STIMULATION OF GASTRIC-ACID SECRETION BY CAMP IN A NOVEL ALPHA-TOXIN-PERMEABILIZED GLAND MODEL

It is generally believed that histamine-stimulated gastric acid secret ion involves a transient elevation of intracellular Ca2+ and the adeno sine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) cascade through phosphorylation, whose actions ultimately effect the f usion of H+-K+-adenosinetriphosphatase (ATPase)containing vesicles to the apical plasma membrane of parietal cells. To dissect the signaling events underlying gastric acid secretion, we have developed a permeab ilized gastric gland model using Staphylococcus alpha-toxin. The advan tage of this model is its ability to retain cytosolic components that are required for the secretory machinery. Here we show that acid secre tion in alpha-toxin-permeabilized glands is a cAMP-dependent process, reaching a maximal stimulation at 100 mu M cAMP. The cAMP-elicited aci d secretion, as monitored by the accumulation of the weak base aminopy rine (AP), required functional mitochondria or exogenously supplied AT P. Maximal stimulation elicited by cAMP for AP uptake by permeabilized glands was 51-85% of intact glands. Moreover, secretory activity was potentiated by 0.1 mMATP. The recruitment of H+-K+-ATPase-rich tubulov esicles into the apical plasma membrane was measured using biochemical and morphological assays, thus validating the cell activation process es in response to cAMP. From this permeabilized model, [gamma-P-32]ATP was used to directly phosphorylate target proteins. A number of prote ins whose phosphorylation-dephosphorylation is specifically modulated by cAMP were found. These studies establish the first permeabilized gl and model in which the resting-to-secreting transition can be triggere d and show that cAMP-mediated phosphorylation is correlated with secre tory activity.