Abstract 1302: NADPH Oxidase-Derived Reactive Oxygen Species Reduce Cytochrome P450-Mediated Dilation of Human Coronary Arterioles
Coronary artery disease (CAD) is characterized by excess generation of reactive oxygen species (ROS) that reduce bioavailability of the vasodilator nitric oxide (NO). Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived vasodilator metabolites of arachidonic acid (AA) that can compensate for loss of NO; however, the effect of ROS on EET formation is unclear. We investigated whether NADPH oxidase, a source of vascular ROS, modulates P450-mediated dilation in the coronary circulation of patients with CAD. Arterioles (max. diameter 146±12 μm) from atrial appendages were cannulated, pressurized, and diameter changes were measured by videomicroscopy in the presence of N-nitro-L-arginine methyl ester (10-4 M, nitric oxide synthase inhibitor) and indomethacin (10-5 M, cyclooxygenase inhibitor). Relative production of superoxide and H2O2 was determined using the fluorescent dyes HE and DCFH-DA, respectively. Metabolism of 14C-AA by human P450 2C9-expressing microsomes was assayed by HPLC. In vessels constricted 30–50% with endothelin-1, bradykinin (BK) induced a concentration-dependent dilation that was enhanced by apocynin or diphenyliodonium (3 mM and 5 μM, NADPH oxidase inhibitors, 50±10% vs. 88±6% and 77±9% vs. 97±1% at 10−7 M, n = 6 and 5, respectively, p<0.05). BK-induced dilation was partially reduced by 17-octadecynoic acid (17-ODYA, 10−5 M, P450 inhibitor, 78±13% vs. 62±10% at 10−7 M, n = 6, p=ns); however, in the presence of apocynin, 17-ODYA abolished BK-induced dilation (85±10% vs. 20±13% at 10−7 M, n = 6, p<0.05). Apocynin did not alter endothelium-independent dilation to papaverine. In vessels incubated with HE and DCFH-DA, BK induced superoxide and H2O2 formation (fluorescence ratio vs. vehicle 1.54±0.09 and 2.50±0.34, respectively, n = 5, p<0.05) that was prevented by apocynin or endothelial denudation (0.90±0.18 and 0.59±0.20, 0.81±0.16 and 0.83±0.13, respectively, n = 5 and 4, p<0.05 vs. BK alone). Microsomal metabolism of 14C-AA to EETs was diminished in the presence of H2O2 (10−5 M), as observed by HPLC. We conclude that NADPH oxidase-derived ROS inhibit the P450-mediated component of BK-induced dilation. These findings suggest that an inhibitory interaction exists between EETs and ROS in the human coronary circulation.