Rabbit gastric parietal cells will be used as a model system for the study of the processes underlying the regulation of H- secretion. Rabbit was chosen because it has been extensively studied in the past. It closely resembles human gastric mucosa in response to secretagogues and in response to therapeutic agents. Preliminary studies indicate that histamine receptors present on the isolated cells activate cAMP-dependent protein kinase, that histamine treatment results in changes in morphology, metabolism, and ion transport that are hallmarks of secretory cell activity, that a number of polypeptide species become phosphorylated by the cAMP-dependent protein kinase in parallel with histamine stimulation of H- secretion and morphological changes, and that some of the phosphoproteins associate with membranes under certain conditions of isolation. Using 1- and 2-dimensional gel electrophoresis and quantitative autoradiography of the phosphoproteins from intact cell lysates and cell fractions, we will 1) separate, 2) quantitate, and 3) localize the phosphoproteins intimately involved in the maintenance of the stimulated state of H- secretion. Immunoblotting, immunoprecipitation, chemical modification using affinity probes, peptide mapping, biochemical separation and physical studies will be used to identify and characterize specific phosphoproteins involved in stimulus-secretion coupling. Direct binding of isolated, purified components and affinity chromatography of the isolated components will be used to assess the role of these proteins in the morphological and ion permeability changes of the secretory membrane upon stimulation of H- secretion.
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