Fig 1.
Histological characterization of the gastric mucosa of WT and ClC-2-/- mice.
A. 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. Height of the gastric gland region was measured in WT and ClC-2-/- mice. Results are plotted as mean ± SE (n = 6). *P<0.001 versus WT. C. 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.
Fig 2.
Immunolocalization and expression of H/K ATPase-β subunit in WT and ClC-2-/- mouse gastric mucosa.
Gastric mucosal sections from WT and ClC-2-/- mice were stained for H/K ATPase β subunit by immunohistochemistry (A) and immunofluorescence (C). H/K ATPase β subunit positive cells were orange/brown in (A) and green in (C). Bar in (A) = 25 μm; bar in (B) = 10 μm, representative figures from n = 10–20 regions examined. (B) Quantitation of H/K ATPase-containing cells/gland of WT and ClC-2-/- gastric mucosa, n = 6. #P<0.05 versus WT. (D) Western blot of H/K ATPase in WT and ClC-2-/- mouse gastric mucosa, with β-actin as loading control. (E) Quantitation of H/K ATPase western blot by densitometry, normalized to β-actin, n = 3. *P<0.001 versus WT. Data in (B) and (E) are plotted as mean ± SE.
Fig 3.
Ultrastructure of parietal cells in WT and ClC-2-/- mouse gastric mucosa.
Nucleus (N), mitochondria (M) and tubulovesicles (TV) are indicated. Electron dense bodies in the ClC-2-/- panel are presumed to be fragments of mitochondria. Representation of n = 4 with at least 10 parietal cells examined from 3 different areas of each sample. Bar = 1 μm.
Fig 4.
Immunolocalization and expression of ClC-2 in parietal cells of WT and ClC-2-/- mouse gastric mucosa.
(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.
Fig 5.
(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.
(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). *P<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, *P<0.005 versus WT. For (C) WT & ClC-2-/- n = 7, NS, not significant versus WT.