The contribution of the sarcoplasmic reticulum Ca2+ transport ATPase to caffeine induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca2+-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca2+ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca2+ concentration of 0.36 mu M to load the SR with Ca2+. Release of Ca2+ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = -log(10)[Ca2+]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca2+ transient. The calculated Ca2+ transients were fitted by a double exponential function: Y-0 + A(1) . exp (- t/t(1)) + A(2) . exp(t/t(2)), with A(1) < 0 < A(2), t(1) < t(2) and Y-0, A(1), A(2) in micromolar. Ca2+ transients in the presence of the SR Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca2+-ATPase during caffeine-induced Ca2+ release causes an increase in the peak Ca2+ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 +/- 3 mu M CPA and a Hill slope of 1.1 +/- 0.2, saturating at 100 mu M. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. III terms of this model, the SR Ca2+-ATPase influences the Ca2+ gradient across the SR membrane in particular during the early stages of the Ca2+ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca2+ into the bathing solution.