The manifestations of peptic ulcer disease constitute some of the commonest medical complaints. However, while potent pharmacological therapies targeted at the H2-histamine receptor have evolved over the past decade, the intracellular mechanisms responsible for acid secretion remain elusive. The role of cAMP as a mediator of histamine stimulated acid secretion from parietal cells is well recognized. Nevertheless, the mechanisms actually responsible for the translation of the cAMP signal into the physiological response of secretion remain poorly understood. We have recently found that the dephosphorylation of a subclass of Type II regulatory subunit of cAMP-dependent protein kinase may be a critical step in the processing of the cAMP signal in the parietal cell. It is our working hypothesis that cAMP may act through a coordinated activation of both kinase and phosphatase activities. Inherent in this hypothesis is the belief that cAMP-dependent protein kinase activation II regulatory subunit (RII) of cAMP-dependent protein kinase as a cAMP sensor protein. This proposal will pursue five major lines of investigation: First, the role of protein phosphatase and kinase activities in mediating the histamine stimulation on the phosphorylation of RII in situ will be evaluated in the presence of both a protein phosphatase inhibitor (okadaic acid) and a cAMP-dependent protein kinase inhibitor (H-8). Second, distinct isotypes of RII in membrane-bound and cytosolic compartments of parietal cells will be characterized and the protein kinase systems affecting these proteins will be delineated. Third, the presence of isotypes of RII in distinct subcellular compartments will be established using subcellular fractionation and immunocytochemistry. Fourth, RII protein phosphatase activities in membrane fractions will be isolated and characterized as focal points for histamine action. Fifth, since the effects of cAMP on parietal cell function appear to be in part mediated through activities other than cAMP-dependent protein kinase, the association of RII with other cellular proteins and, in particular, with components of the cytoskeleton will be investigated. Sixth, since secretion by parietal cells is stimulated by both cAMP-dependent and cAMP-independent mechanisms, the common features of histaminergic and cholinergic stimulation will be investigated to determine whether protein phosphatase activities might represent a common mediator. These investigations should elucidate some of the basic mechanisms by which the secretion of acid from the parietal cell is controlled. Expansion of our understanding of the intracellular processes responsible for acid secretion may lead to new methods for the treatment of peptic ulcer disease and other secretory disorders.
