title: Comparison of phospholipase D activity in vasopressin- and phorbol ester-stimulated fibroblasts.
abstract: Phospholipase D (PLD) activation by vasopressin (VP) was compared to activation by TPA in REF52 cells prelabeled with [3H]glycerol and [14C]myristic acid. Upon VP-treatment, the formation of [3H] and [14C]phosphatidic acid (PA) and phosphatidylethanol (PEt) was accompanied by the loss of radioactivity from PC and PI. However, upon TPA-treatment, radioactivity was lost from PC only. No significant changes of phosphatidylethanolamine and phosphatidylserine were detected in the same samples. The inclusion of 5 microM staurosporine for 10 min diminished the production of [3H]PEt and [14C]PEt by 27% and 53% in VP-treated cells, and by 100% and 75% in TPA-treated cells, respectively. Adding 1 mM EGTA to chelate extracellular Ca2+ inhibited [3H]PEt by approximately 31% and [14C]PEt by 17% after VP-stimulation. In contrast, EGTA had no effect on TPA-stimulation. The data suggest that REF52 cells contain dual PLD activities. The first is stimulated only by VP, requires Ca2+ and hydrolyzes PI. The second is stimulated by both TPA and VP, activated by protein kinase C and hydrolyzes PC.
authors.affiliation: Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157-1016.
authors.forename: C
authors.initials: C
authors.lastname: Huang
authors.forename: R L
authors.initials: RL
authors.lastname: Wykle
authors.forename: M C
authors.initials: MC
authors.lastname: Cabot
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000201:enzymology*; D005990:Glycerol / Q000378:metabolism; D020404:Glycerophospholipids; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014667:Vasopressins / Q000494:pharmacology*
chemical_list: D000470:Alkaloids; D020404:Glycerophospholipids; D009227:Myristic Acids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; C051521:phosphatidylethanol; D019814:Myristic Acid; D014667:Vasopressins; D010739:Phospholipase D; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D005990:Glycerol; D002118:Calcium