A transient and a persistent calcium release are induced by chlorocresol in cultivated mouse myotubes

The effect of 4-chloro-m-cresol (4-CmC), a stabilizing agent used in commercial preparations of the muscle relaxant succinylcholine, on intracellular free calcium levels in cultivated mouse myotubes was studied. Calcium signals were monitored with an inverted microscope equipped for fluorescence photometry using fura-2 as the calcium indicator. Upon bath application of 500 mu M 4-CmC for 90 s, two separate calcium signals, a transient and a sustained one, could be regularly discriminated. First, with a delay of 2 s, the intracellular calcium concentration increased from 41 +/- 13 to 541 +/- 319 nM, peaked after 2-5 s and declined within 10 s to nearly resting values (n = 36). Then, after a delay of up to 20 s, intracellular calcium rose quickly again to almost the same value and stayed elevated as long as the drug was applied. Upon drug removal, intracellular calcium rapidly decreased to a new level that was always slightly higher than the original base line. At 250 mu M 4-CmC, the response was small, whereas at 500 mu M it was at its maximum. Thus, the concentration-response curve was very steep. Replacement of extracellular calcium by EGTA and application of calcium channel blockers revealed that, for both the transient and the sustained response, calcium was released from intracellular stores. Pre-treatment with thapsigargin (0.1 mu M) or ryanodine (10 mu M) abolished both signal components. Repeated short-term applications of 4-CmC suggest that the two components may arise from different systems.