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

Abstract

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+]ireversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]iwas 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+content. Tetanic stimulations (100 Hz) increased [Ca2+]ifrom 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+]ito a peak of 3520 ± 540 nM (n= 8). Both force and [Ca2+]ilevels (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 30 mM 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.