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 commer cial preparations of the muscle relaxant succinylcholine, on intracellular free calcium levels in cultivated mouse myotubes was studied. Calcium signa ls were monitored with an inverted microscope equipped for fluorescence pho tometry 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 susta ined one, could be regularly discriminated. First, with a delay of 2 s, the intracellular calcium concentration increased from 41 +/- 13 to 541 +/- 31 9 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 qui ckly 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. A t 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. Replacemen t of extracellular calcium by EGTA and application of calcium channel block ers revealed that, for both the transient and the sustained response, calci um was released from intracellular stores. Pre-treatment with thapsigargin (0.1 mu M) or ryanodine (10 mu M) abolished both signal components. Repeate d short-term applications of 4-CmC suggest that the two components may aris e from different systems.