Cytological transformations associated with parietal cell stimulation: critical steps in the activation cascade

Cultured rabbit parietal cells were used to evaluate morphological response s to activators and inhibitors of HCl secretion. Immunofluorescence was use d to localize the proton pump protein, H,K-ATPase, and the apical membrane- cytoskeletal linker protein, ezrin; fluorescent-labeled phalloidin was used as a marker of F-actin, Treatment of healthy control parietal cells with s ecretagogues resulted in exaggerated swelling of apical membrane vacuoles, presumably with the accumulation of HCl and water. Thus stimulation-associa ted swelling of apical vacuoles was blocked by inhibitors that work at vari ous steps in the secretion-activation cascade. When secretion was blocked b y agents that prevent the translocation of H,K-ATPase-rich tubulovesicles t o apical membrane vacuoles (such as H2-receptor antagonists and protein kin ase A inhibitors), the general resting morphology was maintained. ME-3407 ( a functional analogue of wortmannin) was unique in preventing H,K-ATPase re distribution and effecting the delocalization of ezrin from apical membrane vacuoles, When secretion was blocked by agents that inhibit the H+ pump or induce H+ backflux, the translocation of H,K-ATPase to apical membrane vac uoles occurred but the large vacuolar swelling associated with HCl and H2O accumulation was greatly diminished. These data support the membrane recycl ing/recruitment hypothesis of HCl secretion in which H,K-ATPase-rich tubulo vesicles are recruited from a cytoplasmic domain to the apical surface, and they are inconsistent with models proposing that the tubulovesicles, regar dless of shape, are contiguous with the apical plasma membrane. These studi es also demonstrate the utility of the parietal cell culture model in disti nguishing a general site of action for various inhibitors and antisecretory agents.