Penghong Song

Deletion of the chloride transporter Slc26a9 causes loss of tubulovesicles in parietal cells and impairs acid secretion in the stomach

Slc26a9 is a recently identified anion transporter that is abundantly expressed in gastric epithelial cells. To study its role in stomach physiology, gene targeting was used to prepare mice lacking Slc26a9. Homozygous mutant (Slc26a9−/−) mice appeared healthy and displayed normal growth. Slc26a9 deletion resulted in the loss of gastric acid secretion and a moderate reduction in the number of parietal cells in mutant mice at 5 weeks of age. Immunofluorescence labeling detected the H-K-ATPase exclusively on the apical pole of gastric parietal cells in Slc26a9−/− mice, in contrast to the predominant cytoplasmic localization in Slc26a9+/+ mice. Light microscopy indicated that gastric glands were dilated, and electron micrographs displayed a distinct and striking absence of tubulovesicles in parietal cells and reductions in the numbers of parietal and zymogen cells in Slc26a9−/− stomach. Expression studies indicated that Slc26a9 can function as a chloride conductive pathway in oocytes as well as a Cl−/HCO3− exchanger in cultured cells, and localization studies in parietal cells detected its presence in tubulovesicles. We propose that Slc26a9 plays an essential role in gastric acid secretion via effects on the viability of tubulovesicles/secretory canaliculi and by regulating chloride secretion in parietal cells.

Deletion of the chloride transporter Slc26a7 causes distal renal tubular acidosis and impairs gastric acid secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl−/HCO3− exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.

Localization, Trafficking, and Significance for Acid Secretion of Parietal Cell Kir4.1 and KCNQ1 K+ Channels

BACKGROUND & AIMS K(+) recycling at the apical membrane of gastric parietal cells is a prerequisite for gastric acid secretion. Two K(+) channels are currently being considered for this function, namely KCNQ1 and inwardly rectifying K(+) channels (Kir). This study addresses the subcellular localization, trafficking, and potential functional significance of KCNQ1 and Kir4.1 channels during stimulated acid secretion. METHODS The effect of pharmacologic KCNQ1 blockade on acid secretion was studied in cultured rat and rabbit parietal cells and in isolated mouse gastric mucosa. The subcellular localization of KCNQ1 and Kir4.1 was determined in highly purified membrane fractions by Western blot analysis as well as in fixed and living cells by confocal microscopy. RESULTS In cultured parietal cells and in isolated gastric mucosa, a robust acid secretory response was seen after complete pharmacologic blockade of KCNQ1. Both biochemical and morphologic data demonstrate that Kir4.1 and KCNQ1 colocalize with the H(+)/K(+)-ATPase but do so in different tubulovesicular pools. All Kir4.1 translocates to the apical membrane after stimulation in contrast to only a fraction of KCNQ1, which mostly remains cytoplasmic. CONCLUSIONS Acid secretion can be stimulated after complete pharmacologic blockade of KCNQ1 activity, suggesting that additional apical K(+) channels regulate gastric acid secretion. The close association of Kir4.1 channels with H(+)/K(+)-ATPase in the resting and stimulated membrane suggests a possible role for Kir4.1 channels during the acid secretory cycle.

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion

Parietal cell (PC) proton secretion via H+/K+‐ATPase requires apical K+ recycling. A variety of K+ channels and transporters are expressed in the PC and the molecular nature of the apical K+ recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7‐ to 8‐day‐old KCNQ1+/+, +/– and −/− mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1−/− and +/+ mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin‐stimulated acid output reduced by ∼90% in KCNQ1−/− gastric mucosa. Application of a high K+ concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1−/− mucosa. The study demonstrates that the KCNQ1 channel provides K+ to the extracellular K+ binding site of the H+/K+‐ATPase during acid secretion, and no other gastric K+ channel can substitute for this function.

Kir4.1 channel expression is essential for parietal cell control of acid secretion.

Kir4.1 channels were found to colocalize with the H+/K+-ATPase throughout the parietal cell (PC) acid secretory cycle. This study was undertaken to explore their functional role. Acid secretory rates, electrophysiological parameters, PC ultrastructure, and gene and protein expression were determined in gastric mucosae of 7–8-day-old Kir4.1-deficient mice and WT littermates. Kir4.1−/− mucosa secreted significantly more acid and initiated secretion significantly faster than WT mucosa. No change in PC number but a relative up-regulation of H+/K+-ATPase gene and protein expression (but not of other PC ion transporters) was observed. Electron microscopy revealed fully fused canalicular membranes and a lack of tubulovesicles in resting state Kir4.1−/− PCs, suggesting that Kir4.1 ablation may also interfere with tubulovesicle endocytosis. The role of this inward rectifier in the PC apical membrane may therefore be to balance between K+ loss via KCNQ1/KCNE2 and K+ reabsorption by the slow turnover of the H+/K+-ATPase, with consequences for K+ reabsorption, inhibition of acid secretion, and membrane recycling. Our results demonstrate that Kir4.1 channels are involved in the control of acid secretion and suggest that they may also affect secretory membrane recycling.

Helicobacter pylori vacuolating cytotoxin inhibits duodenal bicarbonate secretion by a histamine-dependent mechanism in mice

BACKGROUND The pathogenic mechanisms involved in Helicobacter pylori-induced duodenal mucosal injury are incompletely understood. In the present study, we sought to investigate the effect of H. pylori vacuolating cytotoxin (VacA) on duodenal mucosal bicarbonate (HCO3-) secretion. METHODS Concentrated bacterial culture supernatants from an H. pylori wild-type strain producing VacA with s1/m1 genotypes (P12) and from an isogenic mutant lacking VacA (P12DeltavacA) were used. HCO3- secretion by murine duodenal mucosa was examined in vitro in Ussing chambers. Duodenal mucosal histamine release was measured using enzyme-linked immunosorbent assay. The expression of histamine H2 receptor was examined by immunohistochemical analysis. RESULTS In a dose-dependent manner, the VacA-positive supernatant P12 reduced prostaglandin E2 (PGE2)-stimulated duodenal mucosal HCO3- secretion to a maximum of 49% (P<.0001), whereas P12DeltavacA did not result in significant inhibition (P>.05). Purified VacA had a similar effect. Histamine H2 receptor antagonists attenuated the effect of P12 on PGE2-induced HCO3- secretion. P12 stimulated duodenal histamine release in a dose-dependent manner, and exogenous histamine inhibited PGE2-stimulated duodenal HCO3- secretion. H2 receptor expression was found in duodenal epithelial cells, the enteric nerve plexus, and lymphocytes in Peyers patch. CONCLUSIONS H. pylori VacA inhibits PGE2-stimulated duodenal epithelial HCO3- secretion by a histamine-dependent mechanism. This effect likely contributes to the damaging effect of H. pylori in the duodenal mucosa.

36 Expression and Function of Slc26a9 Member of the SLC26 Anion Transporter Family in the Gastrointestinal Tract

Background: We recently identified Slc26a9 as an anion conductance that is upregulated in airway inflammation and prevents bronchial mucus obstruction (Anagnostopoulou et al. JCI 2012). Slc26a9 variants were recently found associated with meconium ileus in cystic fibrosis infants (Sun et al. Nature 2012). Aim: The association with meconium ileus raises the question where Slc26a9 is expressed in the gastrointestinal tract and what is its function. Methods: Acid, HCO3, short circuit current (Isc) measurements were performed in isolated mucosa and acid, HCO3 , and fluid absorptive and secretory rates were measured by single pass perfusion and back titration in anesthetized Slc26a9 KO and WT mice by inhalation of 2.0% isoflurane. Slc26a9 cellular expression was studied by laser dissection and qPCR, and quantitative morphometry was performed in the different segments of the murine gastrointestinal tract. Results: Slc26a9 was found highly expressed in the mucosae of the upper gastrointestinal tract, with abrupt decrease of expression levels to virtually undectable levels beyond the duodenum. As previously reported, Slc26a9 KO mice had completely lost the ability to secrete acid in adulthood. However, Slc26a9 was found highly expressed along the whole gastric gland, even in areas without H+,K+-ATPase expression. Proximal duodenal bicarbonate and fluid secretory rates, which are higher in the proximal than the distal duodenum in WT mice, as well as the ability to stimulate these rates with forskolin, were reduced in the absence of Slc26a9 expression. The gastric antrum, as well as the fundus (after omeprazole treatment to rule out any residual acid secretory capacity) was studied to test whether Slc26a9 transports HCO3 itself. Gastric antrum, while expressing high Slc26a9 levels in WT mice, had lower basal and forskolin-stimulated HCO3 rate as well as lower Isc response in WT than KO mice, arguing against a role of Slc26a9 as a HCO3 transporter. Morphometry revealed strongly elongated fundic as well as antral glands, and slightly elongated proximal duodenal villi as well as crypts. Conclusions: Slc26a9 expression is necessary for normal gastric acid and proximal duodenal bicarbonate secretion, but it is not expressed inmore distal parts of the gastrointestinal mucosa. The increased risk for meconium ileus may be due to loss of digestive function of the stomach and proximal duodenum.

M1684 Genistein Stimulates Duodenal HCO3- Secretion Through PI3K Pathway in Mice

G A A b st ra ct s rolipram significantly enhanced the cAMP formation induced in the isolated mouse stomach by histamine and PACAP. Conclusion: These results suggest that among various PDE isozymes only PDE4 is involved in the local regulatory mechanism of gastric acid secretion through degradation of cAMP at different cellular levels, and the PDE4 inhibitor roliprum modulates the acid response by potentiating acid production in the parietal cells and enhancing histamine release from the enterochromaffin-like (ECL) cells.

S1286 Enhanced Expression of Vacuolar H+-ATPase in Native Human Hepatic Carcinoma

Background&Aims: Maintenance of intracellular pH is crucial to normal cell function, as many cellular processes have a narrow pH optimum. Tumor cells possess high-glycolytic activity and produce acidic metabolites. Moreover, tumor cells often exist in a hypoxic microenvironment with a lower extracellular pH than that of surrounding normal cells. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. In this study, we made an investigation on expressions of H+-regulated channels/ exchangers in native human hepatic carcinoma. Methods: The mRNA expressions of Na+/ H+ exchanger (NHE), H+-K+ ATPase, and vacuolar H+-ATPase (V-ATPase) subunit c were examined by real time PCR. The protein expressions were analyzed by Western Blot. The protein expression and intracellular distribution of V-ATPase subunit c was further analyzed by immunohistochemistry. Results: The expressions of mRNA and protein of H+-K+ ATPase were not detected in native human hepatic carcinoma and normal hepatic tissues. The mRNA and protein expression levels of NHE1 and NHE3 were similar in native human hepatic carcinoma and normal hepatic tissues. However, the mRNA and protein expression levels of V-ATPase subunit c in native human hepatic carcinoma were markedly increased, compared with normal hepatic tissues. Immunohistochemistry analysis further confirmed the enhanced expression of V-ATPase subunit c on the plasma membrane of hepatic carcinoma cells. Conclusion: The results demonstrated that the expression of V-ATPase in human hepatic carcinoma is markedly increased, indicating V-ATPase is possibly involved in the regulation of intracellular pH of hepatic carcinoma cells. V-ATPase may represent a potential target for a novel pharmacotherapy of hepatic carcinoma.

787 Deletion of the Chloride/Base Exchanger Slc26a7 Impairs Gastric Acid Secretion and Causes Distal Renal Tubular Acidosis

G A A b st ra ct s located either in the proximal third of the corpus or in the fundus. Cardia tumors were classified according to the AEG-classification by Siewert and colleagues (AEG = adenocarcinoma of the esophagogastric junction; Siewert Br J Surg 1998). Included were tumors with their main tumor mass located directly at the cardia or subcardial with involvement of the cardia (AEG 2 and 3). Barrett carcinomas and supracardial cancers (AEG1) have been excluded. Patients were compared concerning the influence of H. pylori infection and the presence of histopathological changes. The chi-square test and consecutively Fishers exact test were used for statistical analysis. RESULTS: There was no difference in the prevalence of H. pylori in proximal versus distal GC. The histological differentiation between intestinal versus diffuse type cancers revealed significantly more intestinal type tumors at proximal location compared to distal GC (65 vs 46%; P = 0.022). Individual parameters of the proximal versus distal GC are reported in the table. Subanalysis of adenocarcinomas at the esophagogastric junction revealed no significant difference of H. pylori prevalence between cardial (AEG2) and subcardial (AEG3) tumors. CONCLUSION: There is no difference in the prevalence of H. pylori infection in proximal as compared to distal gastric cancer provided the allocation of the tumor is correctly assessed. To obtain correct prevalence data it is essential to exclude Barretts carcinomas (AEG1) from other adenocarcinomas at the esophagogastric junction. There was no distinct association of H. pylori with any of the histological types according to Lauren. Prevalence for H. pylori, CagA, IM and glandular atrophy by histolgical type and tumor location.