title: Inhibition of platelet arachidonic acid liberation by endothelium-derived relaxing factor (EDRF) as studied with sin-1, a nitric oxide generating drug. Evidence for calcium-dependent and calcium-independent mechanisms.
abstract: In order to investigate possible effects of endothelium-derived relaxing factor (EDRF or NO.) on platelet phospholipase A2 activity, human platelets labelled with [3H]arachidonic acid ([3H]AA) were stimulated with thrombin (0.5 IU/ml) in the absence or in the presence of sin-1, a vasodilator and platelet inhibitor releasing NO. by spontaneous decomposition at physiological pH. Sin-1 promoted a dose-dependent inhibition of [3H]AA liberation, which was identical in the presence or in the absence of 1 mM Ca2+ in the external medium, suggesting that a reduction of Ca2+ influx was not responsible for this metabolic effect. Using fura-2 as a fluorescent Ca2+ indicator, sin-1 was found to inhibit similarly both Ca2+ influx and Ca2+ mobilization, the latter effect being directly related to a reduction of inositol 1,4,5-tris phosphate production by phospholipase C. However, comparison of cytoplasmic free calcium concentrations ([Ca2+]i) and of [3H]AA liberation attained by platelets treated under various experimental conditions indicated the lack of a direct relationship between [Ca2+]i and platelet phospholipase A2 activity. The effects of sin-1 on [3H]AA liberation could be reproduced by a membrane-permeant analogue of cGMP (8-bromo cyclic GMP), with no evidence of additional effects of sin-1 under these conditions. These data bring further support to the view that Ca2+, although being a necessary cofactor of intracellular phospholipase A2, is not the only regulator of the enzyme. Owing to the multiple effects of this drug on various events involved in membrane-signal transduction (Ca2+ influx, phospholipase C and phospholipase A2 activation), it is suggested that sin-1 inhibits platelet function at an early step of signal transduction, probably by elevating cGMP through a direct effect of NO. on cytosolic guanylate cyclase.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, Phospholipides membranaires, Signalisation cellulaire et Lipoprotéines, Toulouse, France.
authors.forename: A
authors.initials: A
authors.lastname: Gassama-Diagne
authors.forename: M F
authors.initials: MF
authors.lastname: Simon
authors.forename: H
authors.initials: H
authors.lastname: Chap
mesh_terms: D016718:Arachidonic Acid / Q000097:blood*; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000097:blood / Q000502:physiology*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000097:blood; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D010741:Phospholipases A / Q000097:blood*; D054467:Phospholipases A2; D013917:Thrombin / Q000494:pharmacology
chemical_list: D016718:Arachidonic Acid; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; C002385:linsidomine; D015544:Inositol 1,4,5-Trisphosphate; D008981:Molsidomine; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin; D006152:Cyclic GMP; D002118:Calcium