| Co-Investigator(Kenkyū-buntansha) |
YAMASHITA Nobushige Osaka University Affiliated Hospital., Medical Staff, 医学部・附属病院, 医員
NISHIDA Masashi Osaka University Medical School., Assistant Professor, 医学部, 助手 (40283783)
HOSHIDA Shiro Osaka University Medical School., Assistant Professor, 医学部, 助手 (80238732)
TADA Michihiko Osaka University Medical School., Professor Medicine, 医学部, 教授 (90093434)
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| Research Abstract |
We studied whether or not nitric oxide (NO) produced by cardiac myocytes augments myocardial injury on ischemia-reperfusion using a hypoxia-reoxygenation model of cultured cardiac myocytes. Rat neonatal cardiac myocytes were cultured under hypoxic atmosphere (O2=1%, 3h), followed by reoxygenation (O2=20%, 1h). Myocyte cultures exhibited basal nitrite production on the addition of 4mM L-Arginine (L-Arg) (2.1 (]SY.+-[) 0.2 vs.6.2 (]SY.+-[) 0.3 nmol/mg protein), which resulted in significant augmentation in creatine kinase (CK) release (14.5 (]SY.+-[) 2.3 vs.31.2 (]SY.+-[) 3.8 mU/mg protein). Treatment with interleukin 1beta (IL-1 beta, 24h) increased the levels of inducible NO synthase (iNOS) mRNA and protein. The addition of L-Arg to IL-1b treated myocytes markedly increased nitrite production (3.1 (]SY.+-[) 0.2 vs.23.7 (]SY.+-[) 0.6 nmol/mg protein) and CK release (14.4 (]SY.+-[) 0.9 vs.45.1 (]SY.+-[) 6.0 mU/mg protein). The addition of 8-bromo-3', 5'-cyclic-guanosine-monophosphate did not augment CK release by myocyte cultures after hypoxia-reoxygenation. However, the activity of glutathione peroxidase (GPX), an intrinsic reactive oxygen species scavenger of myocytes, was significantly attenuated [U/mg protein : 41.6 (]SY.+-[) 3.8 (without L-Arg or IL-1beta) vs.32.3 (]SY.+-[) 1.8 (with L-Arg, but without IL-1 beta), 26.4 (]SY.+-[) 1.6 (with L-Arg and IL-1 beta)] together with nitrite production. Nw-nitro-L-arginine-methyl-ester, an NOS inhibitor, inhibited the attenuation of GPX activity (40.2 (]SY.+-[) 8.6 U/mg protein), resulting in decrease in CK release (21.6 (]SY.+-[) 3.9 mU/mg protein). These results suggest that NO derived from cardiac myocytes augments myocardial injury at ischemia-reperfusion not through guanylate cyclase ativation, but through inactivation of GPX.
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