In skeletal muscle fibres, potassium contractures have been widely used as a convenient experimental model for the study of depolarisationÐcontraction coupling. It is generally recognised that the voltage dependence of tension during steady-state depolarisation depends on activation of the voltage sensor in transverse tubule membrane. Moreover, the slow decay in tension during prolonged depolarisation is assumed to depend exclusively on inactivation of excitation-contraction coupling (Dulhunty, 1991). Disulfiram was found to stimulate the sarcoplasmic reticulum Ca²⁺-ATPase activity of skeletal muscle (Starling et al. 1996). Thus the aim of the present study was to use disulfiram to estimate the role of Ca²⁺ uptake by the sarcoplasmic reticulum on the activation and inactivation of steady-state tension in mammalian slow-twitch muscle.

The experiments were performed at 19Ð20 °C on soleus small bundles (2Ð4 cells) isolated from adult male Wistar rats anaesthetised by an ether vapour flow and then killed by cervical dislocation. Preparations were mounted in the experimental chamber as described by Joumaa et al. (2002). The application of an elevated potassium concentration (20Ð146 mM [K⁺]_o) allowed us to obtain the activation curve, and the inactivation curve of [K⁺]_o contracture was obtained by measuring test 146 mM [K⁺]_o contracture amplitude after submaximal depolarisation for 2 min in a conditioning [K⁺]₀. In this solution, the [K⁺][Cl^–] product was kept constant to allow rapid recovery of resting membrane potential and restoration of the amplitude of tension response. Membrane potentials were measured in the usual way (Joumaa et al. 2002). The values of membrane potential for half-maximal activation (E_a) and inactivation (E_i) of contraction were obtained by fitting the Boltzman equation for each fibre in the experiment.

Disulfiram (3.1Ð62.5 mM) reversibly reduced the amplitude (control: 1.5 ± 0.3 mN, 27 mM disulfiram: 1.0 ± 0.1 mN, n = 12, P < 0.05, mean ± S.E.M., Student’s paired t test), the time to peak tension (control: 14.1 ± 0.5 s, 27 mM disulfiram: 9.8 ± 0.8 s, n = 12, P < 0.05) and the time constant of relaxation (control: 8.5 ± 0.2 s, 27 mM disulfiram: 5.9 ± 0.3 s, n = 12, P < 0.05) of 146 mM [K⁺]_o contracture. In the presence of 27 mM disulfiram, the relationship between the amplitude of potassium contractures and the membrane potential was shifted to less negative potentials (control: E_a = -43.5 ± 1.1 mV, disulfiram: E_a = -29.7 ± 2.3 mV, n = 8, P < 0.05), whereas the steady-state inactivation curve was unchanged (control: E_i = -39.9 ± 0.6 mV, disulfiram: E_i = -38.6 ± 1.7 mV, n = 8, P < 0.05), suggesting that disulfiram has no effect on voltage sensor. The difference presently found between potassium contractures in the absence and presence of disulfiram implies that, as previously reported in frog muscle (Mme & Léoty 1999), the peak amplitude and the slow relaxation of tension during prolonged steady-state depolarisation depends not only on inactivation of the process regulating Ca²⁺ release from the sarcoplasmic reticulum, but also on the ability of the sarcoplasmic reticulum to pump Ca²⁺.

All procedures accord with current National guidelines.