A CA2-INDUCED CA2+ RELEASE MECHANISM INVOLVED IN ASYNCHRONOUS EXOCYTOSIS AT FROG MOTOR-NERVE TERMINALS()

The extent to which Ca2+-induced Ca2+ release (CICR) affects transmitt er release is unknown. Continuous nerve stimulation (20-50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequ ency (MEPP-hump) and intracellular free Ca2+ ([Ca2+](i)) in presynapti c terminals (Ca2+-hump) in frog skeletal muscles over a period of minu tes in a low Ca2+, high Mg2+ solution. Mn2+ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by ope ning of voltage-dependent Ca2+ channels. MEPP-hump depended on extrace llular Ca2+ (0.05-0.2 mM) and stimulation frequency. Both the Ca2+- an d MEPP-humps were blocked by 8-(N,N-diethylamino) octyl3,4,5-trimethox ybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enha nced by CN-. Thus, Ca2+-hump is generated by the activation of CICR vi a ryanodine receptors by Ca2+ entry, producing MEPP-hump. A short inte rruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP fre quency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous o r higher level. Thus, the steady/equilibrium condition balancing CICR and Ca2+ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP -hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca2+-hump affected little MEPP frequency or [Ca2+](i), but caused a quick increase (faster than MEPP - or Ca2+-hump) after the pause, whose magnitude increased with an inc rease in pause duration (<1 min), suggesting that Ca2+ entry-dependent inactivation, but not depriming process, explains the decay of the hu mps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endo wed with Ca2+ entry-dependent slow priming and fast inactivation mecha nisms, as well as Ca2+ entry-dependent activation, and involved in asy nchronous exocytosis. Physiological significance of CICR in presynapti c terminals was discussed.