It has been extensively reported that free radical species are generated by contracting skeletal muscle following different exercise protocols (O’Neill et al. 1996; Reznick et al. 1996; McArdle et al. 2001), including non-damaging contractions (McArdle et al. 2001). However, numerous non-muscle cells are found in skeletal muscle in vivo and the presence of these cells has complicated the interpretation of these data.

Previous studies have shown that skeletal muscle cell cultures, which were electrically stimulated to contract, are a major source of superoxide radicals (McArdle et al. 2001). However, the site of production of these species remains unresolved. Production of superoxide release has been postulated to be from a number of sources (O’Neill et al. 1996; McArdle et al. 2001).

The aim of this study was to determine whether free radical species are generated in stimulated skeletal muscle myotubes in response to non-damaging contractions and to examine the possible source of this increased production of free radicals. H₂K-derived mouse myotubes (Harris et al. 1999) were electrically stimulated to contract in culture for 15 min (50 Hz frequency with pulses of 2 ms duration and 30 V well^-1) using platinum electrodes. The generation of hydroxyl and superoxide radicals was determined as previously described (McArdle et al. 2001; Pattwell et al. 2001). Data were analysed with Student’s unpaired t test, n = 12, and data are presented as means ± S.E.M.

Data indicate that 15 min of stimulation resulted in significant increases (P < 0.05) in superoxide (36 ± 6 cf. control 1 ± 0.01 nmol (15 min)^-1) and hydroxyl (0.60 ± 0.09 cf. 0.04 ± 0.01 nmol (15 min)^-1) radical production. Stimulation of the myotubes in the presence of the nitric oxide inhibitor, L-NAME, resulted in a further increase in superoxide production (45 ± 7 nmol (15 min)^-1, P < 0.05). Superoxide production was significantly decreased (P < 0.05) by cells that were stimulated to contract in the presence of diphenyleneiodium (DPI), an NADPH oxidase inhibitor or in the presence of superoxide dismutase (SOD) (DPI: 19.7 ± 4.3 nmol (15 min)^-1; SOD: 24 ± 6.7 nmol (15 min)^-1), respectively. These data indicate that free radical species are released by skeletal muscle cells during contraction and indicate possible sites of production of these radical species.

H₂K cells were a kind gift from Professor T.A. Partridge and Dr J. Morgan, Imperial School of Medicine, Hammersmith Hospital, London, UK. This work was supported by The Wellcome Trust.