Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+](i)) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+](i) reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+](i) was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+](i) from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+](i) to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+](i) levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca2+-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.