Abstract 1288: 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 in animals and humans 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 to synthesize EDHF/H2O2 by dismutating eNOS-derived superoxide anions. However, some EDHF-mediated responses remain in singly eNOS−/− mice and are also sensitive to catalase. It is widely known that 3 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 3 NOS isoforms (triply NOSs−/− mice) that we have recently developed.
Methods and Results: We used male wild-type (WT) 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 triply NOSs−/− mice than in the WT mice. In the WT mice, endothelium-dependent relaxations to acetylcholine (ACh) 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 were totally absent in the triply NOSs−/− mice, and were not improved by hydralazine. In the triply NOSs−/−mice, endothelium-dependent hyperpolarizations were also markedly reduced (n=6), whereas endothelium-independent relaxations to sodium nitroprusside were significantly enhanced and those to NS-1619 (a direct opener of KCa channels) were preserved (n=7 each). Laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that endo-thelial H2O2 production in response to ACh was markedly reduced in triply NOSs−/− mice. Furthermore, the inhibition of intracellular sources of superoxide anions other than eNOS did not affect EDHF-mediated responses in WT mice.
Conclusions: These results provide the first evidence that EDHF-mediated responses are totally dependent on the endothelial NOSs system.