Volume 127, Issue 1, Pages 145-154 (July 2004) Gastric parietal cell acid secretion in mice can be regulated independently of H+/K+ ATPase endocytosis  Nhung V. Nguyen, Paul A. Gleeson, Nathalie Courtois-Coutry, Michael J. Caplan, Ian R. van Driel  Gastroenterology  Volume 127, Issue 1, Pages 145-154 (July 2004) DOI: 10.1053/j.gastro.2004.04.016

Figure 1 Immunoblot analysis of H+/K+ ATPase. Gastric units from fasted mice of the genotypes indicated were isolated and proteins separated by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were sequentially incubated with monoclonal anti-H/Kβ or a rabbit anti-H/Kα antibody as indicated, biotinylated-secondary antibodies, and streptavidin-horseradish peroxidase. Bound antibody was visualized by chemiluminescence. Mr × 10−3 of molecular weight markers are shown. Membranes that were incubated with isotype-matched control antibodies displayed negligible staining. Gastroenterology 2004 127, 145-154DOI: (10.1053/j.gastro.2004.04.016)

Figure 2 Localization of H+/K+ ATPase subunits in gastric mucosae. (A) Stomachs from fasted mice of the genotypes indicated were fixed in formalin, embedded in paraffin, and sectioned. Sections were sequentially incubated with anti-H/Kα or anti-H/Kβ antibodies (as indicated), biotinylated anti-mouse immunoglobulin, and streptavidin-horseradish peroxidase. Bound antibodies were visualized by incubating with DAB, which results in a dark precipitate. Sections that were incubated with isotype-matched control antibodies displayed negligible staining. Bar in H = 10 μm and applies to all panels. (B) Stomachs from fasted Y20A/Atp4bo/o mice were stained with anti-H/Kα or anti-H/Kβ antibodies (as indicated) as described in A. Bar = 10 μm and applies to both panels. Gastroenterology 2004 127, 145-154DOI: (10.1053/j.gastro.2004.04.016)

Figure 3 Localization of H+/K+ ATPase and actin. Gastric units from fasted mice of the genotypes indicated were isolated and incubated with a monoclonal antibody specific for H/Kα, followed by anti-mouse immunoglobulin-FITC and phalloidin-TRITC. Phalloidin binds to F-actin, which is present on the secretory canalicular membranes and basolateral membranes but not tubulovesicular membranes. TRITC (red) and FITC (green) fluorescence was separately visualized by confocal microscopy and digitally captured. Digital files were then merged. Colocalization of the TRITC and FITC fluorescence is indicated by yellow. Bar = 10 μm and applies to all panels. Gastroenterology 2004 127, 145-154DOI: (10.1053/j.gastro.2004.04.016)

Figure 4 Ultrastructure of parietal cells. Stomachs from fasted Atp4bw/o (A), Y20A/Atp4bw/o (B), Atp4bo/o (C), and Y20A/Atp4bo/o (D-F) mice were fixed, embedded, and sectioned, and sections were imaged by transmission electron microscopy. Mice were treated with cimetidine (A-E) or histamine (F) 30 minutes before collection of tissue. T, tubulovesicles, S, secretory canaliculi. Bar in F = 1 μm and applies to all panels. Gastroenterology 2004 127, 145-154DOI: (10.1053/j.gastro.2004.04.016)

Figure 5 Gastric acid secretion in whole stomachs. Atp4bw/o (n = 4), Y20A/Atp4bw/o (n = 3), Atp4bo/o (n = 3), and Y20A/Atp4bo/o (n = 3) mice were fasted overnight, anesthetized, and administered histamine (H), cimetidine (C), and omeprazole (O) at the times indicated. Gastric acid production was determined every 15 minutes. Each data point represents the average values of all mice ± SEM. Gastroenterology 2004 127, 145-154DOI: (10.1053/j.gastro.2004.04.016)