Oxidant-mediated activation of phospholipase A2 in pulmonary endothelium.
Exposure of bovine pulmonary arterial endothelial cells to the oxidant tert-butyl hydroperoxide (t-bu-OOH) caused a dose-dependent increase in the release of [14C]arachidonic acid and synthesis of the cyclooxygenase products, thromboxane, prostaglandin E2, prostaglandin D2, and prostacyclin. There was no detectable production of peptide leukotrienes before or after administration of t-bu-OOH. Pretreatment with the oxygen radical scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidino radical (HTP) or the antioxidants vitamin E and dithiothreitol prevented the increased arachidonic acid (AA) release caused by t-bu-OOH. t-bu-OOH increased the activity of phospholipase A2 by increasing its apparent maximum velocity without affecting its Michaelis constant. The increased AA release caused by t-bu-OOH did not appear to require new RNA or protein synthesis, because pretreatment of the cells with actinomycin D or cycloheximide did not reduce the increased release of AA or activation of phospholipase A2 caused by t-bu-OOH. Dexamethasone pretreatment of the cells prevented the increase in phospholipase A2 activity, and AA release produced by t-bu-OOH. t-bu-OOH increased the activity of phospholipase A2 and release of AA in both the presence and absence of extracellular calcium (Ca2+). Pretreatment with a nominal Ca2+-free buffer, the Ca2+ chelator ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, nifedipine, or verapamil did not reduce t-bu-OOH-stimulated AA release. In contrast, treatment with the intracellular Ca2+ chelator 8-N,N-diethyamino octyl 3,4,5-trimethoxybenzoate (TMB-8) prevented t-bu-OOH-stimulated AA release in both the presence and absence of extracellular Ca2+. Treatment with calmodulin antagonists also prevented the increased release of AA caused by t-bu-OOH.
Chakraborti
S
S
Division of Respiratory and Critical Care Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205.
Gurtner
G H
GH
Michael
J R
JR
D015121:6-Ketoprostaglandin F1 alpha / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D001241:Aspirin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D007700:Kinetics; D010545:Peroxides / Q000494:pharmacology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D015230:Prostaglandin D2 / Q000378:metabolism; D011651:Pulmonary Artery / Q000502:physiology*; D013929:Thromboxane B2 / Q000378:metabolism; D014810:Vitamin E / Q000494:pharmacology; D020122:tert-Butylhydroperoxide
D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D010545:Peroxides; D014810:Vitamin E; D003609:Dactinomycin; D016718:Arachidonic Acid; D004533:Egtazic Acid; D013929:Thromboxane B2; D015121:6-Ketoprostaglandin F1 alpha; D003907:Dexamethasone; D020122:tert-Butylhydroperoxide; D003513:Cycloheximide; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D015232:Dinoprostone; D001241:Aspirin; D015230:Prostaglandin D2; D002118:Calcium