The 2003 Annual Meeting of OASYS_NEW

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Expression and Distribution of Nos Gene and Protein in Reperfused Skeletal Muscle

Qi WN1, Chen LE1, Seaber AV1, and Urbaniak JR2. (1) Surgery, Duke University, PO Box 3093, Durham, NC, USA, (2) Division of Orthopaedic Surgery, Duke University Medical Center, Box 2912, Durham, NC, USA

Introduction. Our previous studies suggest an ischemia/reperfusion (I/R)-induced NOS change in skeletal muscle. However, it has not been documented which of the three NO synthesis pathways (neuronal, inducible, and endothelial NOS, i.e. nNOS, iNOS, and eNOS) is involved and at what level this change happens. This study was designed to identify gene and protein expression of individual NOS isoenzymes and to characterize their distribution in the rat skeletal muscle subjected to I/R injury. Methods. One side of the extensor digitorum longus (EDL) muscles from 20 adult rats underwent 3h/3h (I/R). The opposite EDL was used as a control. The opposite EDL muscle of each rat was used as a normal control. Quantitative RT-PCR and Western blot technique was used to determine expression of NOS mRNA and protein. The relative expression of NOS mRNA and protein in the reperfused EDL muscle was determined by comparing with the normal (as a 100%). Statistical significance was determined by ANOVA. Results. RT-PCR showed that neutonal (n-), inducible (i-), and endothelial (e-) NOS mRNAs were each expressed in the normal EDL. I/R up-regulated nNOS to 116±22% (mean±SD) of normal and iNOS to 5682±180%, while eNOS remained at normal level (100±22%) in the EDL. There was significant upregulation in iNOS mRNA. Western blot detected greater eNOS (136±28%) and iNOS (384±137%), but less nNOS (63±15%) in the I/R EDL, with a significant difference when compared to normal. Immunolocalization showed nNOS antigens were prominent in the muscle sarcolemma in both normal and reperfused EDLs and also in the cytoplasm in reperfused EDL. eNOS expressed in vascular endothelial cells in normal and reperfused EDL, and also expressed in the cytoplasm of some muscle fibers in reperfused EDL. iNOS antigens were not found in normal EDL, but were present in the muscle cytoplasm in reperfused EDL. Discussion. The results demonstrated that I/R mediated not only expression changes at transcriptional and translational levels, but also distribution changes of proteins of all three NOS isoforms in rat skeletal muscle. Because each NOS has distinct function, these changes may imply the mechanisms of I/R injury. Huge increased iNOS mRNA and protein during reperfusion could affect many cellular functions to lead to cellular damage or necrosis. It is conceivable that an understanding of this modulation on a cellular and molecular level under different conditions may lead to understanding of I/R injury observed in vivo and improvement of clinical outcome of reperfusion injury.