Thrombin stimulation of human endothelial cell phospholipase D activity. Regulation by phospholipase C, protein kinase C, and cyclic adenosine 3'5'-monophosphate.
The activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether alpha-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [32Pi] and PLD activity determined by formation of the PLD metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10 nmol/L) and gamma-thrombin (1 mumol/L), but not inactive DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of Ca2+ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca2+ ionophores A23187 and ionomycin (1 mumol/L, three-fold to fourfold increase in PEt). Alpha-Thrombin-stimulated PEt formation was abolished (greater than 90% inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced alpha-thrombin-induced PEt formation in a dose-dependent manner (10 mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of alpha-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered alpha-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes) consistently augmented alpha-thrombin-stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, alpha-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT TRUNCATED AT 400 WORDS)
Garcia
J G
JG
Department of Medicine, Indiana University School of Medicine, Indianapolis.
Fenton
J W
JW
Natarajan
V
V
D037361:Adenylate Cyclase Toxin; D000470:Alkaloids / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D010703:Phorbol Esters / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D019311:Staurosporine; D013917:Thrombin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism*; D014471:Umbilical Veins; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000470:Alkaloids; D010703:Phorbol Esters; D010566:Virulence Factors, Bordetella; D000001:Calcimycin; D015759:Ionomycin; D003994:Bucladesine; D012969:Sodium Fluoride; D002772:Cholera Toxin; D000242:Cyclic AMP; D011493:Protein Kinase C; D010738:Type C Phospholipases; D010739:Phospholipase D; D013917:Thrombin; D019311:Staurosporine