on October 20, 2005
Published online before print October 20, 2005, doi: 10.1161/01.ATV.0000191640.73313.ad
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Submitted on July 20, 2005
Accepted on October 10, 2005
Reticulocyte 15-Lipoxygenase-I Is Important in Acetylcholine-Induced Endothelium-Dependent Vasorelaxation in Rabbit Aorta
Xin Tang ;
From the Department of Pharmacology and Toxicology (X.T., B.B.H., K.N., C.J.H., W.B.C.), Medical College of Wisconsin, Milwaukee; and the Institute of Biochemistry (H.K.), University Medicine Berlin-Charité, Berlin, Germany.
* To whom correspondence should be addressed. E-mail: wbcamp{at}mcw.edu.
Objective--Aortic 15-lipoxygenase (15-LO) metabolizes arachidonic acid (AA) to 15-hydroperoxyeicosatetraenoic acid, which is then converted to the vasodilators 15-hydroxy-11,12-epoxyeicosatrienoic acid and 11,12,15-trihydroxyeicosatrienoic acid. These metabolites contribute to endothelium-dependent relaxations of rabbit aorta to AA and acetylcholine. We investigated the identity of rabbit aortic 15-LO and studied its importance in the regulation of vascular tone.
Methods and Results--RT-PCR using 12-lipoxygenase/15-LO specific primers resulted in a 572-bp product with a sequence identical to 15-LO-I from rabbit aorta. A RT-PCR/restriction digest strategy excluded expression of 12-lipoxygenase. Immunoblotting revealed 15-LO-I expression in rabbit endothelial and smooth muscle cells. Aortic homogenates and cytosolic fractions metabolize AA to 15(S)-hydroxyeicosatetraenoic acid and linoleic acid to 13(S)-hydroxyoctadecadienoic acid. This activity was blocked by LO inhibitors. The kinetic characteristics (Michaelis constant of aortic 15-LO is 2.2±0.3 µmol/L for AA and 23.5±3.3 µmol/L for linoleic acid) of aortic 15-LO were similar to those of the purified 15-LO-I. An antisense oligonucleotide inhibited 15-LO-I expression in rabbit aorta. Indomethacin and nitro-L-arginine-resistant relaxations to acetylcholine were inhibited by 15-LO-I antisense oligonucleotide but not by the scrambled oligonucleotide.
Conclusions--15-LO-I is expressed in rabbit aortic endothelium and is important in endothelium-dependent regulation of vascular tone.
Key words: endothelium • arachidonic acid • 15-lipoxygenase • endothelium-derived hyperpolarizing factor
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