Abstract
Nitric oxide (NO)-derived species could potentially react with arachidonic acid to generate novel vasoactive metabolites. We studied the reaction of arachidonic acid with nitrogen dioxide (NO2), a free radical that originates from NO oxidation. The reaction mixture contained lipid products that relaxed endothelium-removed bovine coronary arteries. Relaxation to the lipid mixture was inhibited ∼20% by indomethacin and ∼70% by a soluble guanylate cyclase (sGC) inhibitor (ODQ). Thus, novel lipid products, which activate sGC presumably through a mechanism involving NO, appeared to have contributed to the observed vasorelaxation. Lipids that eluted at 9 to 12 min during high-performance liquid chromatography fractionation accounted for about one-half of the vasodilator activity in the reaction mixture, which was inhibited by ODQ. Lipid products in fractions 9 to 12 were identified by electrospray tandem mass spectrometry to be eight isomers having molecular weight of 367 and a fragmentation pattern indicative of arachidonic acid derivatives containing nitro and hydroxy groups and consistent with the structures of vicinal nitrohydroxyeicosatrienoic acids. These lipids spontaneously released NO (183 ± 12 nmol NO/15 min/μmol) as detected by head space/chemiluminescence analysis. Mild alkaline hydrolysis of total lipids extracted from bovine cardiac muscle followed by isotopic dilution gas chromatography/mass spectrometry analysis detected basal levels of nitrohydroxyeicosatrienoic acids (6.8 ± 2.6 ng/g tissue;n = 4). Thus, the oxidation product of NO, NO2, reacts with arachidonic acid to generate biologically active vicinal nitrohydroxyeicosatrienoic acids, which may be important endogenous mediators of vascular relaxation and sGC activation.
Footnotes
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This study was supported by National Institutes of Health Grants R01 GM62453, S10 RR12993 (to M.B.); HL31069, HL43023, and HL66331 (to M.S.W.); and a grant from the American Heart Association New York State Affiliate (9850104 to M.B.). This work was presented at the 72nd Scientific Sessions of the American Heart Association in Atlanta GA [(1999) Circulation100:I814].
- Abbreviations:
- NO
- nitric oxide
- NO2
- nitrogen dioxide
- PG
- prostaglandin
- ODQ
- 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one
- NO2AA
- nitroeicosatetraenoic acid
- HPLC
- high-performance liquid chromatography
- GC/MS
- gas chromatography/mass spectrometry
- ESI
- electrospray ionization
- LC/MSn
- liquid chromatography/tandem mass spectrometry
- PFB
- pentafluorobenzyl
- TMS
- trimethylsilyl
- BCA
- bovine coronary artery
- sGC
- soluble guanylate cyclase
- NO2AAOH
- vicinal nitrohydroxyeicosatrienoic acids (mixture of isomers)
- EET
- epoxyeicosatrienoic acids
- diHETrE
- vicinal dihydroxyeicosatrienoic acids
- Received May 15, 2001.
- Accepted July 20, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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