20‐hydroxyeicosatetraenoic acid brings about endothelial dysfunction via eNOS uncoupling

Abstract

Nitric oxide (NO), generated from L-arginine by endothelial NO synthase (eNOS), is a key factor whose bioavailability is essential to the integrity of the endothelium. Dysfunctional endothelium is characterized by loss of NO bioavailability. We have recently reported that renal arteries from rats transduced with a cytochrome P450 (CYP) 4A2-expressing adenovirus had increased vascular CYP4A expression and 20-hydroxyeicosatetraenoic acid (20-HETE) production, and displayed endothelial dysfunction exemplified by reduced acetylcholine-induced relaxations, reduced levels of NO, and increased levels of superoxide anion. Because endothelial dysfunction in arteries from CYP4A2-transduced rats was abrogated by inhibition of 20-HETE synthesis, we examined the effect of 20-HETE on NO production and bioavailability in vitro. Addition of 20-HETE (5 nM) to cultured bovine aortic endothelial cells reduced calcium ionophore (5 μM)-stimulated NO production by 60%. This reduction was associated with significant increases in superoxide (32%) and peroxynitrite (40%) levels measured by fluorescent dyes which were offset by L-NAME pretreatment, suggesting that uncoupled eNOS is partly the source for superoxide. 20-HETE had no effect on ionophore-stimulated eNOS phosphorylation; however, it markedly inhibited HSP90 association with eNOS, a critical step in eNOS activation. Hence, removal of this association by 20-HETE may underlie the mechanism by which increased CYP4A expression and activity cause endothelial dysfunction. NIH grant HL34300