Abstract
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 IC50 value of 5.8 ± 1.2 μM. 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 HCO3−/Cl− anion exchanger. Treatment of platelets with (−)-epigallocatechin gallate (2 μM), a tea polyphenol and inhibitor of tyrosine nitration, abolished the inhibitory effect of ONOO− on arachidonate-induced aggregations by 88%. ONOO− (50–300 μM), added to platelets 1 min before arachidonic acid, inhibited (20–100%) formation of platelet cyclooxygenase (COX) products thromboxane A2 and 12-hydroxyheptadecatrienoic acid. Interestingly, simultaneous addition of ONOO− and arachidonic acid stimulated eicosanoid production by 20 to 60%. The inhibition of thromboxane A2 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 H2 when ONOO− (50 μM) was added 1 min before arachidonic acid. However, the addition of ONOO− (50–100 μM) simultaneously with arachidonic acid increased prostaglandin H2 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 F2α, epoxyhydroxyeicosatrienoic acid, and trans-arachidonic acids, suggesting that OH and NO2 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.
Footnotes
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Send reprint requests to: Michael Balazy, Ph.D., Department of Pharmacology, New York Medical College, Valhalla, NY 10595. E-mail:Michael_Balazy{at}nymc.edu
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↵1 This research was supported by American Heart Association, New York State Affiliate, Grant 9850104 and in part by National Institutes of Health Grant HL34300.
- Abbreviations:
- NO
- nitric oxide
- COX
- cyclooxygenase
- ONOO−
- peroxynitrite (this refers to the sum of peroxynitrite anion ONOO− and its conjugated acid, peroxynitrous acid ONOOH)
- O⨪2
- superoxide
- DIDS
- 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid
- SOD
- superoxide dismutase
- DCF-DA
- 2′,7′-dichlorodihydrofluorescein diacetate
- DCFH
- 2′,7′-dihydrodichlorofluorescein
- 12-HETE
- 12-hydroxyeicosatetraenoic acid
- 12-HpETE
- 12-hydroperoxyeicosatetraenoic acid
- 12-HHT
- 12-hydroxyheptadecatrienoic acid
- PG
- prostaglandin
- EpHETrE
- epoxyhydroxyeicosatrienoic acid
- TNM
- tetranitromethane
- TX
- thromboxane
- PFB
- pentafluorobenzyl
- GC
- gas chromatography
- MS
- mass spectrometry
- MS/MS
- tandem MS
- GSH-Px
- glutathione peroxidase
- Received July 15, 1999.
- Accepted January 5, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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