Role of superoxide production by NOS in skeletal muscles in the development of shock after ischemia/reperfusion.

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15590582
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Legal medicine
• Research Institution: Fukushima Medical University
• Principal Investigator: KURISAKI Emiko Fukushima Medical University, Dept of Legal Medicine, Lecturer, 医学部, 講師 (30106356)
• Co-Investigator(Kenkyū-buntansha): ADACHI Junko Kobe University, Graduate School of Medicine, Associate professor, 大学院・医学研究科, 助教授 (40030887)
• Project Period (FY): 2003 – 2004
• Keywords: ischemia / repeefusion / shock / nitric oxide synthase (NOS) / superoxide / cholesterol peroxides / histochemistry / HPLC / HPLC

Research Abstract

To clarify the role of superoxide (O_2-) produced by NOS after ischemia/reperfusion (I/R), a hind limb tourniquet ischemia animal model was used. A rubber band was applied to the inguinal region of the hind limb for 3 hours under isoflurane anesthesia followed by reperfusion. O_2- production in skeletal muscle cells was observed in mice using an in situ detection technique with hydroethidine. Five minutes after the start of reperfusion, burst production of O_2- was detected in the cytoplasm as well as on and/or near the membrane of muscle cells subjected to I/R. Remarkably increased NADPH diaphorase activiry, and immunoreactivities using an anti-NOS antibody and anti-4-hydroxy-nonenal (HNE) antibody were also observed in I/R muscle, whereas there was no significant increase of immunoreadrivity using anti-nitrotyrosine (NT). These findings suggested that O_2- production by NOS as well as xanthine oxides was involved in the mechanism of O_2- production after reperfusion. Positive imunoreactivity using anti-HNE was observed in the liver and the kidney and immunoreactivity using anti-NT was positive in the heart after reperfusion. Cholesterol peroxides and NO metabolites were determined by HPLC. Levels of cholesterol peroxides increased in the muscle subjected to I/R and in the kidney after reperfusion. Levels of NO metabolites in plasma also increased between 15 and 60 min after reperfuiion. HPLC results were consistent with histochemical findings. These results suggested that O2-and/or NO produced by NUS was responsible for not only local injury but also remote organ injury after I/R. In the present study, we showed that O_2- production by NOS plays a role as an initiator of the development of shock after I/R.