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

Abstract

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(-)/HCO(3)(-) 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.

Fig. 1.

Generation of Slc26a9^−/− mutant mice. (A) A schematic of the Slc26a9 targeting construct. The Neo cassette replaces 4.0 kb of the gene, including exons 2–5 (and including the ATG start codon). (B) Slc26a9 targeting allele and delineation of locations of primers. Using primers designated A1, A2, and A3, which are downstream (3′) of the short homology arm (SA), PCRs were performed in conjunction with a primer at the 5′ end of the Neo cassette (referred to as N1). These reactions were expected to amplify 2.1-, 2.2-, and 2.3-kb fragments, respectively. The control PCR was done using AT1 and AT2, which are at the 5′ end of the SA inside the region used to create the targeting construct. This amplifies a band of 2.0 kb. The sequence for each primer is as follows: A1, 5′-tcacatgtgacttctggtcccattgg-3′; A2, 5′-tcacatagtggccatagaacac-3′; A3, 5′-tttcccagatcccactgtcttgc-3′; AT1, 5′-caccacaatcatctctgtagg-3′; AT2, 5′-tctgatggagctatcttgacc-3′ and N1, 5′-tgcgaggccagaggccacttgtgtagc-3′. (C) Identification of homologous recombinant clones. PCR analysis of DNA isolated from 200 surviving colonies identified an individual clone (clone 114) that showed homologous recombination. Southern blotting confirmed the results. The positive control was performed using primers AT1/N1, which gave the expected fragment size of 1.8 kb. (D) Generation of Slc26a9-null mice. Crossing of male chimera with female wild-type mice resulted in the generation of several heterozygote animals. Crossing of male and female Slc26a9 heterozygote mice (+/−) resulted in the generation of Slc26a9 KO mice. Northern hybridization on RNA isolated from the stomachs and lungs of Slc26a9 +/+, +/−, and −/− mice is shown. The expression of Slc26a9 is completely absent in the Slc26a9-null mouse. Slc26a9^−/− mice exhibited normal growth, fertility, and survival compared with the wild-type littermates.

Fig. 2.

Histopathologic and ultrastructural changes in Slc26a9-null gastric mucosa. (A) H&E staining shows gastric glands in the Slc26a9^−/− mouse as dilated (D indicates dilated gastric gland) and containing lucent contents, which did not appear eosinophilic or basophilic, and small amounts of cellular debris (Center and Right) vs. Slc26a9^+/+ mice (Left). Parietal cell numbers were decreased in Slc26a9^−/− mice (blue arrows). Zymogen cells were significantly reduced in Slc26a9^−/− stomach (Right, white arrow). Mucous cells in Slc26a9^−/− stomach contained a significant amount of hyaline granules (Right, dark blue). (B) Electron microscopy shows parietal cell ultrastructure. There was a distinct and striking absence of tubulovesicles (TV) in parietal cells in Slc26a9^−/− (Right) vs. Slc26a9^+/+ stomach (Left). In place of tubulovesicles, parietal cells in Slc26a9^−/− mice had abundant amounts of round vesicles (RV), which could be a premature or undeveloped form of tubulovesicle. Insets show a low-magnification image of part of a parietal cell, including nucleus (N), mitochondria, and areas of secretory membranes [canaliculi (black arrows) and tubulovesicles in the WT, and canaliculi (black arrows) and round vesicles in the KO]. The higher-magnification images of secretory membranes of the WT and KO are labeled similarly. The flattened tubulovesicles typical of the WT parietal cell were replaced by round vesicles in the KO. The white circle in each of the main figures surrounds a cross-section of a microvillus. In the WT the actin filaments in the microvillus are largely found peripherally and in close proximity to the plasma membrane, but in the KO the actin filaments are centrally located in a core. (Scale bars: A, 100 μm for light micrographs; B, 1 μm for electron micrograph.)

Fig. 3.

Gastric acid secretion in Slc26a9^+/+ and Slc26a9^−/− mice. Animals were fasted overnight, and gastric acid secretion was measured after s.c. injection of histamine according to Experimental Procedures. (A) Gastric acid pH in Slc26a9^+/+ and Slc26a9^−/− mice (5–6 weeks old). The pH of the gastric secretions was significantly more alkaline in 5- to 6-week-old Slc26a9^−/− mice. (B) Gastric acid secretion in Slc26a9^+/+ and Slc26a9^−/− mice (5–6 weeks old). Acid secretion was decreased by ≈98% in Slc26a9^−/− vs. Slc26a9^+/+ mice. (C) Western blotting of H-K-ATPase in the stomachs of 5- to 6-week-old Slc26a9^+/+ and Slc26a9^−/− mice. (D) Gastric acid secretion in Slc26a9^+/+ and Slc26a9^−/− mice (17–19 days old). The quantitation of gastric acid secretion in 17- to 19-day-old mice demonstrated ≈49% reduction in Slc26a9^−/− mice.

Fig. 4.

Expression of AE2 and H-K-ATPase and localization of Slc26a9 in parietal cells. (A) Expression of AE2 and gastric H-K-ATPase (low magnification). (Upper) Expression of AE2 (Left) and gastric H-K-ATPase (Right) in wild-type mouse stomach. Center shows a merged image. (Lower) Expression of AE2 (Left) and gastric H-K-ATPase (Right), along with the merged image (Center), in an Slc26a9-null mouse. (B) Expression of AE2 and gastric H-K-ATPase (high magnification). AE2/H-K-ATPase merged images in the stomachs of Slc26a9^+/+ (Left) and Slc26a9^−/− (Right) mice are shown. Contrary to wild-type animals, H-K-ATPase labeling in Slc26a9-null mice was detected almost exclusively on the apical pole of gastric parietal cells, with little intracellular localization (Right). (Original magnifications; A, 100×; B, 400×.) (C) Western blotting of Slc26a9 in tubulovesicles isolated from gastric parietal cells. Western blotting detected a specific band in tubulovesicles isolated from gastric parietal cells. Left lane, immune serum; right, preadsorbed serum.

Fig. 5.

Time course (Left) and peak acid secretory rates (Right) in isolated gastric mucosa. (A and B) The 40- to 45-day-old (A) and 7- to 9-day-old (B) Slc26a9^−/−, Slc26a9^+/+, and Slc26a9^+/− mice. (C) Effect of high-potassium perfusate on acid secretion in Slc26a9^+/+ and Slc26a9^−/− mice. Acid secretion was measured at basal state and after the stimulation with forskolin (10⁻⁵ M) in the gastric mucosa of the mice. Acid secretion was completely absent in adult Slc26a9^−/− mice (C), and peak secretion was significantly lower in Slc26a9^+/− relative to Slc26a9^+/+ mucosa (n = 9 for +/+, 13 for +/−, and 4 for −/− mice) (A). No significant difference was observed between stomachs of 7- to 9-day-old Slc26a9^−/−, +/+, and +/− mice (n = 5 for −/−, 11 for +/+, and 17 for +/−. Acid secretion at basal state and in response to forskolin was examined in the presence of perfusate NaCl or KCl in Slc26a9^+/+ and Slc26a9^−/− mice (C).