Characterization of membrane and cytoskeletal compartments in cultured parietal cells: immunofluorescence and...

Primary cultures of rabbit gastric parietal cells respond to various gastric secretagogues as evidenced by morphological alterations and [14C]aminopyrine uptake. The availability of cultures of > 95% purity has allowed us to utilize immunofluorescence and confocal microscopy to observe the direct effect of histamine upon the distribution of membrane and cytoskeletal proteins in parietal cells. Cells cultured for 3 days were incubated for 45 min with or without 10(-4) M histamine, washed, and fixed with 3% paraformaldehyde. Immunofluorescence was performed with antibodies against H+/K(+)-ATPase, Na+/K(+)-ATPase, ezrin, and beta-tubulin, as well as with Bodipy-phallacidin. Anti-H+/K(+)-ATPase antibody stained resting cells in a vesicular cytoplasmic pattern. Stimulation with histamine resulted in the development of a well-defined linear pattern, outlining the expanded secretory canaliculi. The Na+/K(+)-ATPase was restricted to predominantly the lateral surface in both the resting and stimulated cells, suggesting that the cultured parietal cells retain membrane polarity. Ezrin was visualized outlining the intracellular canaliculi in the resting state, and surrounding the large secretory canaliculi in the stimulated cell. Phallacidin labeling of F-actin localized to an area tightly surrounding the intracellular canaliculi in the resting cell, and was comparable with the staining observed with ezrin. In the stimulated cells this fluorescence pattern became more diffuse and surrounded the expanded secretory surface. In both the resting and stimulated cells, antibodies to beta-tubulin revealed a microtubular pattern located predominantly in the basal portion of the cell. These results demonstrate that the cells are capable of translocating the H+/K(+)-ATPase-containing tubulovesicles to a secretory surface, and that they exhibit organization and maintenance of basolateral and canalicular membrane domains. Furthermore, these studies demonstrate the directed movement of membrane and cytoskeletal proteins upon stimulation of the cultured parietal cells.