concentrations of ATP markedly inhibited both the osmotically induced isotope efflux and whole-cell Cl(-) currents. Inhibition of whole-cell Cl(-) currents, not only by millimolar ATP but also by the purinoceptor antagonists suramin and reactive blue, was observed most prominently at depolarizing holding potentials, suggesting a direct interaction with volume-sensitive Cl(-) channels rather than interaction with purinoceptors. Both ATP and UTP, at submicromolar levels, were found to act as potent activators of Erk-1/2 in intestine 407 cells. Addition of the ATP hydrolase apyrase to the bath greatly reduced the hypotonicity-induced Erk-1/2 activation, but did not affect the swelling-induced isotope efflux or whole-cell Cl(-) currents. Furthermore, pre-treatment with suramin or reactive blue almost completely prevented the hypo-osmotic activation of Erk-1/2. The results indicate that extracellularly released ATP functions as an autocrinic/paracrinic factor that mediates hypotonicity-induced Erk-1/2 activation but does not serve as an activator of volume-sensitive compensatory Cl(-) currents.

Research Article2 January 1996free access Serum-induced membrane depolarization in quiescent fibroblasts: activation of a chloride conductance through the G protein-coupled LPA receptor. F. R. Postma F. R. Postma Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author K. Jalink K. Jalink Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author T. Hengeveld T. Hengeveld Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author A. G. Bot A. G. Bot Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author J. Alblas J. Alblas Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author H. R. de Jonge H. R. de Jonge Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author W. H. Moolenaar W. H. Moolenaar Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author F. R. Postma F. R. Postma Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author K. Jalink K. Jalink Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author T. Hengeveld T. Hengeveld Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author A. G. Bot A. G. Bot Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author J. Alblas J. Alblas Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author H. R. de Jonge H. R. de Jonge Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author W. H. Moolenaar W. H. Moolenaar Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Search for more papers by this author Author Information F. R. Postma1, K. Jalink1, T. Hengeveld1, A. G. Bot1, J. Alblas1, H. R. Jonge1 and W. H. Moolenaar1 1Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. The EMBO Journal (1996)15:63-72https://doi.org/10.1002/j.1460-2075.1996.tb00334.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin-bound lysophosphatidic acid (LPA) acting on its own G protein-coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl- efflux. This depolarizing Cl- current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume-regulated Cl- currents. Activation of the Cl- current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras-related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein-coupled receptor agonists evoke membrane depolarization by activating a new type of Cl- channel through a signalling pathway that is closely associated with phosphoinositide hydrolysis, yet independent of known second messengers. Previous ArticleNext Article Volume 15Issue 11 January 1996In this issue RelatedDetailsLoading ...

Cystic fibrosis (CF) is frequently associated with progressive loss of exocrine pancreas function, leading to incomplete digestion and absorption of dietary fat. Supplementing patients with pancreatic lipase reduces fat excretion, but it does not completely correct fat malabsorption, indicating that additional pathological processes affect lipolysis and/or uptake of lipolytic products. To delineate the role of such (post) lipolytic processes in CF-related fat malabsorption, we assessed fat absorption, lipolysis, and fatty acid uptake in two murine CF models by measuring fecal fat excretion and uptake of oleate- and triolein-derived lipid. Pancreatic and biliary function was investigated by determining lipase secretion and biliary bile salt (BS) secretion, respectively. A marked increase in fecal fat excretion was observed in cftr null mice but not in homozygous DeltaF508 mice. Fecal BS loss was enhanced in both CF models, but biliary BS secretion rates were similar. Uptake of free fatty acid was delayed in both CF models, but only in null mice was a specific reduction in lipolytic activity apparent, characterized by strongly reduced triglyceride absorption. Impaired lipolysis was not due to reduced pancreatic lipase secretion. Suppression of gastric acid secretion partially restored lipolytic activity and lipid uptake, indicating that incomplete neutralization of gastric acid impedes fat absorption. We conclude that fat malabsorption in cftr null mice is caused by impairment of lipolysis, which may result from aberrant duodenal pH regulation.

Nitrovasodilators, such as sodium nitroprusside (SNP), release nitric oxide (NO) and stimulate intestinal electrolyte transport. However, the second messengers involved in this process are unknown. NO stimulates soluble guanylate cyclase activity in other tissues, but stimulation of this enzyme has not previously been described for intestine. We report a 20-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) production by radioimmunoassay in colonic mucosal strips stimulated with SNP. SNP also caused a significant increase in prostaglandin (PG) E2 release but did not stimulate release of the prostanoids thromboxane B2 or 6-keto-PGF1alpha. Stimulation of isolated colonic crypts and the remaining subepithelial mucosa demonstrated that the latter was the major source of the increases in cGMP and PGE2. Immunostaining of colonic mucosa revealed minimal basal cGMP immunoreactivity but large increases in abundance, localizing to the subepithelium, after SNP treatment. Under basal conditions, there was diffuse immunostaining for constitutive NO synthase in both the epithelial and subepithelial compartments, which was corroborated with NADPH diaphorase staining. In conclusion, SNP was an NO donor stimulates production of cGMP and PGE2 from the subepithelium. NO may be an important mediator of colonic secretion and other processes predominantly via its direct effects on cells of the lamina propria.

The modulation of ion transport pathways in filter-grown monolayers of the Cl(-)-secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments. Basolateral addition of 10(-4) M carbachol induced a complex poly-phasic change of the cell potential consisting of (i) a fast and short (30-sec) depolarization of 15 +/- 1 mV from a resting value of -52 +/- 1 mV and an increase of the fractional resistance of the apical membrane (first phase), (ii) a repolarization of 22 +/- 1 mV leading to a hyperpolarization of the cell (second phase), (iii) a depolarization of 11 +/- 1 mV and a decrease of the fractional resistance of the apical membrane (the third phase), (iv) and sometimes, a hyperpolarization of 6 +/- 1 mV and an increase of the fractional resistance of the apical membrane (fourth phase). The transepithelial potential increased with a peak value of 2.4 +/- 0.3 mV (basolateral side positive). The transepithelial PD started to increase (serosa positive), coinciding with the start of the second phase of the intracellular potential change, and continued to increase during the third phase. Ion replacements and electrical circuit analyses indicate that the first phase is caused by increase of the Cl- conductance in the apical and basolateral membrane, the second phase by increased K+ conductance of the basolateral membrane, and the third phase and the fourth phase by increase and decrease, respectively, of an apical Cl- conductance. The first and second phase of the carbachol effect could be elicited also by ionomycin. They were strongly reduced by EGTA. Phorbol dibutyrate (PDB) induced a sustained depolarization of the cell and a decrease of the apical fractional resistance. The results suggest that two different types of Cl- channels are involved in the carbachol response: one Ca2+ dependent and a second which may be PKC sensitive. In the presence of a supramaximal concentration of forskolin, carbachol evoked a further increase of the apical Cl- conductance. It is concluded that the short-lasting carbachol/Ca(2+)-dependent Cl- conductance is different from the forskolin-activated conductance. The increase of the Cl- conductance in the presence of forskolin by carbachol may be due to activation of different Cl- channels or to modulation of the PKA-activated Cl- channels by activated PKC.

The CFTR gene encodes a chloride channel with pleiotropic effects on cell physiology and metabolism. Here, we show that increasing cGMP levels to inhibit epithelial Na(+) channel in cystic fibrosis (CF) respiratory epithelial cells corrects several aspects of the downstream pathology in CF. Cell culture models, using a range of CF cell lines and primary cells, showed that complementary pharmacological approaches to increasing intracellular cGMP, by elevating guanyl cyclase activity though reduced nitric oxide, addition of cell-permeable cGMP analogs, or inhibition of phosphodiesterase 5 corrected multiple aspects of the CF pathological cascade. These included correction of defective protein glycosylation, bacterial adherence, and proinflammatory responses. Furthermore, pharmacological inhibition of phosphodiesterase 5 in tissues ex vivo or in animal models improved transepithelial currents across nasal mucosae from transgenic F508del Cftr(tm1Eur) mice and reduced neutrophil infiltration on bacterial aerosol challenge in Pseudomonas aeruginosa-susceptible DBA/2 mice. Our findings define phosphodiesterase 5 as a specific target for correcting a number of previously disconnected defects in the CF respiratory tract, now linked through this study. Our study suggests that phosphodiesterase 5 inhibition provides an opportunity for simultaneous and concerted correction of seemingly disparate complications in CF.

FEBS LettersVolume 55, Issue 1-2 p. 143-152 Full-length articleFree Access Properties of guanylate cyclase and levels of cyclic GMP in rat small intestinal villous and crypt cells H.R. de Jonge, H.R. de Jonge Department of Biochemistry I, Faculty of Medicine, Erasmus University Rotterdam, P.O. Box 1738, Rotterdam, The NetherlandsSearch for more papers by this author H.R. de Jonge, H.R. de Jonge Department of Biochemistry I, Faculty of Medicine, Erasmus University Rotterdam, P.O. Box 1738, Rotterdam, The NetherlandsSearch for more papers by this author First published: July 15, 1975 https://doi.org/10.1016/0014-5793(75)80980-XCitations: 30AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 J.G. Banwell, H. Sherr, Gastroenterology, 65, (1973), 467– 497. 2 D.V. Kimberg, Gastroenterology, 67, (1974), 1023– 1064. 3 C.J. Schwartz, D.V. Kimberg, H.E. Sheerin, M. Field, S.I. Said, J. Clin. Invest., 54, (1974), 536– 544. 4 E. Ishikawa, S. Ishikawa, J.W. Davis, E.W. Sutherland, J. Biol. Chem., 244, (1969), 6371– 6376. 5 A.A. White, G.D. Aurbach, Biochim. Biophys. Acta, 191, (1969), 686– 697. 6 H. Kimura, F. Murad, J. Biol. Chem., 249, (1974), 6910– 6916. 7 Jonge H.R. de, FEBS Lett., 53, (1975), 237– 242. 8 T.D. Chrisman, D.L. Garbers, M.A. Parks, J.G. Hardman, J. Biol. Chem., 250, (1975), 374– 381. 9 W.G. Iemhoff, den Berg J.W.O. van, Pijper A.M. de, W.C. Hülsmann, Biochim. Biophys. Acta, 215, (1970), 229– 241. 10 W.C. Hülsmann, den Berg J.W.O. van, Jonge H.R. de, S. Fleischer L. Packer Methods in Enzym. XXXII, (1974), Academic Press New York 665– 673. 11 D.D. Harrison, N.L. Webster, Biochim. Biophys. Acta, 93, (1964), 662– 664. 12 Jonge H.R. de, Biochim. Biophys. Acta, 381, (1975), 128– 143. 13 R.M. Smith, R.A. Alberty, J. Amer. Soc., 78, (1956), 2376– 2380. 14 W.J. O'Sullivan, D.D. Perrin, Biochemistry, 3, (1964), 18– 26. 15 V. Dinnendahl, Naunyn-Schmiedebergs Arch. Pharm., 284, (1974), 55– 61. 16 J.F. Kuo, P. Greengard, J. Biol. Chem., 245, (1970), 2493– 2498. 17 K.C. Tovey, K.G. Oldham, J.A.M. Whelan, Clin. Chim. Acta, 56, (1974), 221– 234. 18 A.G. Gilman, Proc. Natl. Acad. Sci. U.S., 67, (1970), 305– 312. 19 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 275. 20 D.K. Parkinson, H. Ebel, D.R. DiBona, G.W.G. Sharp, J. Clin. Invest., 51, (1972), 2292– 2298. 21 J.G. Hardman, E.W. Sutherland, J. Biol. Chem., 244, (1969), 6363– 6370. 22 K.L. Cohen, M.W. Bitensky, J. Pharm. Exp. Ther., 169, (1969), 80– 86. 23 W.J. Thompson, R.H. Williams, S.A. Little, Biochim. Biophys. Acta, 302, (1973), 329– 337. 24 G. Illiano, G.P.E. Tell, M.I. Siegel, P. Cuatrecasas, Proc. Natl. Acad. Sci. U.S., 70, (1973), 2443– 2447. 25 Jonge H.R. de, W.C. Hülsmann, Biochem. Soc. Transact., 2, (1974), 416– 419. 26 N.D. Goldberg, R.F. O'Dea, M.K. Haddox, P. Greengard G.A. Robison Advances in Cyclic Nucleotide Research 3, (1973), Raven Press New York 155– 223. 27 D.L. Garbers, J.G. Hardman, F.B. Rudolph, Biochemistry, 13, (1974), 4166– 4171. 28 Sande J. Van, C. Decoster, J.E. Dumont, Biochem. Biophys. Res. Commun., 62, (1975), 168– 175. 29 J.A. Ferrendelli, E.H. Rubin, D.A. Kinscherf, Fed. Proc., 34, (1975), 231– 30 P.S. Rudland, D. Gospodarowicz, W. Seifert, Nature, 250, (1974), 741– 742. P.S. Rudland, D. Gospodarowicz, W. Seifert, Nature, 250, (1974), 773– 31 J.F. Kuo, Proc. Natl. Acad. Sci. U.S., 71, (1974), 4037– 4041. 32 J.E. Camellie, P. Greengard, Proc. Natl. Acad. Sci. U.S., 71, (1974), 1891– 1895. 33 P.F. Millington, J.B. Finean, J. Cell Biol., 14, (1962), 125– 139. 34 J.P. Gray, G.I. Drummond, Proc. Can. Fed. Biol. Soc., 16, (1973), 79– 35 E. Böhme, R. Jung, I. Mechler, J.G. Hardman B.W. O'Malley Methods in Enzym. XXXVIII, (1974), Academic Press New York 199– 202. 36 L.G. Tilney, M. Mooseker, Proc. Natl. Acad. Sci. U.S., 68, (1971), 2611– 2615. Citing Literature Volume55, Issue1-2July 15, 1975Pages 143-152 ReferencesRelatedInformation

Publisher Summary Isolation methods for intestinal cells can generally be subdivided into “biochemical” and “mechanical” methods, although in some methods these procedures are combined. Isolation of cells from small intestinal epithelium has been accomplished by incubation of the intestine with enzymes such as trypsin and pancreatin, lysozyme, or hyaluronidase. Usually only the morphological intactness of the cells obtained has been evaluated. Other means depended on incubation of intestine with calcium-binding agents, such as citrate or EDTA. Recently also functional integrity and capabilities of cells isolated by this kind of method has been evaluated.

Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin-stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.

Background. Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease.

Nutritional studies are greatly hampered by a paucity of proper models. Previous studies on nutrition have employed conventional cell lines and animal models to gain a better understanding of the field. These models lack certain correlations with human physiological responses, which impede their applications in this field. Enteroids are cultured from intestinal stem cells and include enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and stem cells, which mimic hallmarks of in vivo epithelium and support long-term culture without genetic or physiological changes. Enteroids have been used as models to study the effects of diet and nutrients on intestinal growth and development, ion and nutrient transport, secretory and absorption functions, the intestinal barrier, and location-specific functions of the intestine. In this review, the existing models for nutritional studies are discussed and the importance of enteroids as a new model for nutritional studies is highlighted. Taken together, it is suggested that enteroids can serve as a potential model system to be exploited in nutritional studies.

Ischemia of the bile duct is a common feature in liver disease and transplantation, which represents a major cause of morbidity and mortality, especially after liver transplantation. Detailed knowledge of its pathogenesis remains incomplete due to the lack of appropriate in vitro models.To recapitulate biliary damage induced by ischemia and reperfusion in vitro, human intrahepatic cholangiocyte organoids (ICOs) were grown at low oxygen levels of 1% up to 72 h, followed by re-oxygenation at normal levels.ICOs stressed by ischemia and subsequent re-oxygenation represented the dynamic change in biliary cell proliferation, upregulation of epithelial-mesenchymal transition (EMT)-associated markers, and the evocation of phase-dependent cell death programs similar to what is described in patients. Clinical-grade alpha-1 antitrypsin was identified as a potent inhibitor of both ischemia-induced apoptosis and necroptosis.These findings demonstrate that ICOs recapitulate ischemic cholangiopathy in vitro and enable drug assessment studies for the discovery of new therapeutics for ischemic cholangiopathies.Dutch Digestive FoundationMLDS D16-26; TKI-LSH (Topconsortium Kennis en Innovatie-Life Sciences & Health) grant RELOAD, EMC-LSH19002; Medical Delta program "Regenerative Medicine 4D"; China Scholarship Council No. 201706230252.

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Ibeta or a membrane-bound cGK II/Ibeta chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

The involvement of tyrosine phosphorylation in the regulation of epithelial cell Cl- secretion is unknown. Therefore, the purpose of these studies was to determine if tyrosine kinase activation was involved in the regulation of Cl- secretion, using the tyrosine kinase inhibitors, genistein and tyrphostin 47, and human intestinal epithelial cells (T84 cells) as an intestinal Cl- secretory model. Genistein rapidly but reversibly stimulated sustained apical Cl- secretion in monolayers of T84 cells without increasing intracellular cyclic nucleotides or Ca2+ levels. Tyrphostin 47 also stimulated Cl- secretion in T84 monolayers, although it was short-lived. Transfection experiments in 3T3 fibroblasts and IEC-6 intestinal cells utilizing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) showed that genistein and tyrphostin 47 stimulated 125I efflux only in CFTR-transfected cells and not in CFTR-negative cells. Thus genistein- and tyrphostin 47-stimulated Cl- secretion involved CFTR. Genistein also acted synergistically with the Ca(2+)- and protein kinase C-dependent acetylcholine analogue, carbachol, to stimulate Cl- secretion in T84 monolayers. However, the Cl- secretory response to saturating concentrations of the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin, or the guanosine 3',5'-cyclic monophosphate (cGMP) agonist, Escherichia coli heat-stable enterotoxin, was not further enhanced by genistein. Although the mechanism of activation of Cl- secretion is unclear, these data suggest that tyrosine kinase activity limits basal Cl- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical membrane Cl- secretion, most likely through activation of the CFTR-Cl- channel. Moreover, genistein does not itself act through cAMP or cGMP elevation but appears to share a common Cl- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory responses to a Ca(2+)- and protein kinase C-dependent agonist.

Cyclic guanosine monophosphate (cGMP) was established as an important regulator of intestinal ion transport in the late seventies, when it was shown to be the intracellular mediator of salt and water secretion induced by Escherichia coli heat-stable enterotoxin (STa). The recent finding of a distinctive endogenous activator of intestinal guanylyl cyclase (guanylin) affirms that the intestine possesses a unique cGMP-signaling pathway for the physiological regulation of ion transport. The regulation of fluid secretion by cGMP, however, has been thought to be restricted to the intestine. However, the recent detection of some of the components of the “intestinal cGMP pathway” (GC-C and guanylin) in other tissues suggests that it is expressed more generally than previously assumed. The chapter discusses the role of cGMP pathway in the intestine as a regulator of transepithelial ion and fluid transport.

Forskolin (i.e., cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl(-)-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments. Changes in electrophysiological parameters provoked by serosal addition of 10(-5) M forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9 +/- 0.4 mV), (ii) a transient decrease in transepithelial resistance with 26 +/- 3 omega.cm2 from a mean value of 138 +/- 13 omega.cm2 before forskolin addition, (iii) a depolarization of the cell membrane potential by 24 +/- 1 mV from a resting value of -50 +/- 1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80 +/- 0.02 to 0.22 +/- 0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl- in the medium. Subsequent addition of bumetanide (10(-4) M), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba2+ (1 mM), a known inhibitor of basolateral K+ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K+ conductance. The results suggest that forskolin-induced Cl- secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl- conductance of the apical membrane but does not affect K+ or Cl- conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl- and the basal Ba(2+)-sensitive K+ conductance are sufficiently large to maintain the Cl- efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl- channel blockers NPPB and DPC on basolateral conductance rather than apical Cl- conductance.