Although recent advances in receptor characterization have led to the development of new agents, such as the H2 blockers, for the treatment of ulcers, the intracellular mechanisms of gastric secretion remain more obscure. The goal of this investigation is to elucidate the intracellular mechanisms which underly the stimulation of parietal cell secretion by acetylcholine and histamine. In essence, we intend to use protein phosphorylation as a tool to study the physiology of a polarized secretory cell system - the parietal cell. We will, firstly, study the stimulation pf protein phosphorylation by histamine in isolated rabbit parietal cells, correlating the magnitude and temporal scale of phosphorylation with the course of acid secretion (aminopyrine uptake), intrinsic factor secretion and intracellular cAMP levels. Secondly, we will confirm the role of histamine in the stimulation of cAMP-dependent phosphorylation through the use of direct stimulatory and inhibitory probes. Thirdly, we will investigate carbachol stimulation of protein phosphorylation correlating :he magnitude and temporal course of secretion inositol triphosphate levels and intracellular calcium fluxes. Fourthly, we will compare the patterns of protein phosphorylation stimulated by histamine and carbachol in order to evaluate points of divergence and confluence in their stimulation of secretion. Fifthly, we will investigate the mechanisms of physiological and pharmacological inhibitors of parietal cell secretion through their effects on secretion, phosphorylation and intracellular messengers. Sixthly, we will investigate the role of protein phosphorylation in the initiation and maintenance of the morphological alterations necessary for secretion. Finally, we will undertake the purification of phosphoprotein substrates in order to investigate the precise localization and function of histamine and carbachol stimulated phosphoproteins. This protocol will allow us to elucidate basic mechanisms underlying coordinate stimulatory pathways for activation of parietal cell secretion. Elucidation of the intracellular mechanisms of secretion may yield insights with therapeutic ramifications as significant as those derived from the delineation of H2-receptor agents.
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