title: A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells.
abstract: Many receptors, in response to their specific ligands, trigger activation of phospholipase D (PLD), resulting in the production of phosphatidic acid which, in turn, is acted upon by a specific phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol. We report here that isolated nuclei from Madin-Darby canine kidneys (MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence of ATP. PLD activity was measured in the presence of ethanol, by quantitating the production of phosphatidylethanol. Non-phosphorylating ATP analogs were unable to substitute for ATP in activating PLD, indicating that ATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The protein kinase C inhibitors chelerythrine and calphostin completely suppressed the ATP-induced nuclear PLD, implicating protein kinase C as the kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In the absence of ethanol, phosphatidic acid was detected in ATP-treated nuclei. Accumulation of phosphatidic acid preceded or closely paralleled that of diacylglycerol, suggesting a precursor-product relationship. Consistent with those results, we detected phosphatidate phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of phosphatidic acid and diacylglycerol levels at increasing amounts of ethanol demonstrated that PLD and phosphatidate phosphohydrolase are responsible for generating the majority of the diacylglycerol accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate diacylglycerol from the concerted action of those two enzymes provides a means to regulate nuclear lipid synthesis as well as protein kinase C activity.
authors.affiliation: Department of Pharmacology, University of California at San Diego, La Jolla, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Balboa
authors.forename: J
authors.initials: J
authors.lastname: Balsinde
authors.forename: E A
authors.initials: EA
authors.lastname: Dennis
authors.forename: P A
authors.initials: PA
authors.lastname: Insel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004075:Diglycerides / Q000096:biosynthesis*; D004848:Epithelium; D000431:Ethanol / Q000494:pharmacology; D007668:Kidney; D008954:Models, Biological; D009281:Naphthalenes; D010617:Phenanthridines / Q000494:pharmacology; D010711:Phosphatidate Phosphatase / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010739:Phospholipase D / Q000502:physiology*; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology
chemical_list: C011439:1,2-diacylglycerol; D000470:Alkaloids; D053119:Benzophenanthridines; D004075:Diglycerides; D009281:Naphthalenes; D010617:Phenanthridines; D010712:Phosphatidic Acids; D011083:Polycyclic Compounds; C061465:calphostin complex; D000431:Ethanol; D000255:Adenosine Triphosphate; C016299:chelerythrine; D011493:Protein Kinase C; D010711:Phosphatidate Phosphatase; D010739:Phospholipase D