Diffusion of peroxynitrite into the human platelet inhibits cyclooxygenase via nitration of tyrosine residues.
Peroxynitrite (ONOO(-)), a reactive oxidant produced by the reaction between nitric oxide and superoxide, was found to diffuse into the platelet cytosol and inhibit arachidonic acid-induced platelet aggregations with IC(50) value of 5.8 +/- 1.2 microM. A fluorescence assay established that ONOO(-) diffused into the platelet cytosol in a manner that was inhibited (50-70%) by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of HCO(3)(-)/Cl(-) anion exchanger. Treatment of platelets with (-)-epigallocatechin gallate (2 microM), a tea polyphenol and inhibitor of tyrosine nitration, abolished the inhibitory effect of ONOO(-) on arachidonate-induced aggregations by 88%. ONOO(-) (50-300 microM), added to platelets 1 min before arachidonic acid, inhibited (20-100%) formation of platelet cyclooxygenase (COX) products thromboxane A(2) and 12-hydroxyheptadecatrienoic acid. Interestingly, simultaneous addition of ONOO(-) and arachidonic acid stimulated eicosanoid production by 20 to 60%. The inhibition of thromboxane A(2) generation correlated with the 5- to 10-fold increase in the 3-nitrotyrosine levels of the platelet COX. Experiments with purified COX-1 and COX-2 also showed 9-fold increase of 3-nitrotyrosine levels, which correlated with decreased (93-98%) production of prostaglandin H(2) when ONOO(-) (50 microM) was added 1 min before arachidonic acid. However, the addition of ONOO(-) (50-100 microM) simultaneously with arachidonic acid increased prostaglandin H(2) formation by 30 to 60%. Thus, the inhibitory effect of ONOO(-) involved nitration of COX tyrosine residues, whereas the stimulatory effect was likely to be a result of ONOO(-) functioning as a peroxide activator of eicosanoid signaling. Increasing doses of ONOO(-) not only inhibited platelet COX but also induced formation of unique eicosanoids: iso-prostaglandin F(2alpha), epoxyhydroxyeicosatrienoic acid, and trans-arachidonic acids, suggesting that OH and NO(2) radicals were generated from ONOO(-) in platelets. Formation of ONOO(-) from NO and superoxide may function as a platelet hormone-like COX regulatory mechanism in inflammatory processes in which large amounts of these molecules are produced.
Boulos
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C
Department of Pharmacology, New York Medical College, Valhalla, New York, USA.
Jiang
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Balazy
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D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D004058:Diffusion; D015777:Eicosanoids / Q000096:biosynthesis; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins; D009566:Nitrates / Q000097:blood / Q000494:pharmacology*; D016877:Oxidants / Q000097:blood / Q000494:pharmacology*; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D011451:Prostaglandin-Endoperoxide Synthases / Q000187:drug effects / Q000378:metabolism; D013928:Thromboxane A2 / Q000097:blood; D014443:Tyrosine / Q000031:analogs & derivatives / Q000097:blood / Q000378:metabolism*; D014661:Vasoconstriction / Q000187:drug effects
D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D015777:Eicosanoids; D007202:Indicators and Reagents; D007527:Isoenzymes; D008565:Membrane Proteins; D009566:Nitrates; D016877:Oxidants; C039642:peroxynitric acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D013928:Thromboxane A2; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases