Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylgglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 μM 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 ³²P]phosphoinositide breakdown, the first peak of carbachol (1 μM)-stimulated DAG accumulation was found to be related to the breakdown of ³²P]phosphatidylinositol 4-monophosphate (³²P]PIP) and ³²P]phosphatidylinositol 4,5-biphosphate (³²P]PIP₂). The second peak was found to be related to ³²P]PIP₂ breakdown. Carbachol stimulated the release of ³H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of ³H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of ³H]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-μM)-induced release of ³H]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 ³²P]PIP and ³²P]PIP₂ and that the second peak is predominantly associated with ³²P]PIP₂ breakdown and phosphatidylcholine hydrolysis.