Activation of the nicotinic acetylcholine receptor mobilizes calcium from caffeine-insensitive stores in C2C12 mouse myotubes

In cultured mouse C2C12 myotubes, digital Ca²⁺ imaging fluorescence microscopy using the acetoxymethyl ester of Fura-2, Fura-2-AM, showed that, in the absence of extracellular Ca²⁺, acetylcholine (ACh) and nicotine, but not muscarine, raised the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) by about tenfold. AChinduced Ca²⁺ mobilization was prevented by thapsigargin, a drug known to deplete inositol 1,4,5-trisphosphate (InsP₃)-sensitive stores, and was concomitant with InsP₃ accumulation. Caffeine, which releases Ca²⁺ from the ryanodine-sensitive stores of the sarcoplasmic reticulum, did not interfere with the ACh-induced [Ca²⁺]_i increase. Ca²⁺ mobilization was also inhibited when myotubes were depolarized by high K⁺, or when extracellular Na⁺ was omitted. Nicotinic ACh receptor (nAChR) stimulation lowered intracellular pH with a time course slower than the [Ca²⁺]_i increase. Possible mechanisms linking the current flowing through the nAChR pore to [Ca²⁺]_i increase are discussed.