AJP - Lung Cellular and Molecular Physiology, Vol 257, Issue 6 430-L437, Copyright © 1989 by American Physiological Society

ARTICLES

Oxidant-mediated activation of phospholipase A2 in pulmonary endothelium

S. Chakraborti, G. H. Gurtner and J. R. Michael
Division of Respiratory and Critical Care Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205.

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.

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