Modulation of nitric oxide synthase isoenzymes in reperfused skeletal muscle

OBJECTIVE: To investigate the modulation of nitric oxide synthase (NOS) isoenzymes in skeletal muscle during 3 h ischemia/reperfusion (I/R, 3 h ischemia followed by 3 h reperfusion). METHODS: The extensor digitorum longuses (EDLs) from 20 adult rats were divided into 4 groups: the normal, the sham operation, the ischemia (3 h), and the ischemia/reperfusion group. One normal EDL from each rat was used as the non-operated control, and the opposite ones are distributed into the 3 remaining groups. All the samples were studied with Western blotting technique and immunohistochemistry staining. RESULTS: Three sizes of protein bands verified with the proteins of relative molecule to be of 155000, 140000 and 135000, were detected in the EDL homogenate by Western blotting, which were comparable with the positive controls for nNOS, eNOS and iNOS, respectively. Immunostaining demonstrated that nNOS was present in the muscle fiber, with a similar location of the muscle stria, eNOS was found apparently in microvascular endothelia, but not found in muscle fibers, and iNOS was found in the leukocytes around the muscle fiber and some endothelia cells. Immunostaining paralleled the Western blotting results. CONCLUSIONS: It suggests that the constitutive nNOS and eNOS protein can be regulated by I/R, and I/R results in a down regulation of nNOS and up-regulation of eNOS and iNOS in reperfused skeletal muscle. The fact that nNOS is present around stria suggests that nNOS may have a close relationship with muscle function. The localization of eNOS in endothelial cell indicates its role in regulating blood supply of the muscle. Based on these findings, it is possible that NO produced by distinct NOS may play a different role in I/R injury.