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.