Nitric Oxide Inhibits Flow-Mediated Dilation In Human Coronary Arterioles By Regulating Mitochondrial Production Of Reactive Oxygen Species

We have shown that flow-mediated dilation (FMD) in human coronary arterioles (HCA) with coronary artery disease (CAD) occurs mainly by liberation of reactive oxygen species (ROS), specifically hydrogen peroxide (H 2 O 2 ), acting as an endothelial derived hyperpolarizing factor (EDHF). NO potently inhibits EDHF in vessels where the EDHF is an eicosanoid derived from cytochrome P450, but in HCA, mitochondria are the primary source of the EDHF. NO can alter mitochondrial ROS electron transport, but it is not clear if NO modulates shear-induced mitochondrial production of hydrogen H 2 O 2. We tested the hypothesis that NO inhibits FMD in HCA by scavenging O 2 · − to reduce mitochondrial production of H 2 O 2 through the formation of ONOO − . Methods: HCAs were obtained from patients undergoing cardiac surgery. HCAs were then prepared for study by histofluorescence (HF), immunohistochemistry (IHC), and videomi-croscopic techniques. Results: Enhanced fluorescence intensity was observed in HCAs exposed to flow. This enhancement was diminished through the addition of the NO donor, spermine nonoate (SPNO, 1x10 −8 M) (no flow: 1±0, vs flow: 1.6±0.3 vs flow+SPNO: 0.5±0.1, avg. intensity ratios normalized to no flow, n=5), suggesting that NO reduces flow mediated release of mitochondrial O 2 · − . Concurrent formation of ONOO − was confirmed using IHC with antibodies directed against nitrotyrosine (NT) in HCA treated with SPNO or Sin-1 compared with untreated arterioles, indicating the increased production of ONOO − through NO scavenging of O 2 · − . FMD was markedly reduced in HCA in the presence of SPNO compared to controls (%Max: 10±6 Vs 73±6, respectively, n=6). Furthermore, addition of the peroxynitrite donor, Sin-1 (1x10 −6 M), abolished FMD in HCA (%Max: −20±4 Vs 66±3, respectively, n=6), suggesting the importance of ONOO − in FMD inhibition. Finally, the endothelium-dependent agonist, bradykinin (BK), was used to prove viability in the presence of SPNO or Sin-1 (%Max: C: 54±3 Vs SPNO: 49±6 or C: 79±10 Vs Sin-1: 70±6, n=5). Taken together, these results suggest that NO can inhibit FMD in HCA likely through the scavenging of O 2 · − with resultant formation of ONOO − which reduces the mitochondrial ROS generation and reduces production of the EDHF, H 2 O 2 .