MECHANISM OF CARBACHOL-STIMULATED DIACYLGLYCEROL FORMATION IN RAT PAROTID ACINAR-CELLS

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1-mu-M carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [P-32]phosphoinositide breakdown, the first peak of carbachol (1-mu-M)-stimulated DAG accumulation was found to be related to the breakdown of [P-32]phosphatidylinositol 4-monophosphate ([P-32]PIP) and [P-32]phosphatidylinositol 4,5-bisphosphate ([P-32]PIP2). The second peak was found to be related to [P-32]PIP2 breakdown. Carbachol stimulated the release of [H-3]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [H-3]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [H-3]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-mu-M)-induced release of [H-3]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown of [P-32]PIP and [P-32]PIP2 and that the second peak is predominantly associated with [P-32]PIP2 breakdown and phosphatidylcholine hydrolysis.