Reticulocyte 15-lipoxygenase-I is important in acetylcholine-induced endothelium-dependent vasorelaxation in rabbit aorta.
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.


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 micromol/L for AA and 23.5+/-3.3 micromol/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.
Tang
Xin
X
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Holmes
Blythe B
BB
Nithipatikom
Kasem
K
Hillard
Cecilia J
CJ
Kuhn
Hartmut
H
Campbell
William B
WB
D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000201:enzymology*; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D007537:Isometric Contraction / Q000502:physiology; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*; D014665:Vasodilator Agents / Q000494:pharmacology
D014665:Vasodilator Agents; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D000109:Acetylcholine