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

doi:10.1073/pnas.232409599

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

Departments of ^*Anesthesiology, ^‡Biochemistry and Molecular Genetics, ^§Medicine, and UAB Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35233;^¶ Department of Medical Biochemistry, University of Wales Europe College of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom; and ^∥Department of Medicine, University of California, Davis, CA 95616 USA

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved September 30, 2002 (received for review July 10, 2002)

Abstract

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 (LNO₂). Nitroselenylation of linoleic acid yielded LNO₂ that was purified by solvent extraction, silicic acid chromatography, and reverse-phase HPLC. Structural characterization was performed by IR spectroscopy, ¹⁵N-NMR, LC-negative ion electrospray mass spectroscopy (MS), and chemiluminescent nitrogen analysis. Quantitative MS analysis of cell and vessel LNO₂ metabolism, using L[¹⁵N]O₂ as an internal standard, revealed that LNO₂ 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 (ED₅₀ = 30 μM for RASM, 50 μM for aorta). LNO₂ 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 HbO₂. These results reveal that synthetic LNO₂, 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.

Footnotes

↵ † Permanent address: Department of Anesthesiology, School of Medicine, Kyungpook National University, Daegu 702-701, Korea.
↵ ** To whom correspondence should be addressed. E-mail: bruce.freeman{at}ccc.uab.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:
1. ODQ, 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one
2. GC, guanylate cyclase
3. LNO₂, nitrolinoleate
4. MPO, myeloperoxidase
5. PGHS, prostaglandin H synthase
6. RASMC, rat aortic smooth muscle cells
7. MS, mass spectroscopy