Regulation of vasodilatory eicosanoid synthesis by 15-lipoxygenase I

Rabbit aortic 15-lipoxygenase (15-LO) metabolizes arachidonic acid (AA) into 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which is then converted to vasodilatory compounds 15-hydroxy-11, 12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). THETA and HEETA represent a new class of endothelium dependent hyperpolarization factors (EDHFs). The goal of this thesis is to characterize rabbit aortic 15-LO and study its role in regulation of vascular tone. RT-PCR identified aortic 15-LO mRNA sequence as rabbit reticulocyte 15-LO (15-LO-I). Immunoblotting showed expression of 15-LO-I in rabbit aortic tissue, cultured endothelial cells and smooth muscle cells. Immunohistochemistry located 15-LO-I at the endothelium and the smooth muscle cells adjacent to the endothelium in aortic vascular wall. Aortic 15-LO metabolizes AA to 15-(S)-hydroxyeicosatetraenoic acid (15-HETE), and linoleic acid (LA) to 13-(S)-hydroxyoctadecadienoic acid (13-HODE), which is blocked by LO inhibitors, nordihydroguaiaretic acid (NDGA) and cinnamyl-3, 4-dihydroxy-cyanocinnamate (CDC). AA (Km =1.9+/- 0.3 muM) is the preferred substrate of aortic 15-LO compared with LA (Km =23.3+/- 5.0 muM). Antisense oligonucleotide knockout of 15-LO-I in isolated rabbit aortic rings inhibited relaxation responses to acetylcholine in the presence of indomethacin and N-nitro-L-arginine (LNA).; Regulation of aortic 15-LO by various factors also affects vascular activities. 15-LO-I expression was increased by interleukin-13 (IL-13) in a concentration-dependent and time-dependent manner. IL-13 treatment increased the production of 15-HETE, HEETA and THETA. Indomethacin resistant vasorelaxations to AA were greater in IL-13-treated vessels than in control. The relaxation responses to sodium nitroprusside (SNP) were not changed by IL-13 treatment. Aortic 15-LO expression and activity is age related. In rabbits of 1-16 week old, aortic 15-LO expression decreases with the animal's age. Methylation inhibitor 5-azacytidine (5-AZ, 10 muM) increased aortic 15-LO expression in 16-week-old rabbits. The synthesis of 15-LO pathway products, 15-HETE, THETA, and HEETA by aortic tissue is reduced in the older rabbits. Indomethacin and LNA-resistant relaxation responses to acetylcholine and AA are decreased with animal age, while vasorelaxations to SNP were the same in all age groups. Calcium (Ca2+) regulates aortic 15-LO intracellular location and enzyme activity. The majority of 15-LO-I locates in the cytosol in unstimulated cells. Ca2+ 1-to 1000 muM induces translocation of aortic 15-LO to cell membrane. High Ca2+ (1000 muM) did not affect membrane 15-LO activity but reduced cytosolic 15-LO activity by 65% for AA metabolism and 88% for LA metabolism.; In conclusion, 15-LO-I is expressed in rabbit aorta. Aortic 15-LO is regulated by factors such as cytokines, age, and Ca2+, which regulate the synthesis of vasodilatory eicosanoids such as THETA and HEETA. 15-LO-I in rabbit aorta plays important role in regulation of vascular tone.