Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion.

The present studies were designed to examine the effects of ClC-2 ablation on cellular morphology, parietal cell abundance, H/K ATPase expression, parietal cell ultrastructure and acid secretion using WT and ClC-2-/- mouse stomachs. Cellular histology, morphology and proteins were examined using imaging techniques, electron microscopy and western blot. The effect of histamine on the pH of gastric contents was measured. Acid secretion was also measured using methods and secretagogues previously established to give maximal acid secretion and morphological change. Compared to WT, ClC-2-/- gastric mucosal histological organization appeared disrupted, including dilation of gastric glands, shortening of the gastric gland region and disorganization of all cell layers. Parietal cell numbers and H/K ATPase expression were significantly reduced by 34% (P<0.05) and 53% (P<0.001) respectively and cytoplasmic tubulovesicles appeared markedly reduced on electron microscopic evaluation without evidence of canalicular expansion. In WT parietal cells, ClC-2 was apparent in a similar cellular location as the H/K ATPase by immunofluorescence and appeared associated with tubulovesicles by immunogold electron microscopy. Histamine-stimulated [H+] of the gastric contents was significantly (P<0.025) lower by 9.4 fold (89%) in the ClC-2-/- mouse compared to WT. Histamine/carbachol stimulated gastric acid secretion was significantly reduced (range 84-95%, P<0.005) in ClC-2-/- compared to WT, while pepsinogen secretion was unaffected. Genetic ablation of ClC-2 resulted in reduced gastric gland region, reduced parietal cell number, reduced H/K ATPase, reduced tubulovesicles and reduced stimulated acid secretion

Histological characterization of the gastric mucosa of WT and ClC-2^-/- mice.

<p><b>A.</b> Haematoxylin and eosin staining of the gastric mucosa of WT and ClC-2^-/- mice is shown. Flat pink cells with purple nuclei are parietal cells; dark purple cells are zymogen cells (arrowheads). Brackets indicate the gastric gland layer and * indicates places of glandular dilation. Bar = 50 μm. <b>B.</b> Height of the gastric gland region was measured in WT and ClC-2^-/- mice. Results are plotted as mean ± SE (n = 6). *<i>P</i><0.001 versus WT. <b>C.</b> PAS-AB staining of mucus cells in WT and ClC-2^-/- mouse gastric mucosa. Neutral mucin positive surface mucus cells are dark pink and parietal cells show faint pink staining. Bar = 25 μm.</p

Immunolocalization and expression of ClC-2 in parietal cells of WT and ClC-2^-/- mouse gastric mucosa.

<p>(A) WT gastric mucosa was stained for both H/K ATPase (green) and ClC-2 (red). Nuclei are stained blue, bar = 25 μm. Lower panels show the area delineated by the white boxes magnified 2.5X, bar = 10 μm. (B) H/K ATPase (green) and ClC-2 (red) stained ClC-2^-/- gastric mucosa. Nuclei are stained blue, bar = 25 μm. (C) Western blot of ClC-2 in WT and ClC-2^-/- mouse gastric mucosa, with β-actin as loading control. Molecular weight markers are indicated. (D) Immunogold electron microscopy of ClC-2 in WT and ClC-2^-/- mouse gastric parietal cells. Gold labelling is seen as large black dots (black arrowheads). TV, tubulovesicles, bar = 100nm. Inset shows lower magnification of the parietal cell showing M, mitochondria, TV and N, nucleus for orientation, bar = 1 μm. Representative figures of n = 10–20 regions examined.</p

(A) Effect of histamine on the pH of gastric contents and (B) & (C) effect of histamine/carbachol on acid (B) and pepsinogen (C) secretion in WT and ClC-2^-/- mouse gastric mucosa.

<p>(A) The pH of gastric contents was measured in WT (white column) and ClC-2^-/- (black column) mice after 15 min of histamine stimulation. Data are plotted as mean ± SE (n = 3). *<i>P</i><0.025 versus WT. For (B) & (C) mouse stomachs were perfused and at 30 min subcutaneous histamine/diphenhydramine (HIST, 0.23 mg/h/DPH, 0.03 mg/h) and intraluminal carbachol (CCH, 0.5 mg/ml) were started. 15 min samples of gastric effluent were collected and acid, pH and pepsinogen were measured. Data are plotted as mean ± SE. For (B) WT n = 11 & ClC-2^-/- n = 8, *<i>P</i><0.005 versus WT. For (C) WT & ClC-2^-/- n = 7, NS, not significant versus WT.</p