title: Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells.
abstract: 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 microM 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 microM)-stimulated DAG accumulation was found to be related to the breakdown of [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([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-microM)-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 [32P]PIP and of [32P]PIP2 and that the second peak is predominantly associated with [32P]PIP2 breakdown and phosphatidylcholine hydrolysis.
authors.affiliation: Department of Physiology and Pharmacology, Tokyo College of Pharmacy, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Komabayashi
authors.forename: A
authors.initials: A
authors.lastname: Yakata
authors.forename: T
authors.initials: T
authors.lastname: Izawa
authors.forename: H
authors.initials: H
authors.lastname: Fujinami
authors.forename: K
authors.initials: K
authors.lastname: Suda
authors.forename: M
authors.initials: M
authors.lastname: Tsuboi
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002794:Choline / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis*; D066298:In Vitro Techniques; D008297:Male; D010306:Parotid Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D019311:Staurosporine; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D000470:Alkaloids; D004075:Diglycerides; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D013449:Sulfonamides; C017967:W 7; D002217:Carbachol; D011433:Propranolol; D011493:Protein Kinase C; D019311:Staurosporine; D002794:Choline