Figure 3

Impaired tubulovesicle fusion and H⁺,K⁺-ATPase translocation in ASAP3-deficient parietal cells upon histamine stimulation. (a) Transmission electron microscopy (TEM) analysis on the structures of secretory membrane in histamine-stimulated parietal cells of wild-type (WT) mice (top) and ASAP3-deficient (bottom) mice. Note the impaired tubulovesicle (TV) fusion to the abnormal apical membrane (AP) and collapsed intracellular luminal space upon stimulation in ASAP3-deficient parietal cells. In contrast, the intracellular tubulovesicle proportion decreased and normal secretory canaliculi lined up in stimulated WT parietal cells compared to its resting state. MT, mitochondria. Scale bars are indicated in the TEM images. (b) Immunofluorescent image and colocalization analysis of apical membrane and H⁺,K⁺-ATPase in histamine-stimulated parietal cells of WT mice (top) and ASAP3-deficient (bottom) mice. The apical membrane is labeled with a specific antibody for calcium pump pan PMCA ATPase (red), and H⁺,K⁺-ATPase is stained in green. Cell nucleus was stained by 4′,6-diamidino-2-phenylindole (DAPI) in blue. Note the mutual exclusive distribution of apical membrane and H⁺,K⁺-ATPase in stimulated ASAP3-deficient parietal cells. White arrows indicate apical membrane structures that are negatively stained for H⁺,K⁺-ATPase. The R values of Pearson’s correlation coefficients for red versus green panels are indicated. (c) Decreased gastric acid content from the stomachs of ASAP3-deficient mice in contrast to WT mice as control. Values (mean±s.e.m.) were normalized to body weight. Mice were treated with histamine (n=9 per genotype) to measure stimulated acid contents, respectively (*P<0.01, Student’s t-test). (d) The expression levels of active Arf6-GTP-bound pattern, pan Arf6 in ASAP3-defecient and WT mice upon histamine stimulation were detected by western blot. The GAPDH level was used as loading control. The data are representative of immunoblots from nine littermates per group.