Abstract 3629: Crucial Role of Nitric Oxide Synthases System in Endothelium-Dependent Hyperpolarization in Mice
We have previously demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in mouse and human mesenteric arteries and porcine coronary microvessels. We also have demonstrated that endothelial NO synthase (eNOS) is a major source of EDHF/H2O2, where Cu,Zn-superoxide dismutase (SOD) plays an important role to dismutate eNOS-derived superoxide anions to EDHF/H2O2 in animals and humans. However, the mechanism for the endothelial production of H2O2 as an endogenous EDHF remains to be elucidated. Indeed, some EDHF-mediated responses still remain in singly eNOS−/− mice and the remaining responses are also sensitive to catalase that dismutates H2O2 to form water and oxygen. It is widely known that 3 NOS isoforms (neuronal, inducible, and endothelial) compensate each other. In this study, we examined the effects of genetic disruption of all NOS isoforms (n/i/eNOS−/−) on EDHF responses in mice. We examined the contribution of the whole NOS system to EDHF-mediated responses in eNOS−/−, n/eNOS−/− and n/i/eNOS−/− mice that we have recently generated. Isometric tensions and membrane potentials were recorded by organ chamber experiments and microelectrode technique, respectively. EDHF-mediated relaxations and hyperporalizations in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased (n=6 each), whereas vascular smooth muscle functions were preserved (n=6 each). Expressions of endothelial NOS isoforms in the NOSs−/− mice were compensated by NOS gene that had not been disrupted (n=5 each). Laser confocal microscopic examination demonstrated that endothelial H2O2 and superoxide production was absent in n/i/eNOS−/− mice (n=3–5), whereas antihypertensive treatment with hydralazine failed to improve the EDHF-mediated responses (n= 4). Involvement of NOS uncoupling was ruled out as modulation of BH4 synthesis had no effects (n=6–7) and BH4/BH2 ratio (an index of BH4 bioavailability) was preserved (n=4). These results provide the first direct evidence that EDHF-mediated responses are totally dependent on endothelial NOSs system in mouse mesenteric arteries.