title: Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester.
abstract: Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC or HUVEC with 100 nM PMA for 5-10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5-fold. Pretreatment with 100 nM PMA for 5 min also caused a 2-fold enhancement of thrombin-stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2 alpha, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin-stimulated (1 U/ml) PAF production was enhanced 2.6-fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H-7 and staurosporine ablated the enhancing effect of PMA on thrombin-stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.
authors.affiliation: Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
authors.forename: G B
authors.initials: GB
authors.lastname: Zavoico
authors.forename: J K
authors.initials: JK
authors.lastname: Hrbolich
authors.forename: M A
authors.initials: MA
authors.lastname: Gimbrone
authors.forename: A I
authors.initials: AI
authors.lastname: Schafer
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D001095:Arachidonic Acids / Q000378:metabolism / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D015237:Dinoprost / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D011464:Epoprostenol / Q000378:metabolism*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology*; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010879:Piperazines / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000378:metabolism*; D015230:Prostaglandin D2 / Q000378:metabolism; D011463:Prostaglandins H / Q000378:metabolism / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D000470:Alkaloids; D001095:Arachidonic Acids; D007546:Isoquinolines; D010703:Phorbol Esters; D010879:Piperazines; D010972:Platelet Activating Factor; D011463:Prostaglandins H; D000001:Calcimycin; D015759:Ionomycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D015237:Dinoprost; D011464:Epoprostenol; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin; D019311:Staurosporine; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate; D015230:Prostaglandin D2