Abstract 1306: Endothelium-Dependent Hyperpolarizations Are Totally Dependent on Endothelial Nitric Oxide Synthases System -Lessons from Mice Lacking All Nitric Oxide Synthase Isoforms
Background: We have previously demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) and that endothelial nitric oxide synthase (eNOS) is a major source of EDHF/H2O2, where Cu,Zn-superoxide dismutase (SOD) plays an important role as an EDHF synthase by dismutating eNOS-derived superoxide to EDHF/H2O2. However, some EDHF-mediated responses still remain in singly eNOS−/− mice and the remaining responses are also sensitive to catalase. It is widely known that three NOS isoforms (neuronal, inducible, and endothelial NOS) compensate each other. In this study, we thus examined the contribution of the whole NOS system to EDHF-mediated responses in mice lacking all three NOS isoforms (triply NOSs-/- mice) that we have recently developed.
Methods and Results: We used male wild-type (WT), doubly n/eNOSs−/− and triply NOSs−/− mice (10–16 weeks-old). Isometric tensions and membrane potentials were recorded by organ chamber experiments and microelectrode technique, respectively. Systolic blood pressure was significantly higher in the doubly and triply NOSs−/− mice than in the WT mice. In the WT mice, endothelium-dependent relaxations to acetylcholine of the aorta were markedly inhibited by Nω-nitro-L-arginine (L-NNA, 10−4 M) (n=7), whereas those of mesenteric arteries were resistant to indomethacin (10−5 M) and L-NNA but were highly sensitive to the combination of charybdotoxin (10−7 M) plus apamin (10−6 M) (n=7), indicating a primary role of EDHF. Importantly, EDHF-mediated responses (in the presence of 10−5 M indomethacin and 10−4 M L-NNA) were significantly reduced in the doubly NOSs−/− mice compared with the WT mice and were totally absent in the triply NOSs−/− mice (n=4–7). In the triply NOSs−/− mice, endothelium-dependent hyperpolarizations also were markedly reduced (n=4), whereas endothelium-independent relaxations to sodium nitroprusside, (an NO donor) and NS1619 (a direct opener of KCa channels) were preserved (n=7 each). The absence of EDHF-mediated responses also was noted in female triply NOSs−/− mice (n=6).
Conclusions: These results provide the first evidence that EDHF-mediated responses are totally dependent on the endothelial NOS system in both genders.