Nitrolinoleate, a nitric oxide-derived mediator of cell function: Synthesis, characterization, and vasomotor activity

Nitric oxide ((•)NO) and (•)NO-derived reactive species rapidly react with lipids during both autocatalytic and enzymatic oxidation reactions to yield nitrated derivatives that serve as cell signaling molecules. Herein we report the synthesis, purification, characterization, and bioactivity of nitrolinoleate (LNO(2)). Nitroselenylation of linoleic acid yielded LNO(2) that was purified by solvent extraction, silicic acid chromatography, and reverse-phase HPLC. Structural characterization was performed by IR spectroscopy, (15)N-NMR, LC-negative ion electrospray mass spectroscopy (MS), and chemiluminescent nitrogen analysis. Quantitative MS analysis of cell and vessel LNO(2) metabolism, using L[(15)N]O(2) as an internal standard, revealed that LNO(2) is rapidly metabolized by rat aortic smooth muscle (RASM) monolayers and rat thoracic aorta, resulting in nitrite production and up to 3-fold increases in cGMP (ED(50) = 30 μM for RASM, 50 μM for aorta). LNO(2) induced endothelium-independent relaxation of preconstricted rat aortic rings, which was unaffected by L(G)-nitro-l-arginine methyl ester addition and inhibited by the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one and the (•)NO scavenger HbO(2). These results reveal that synthetic LNO(2), identical to lipid derivatives produced biologically by the reaction of (•)NO and (•)NO-derived species with oxidizing unsaturated fatty acids (e.g., linoleate), can transduce vascular signaling actions of (•)NO.