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Source of superoxide production in mammalian skeletal muscle at elevated temperature

C. Van Der Poel, D.G. Stephenson, Zoology Department, La Trobe University, Melbourne, VIC, Australia

It is well established that at normal physiological temperature there is a marked increase in the production of superoxide (O2-) in skeletal muscle1. However, the major source of O2- production is not clear. For example, there are reports that suggest that O2- production is extracellular while other reports suggest that mitochondria or membrane bound NADH oxidase on the sarcoplasmic reticulum play the major role in O2- production2.

In order to determine whether the main source of O2- production is intracellular or extracellular, different types of muscle fibre preparations in combination with specific mitochondrial inhibitors were used. Long-Evans hooded rats were killed under deep anaesthesia (2% v:v Fluothane), EDL muscles excised, single fibres or intact fibre bundles were prepared and volume measured. O2- was measured using the cytochrome C assay, based on changes in cytochrome C absorbance over the spectrum 540-560nm when cytochrome C becomes reduced by O2- 1.

Results show that removing the surface membrane results in more O2- being measured than when the surface membrane is intact (intact preps produced 40.92 ± 5.01% (n = 5) of membrane removed fibres). This strongly suggests that the source of O2- is mainly intracellular.

Addition of 50µM Rotenone, a mitochondrial inhibitor specific to complex I, caused the amount of O2- produced in skinned fibres to increase by a factor of 8.7 ± 0.7. This suggests that not only is O2- production intracellular but also that the primary source is from the mitochondria.

(1) van der Poel, C. & Stephenson, D.G. (2002) Journal of Physiology 544: 765-776.

(2) Zuo, L., Pasnciuc, S., Wright, V., Merola, A. & Clanton, T. (2003) Antioxidants and Redox Signalling 5: 667-675.