ИСТИНА

It is well known that ACh secretion in mature mammalian motor synapses is triggered by Ca2+ influx into nerve terminals mainly through voltage-dependent Ca2+ channels of the P/Q-type [1]. Nerve terminals possess also Ca2+ channels of L-type – normally “silent”, but when activated, they provide additional Ca2+ influx which leads to increase of quantal content of single evoked endplate potentials [2,3]. In many excitable cells, Ca2+ influx through L-type Ca2+ channels activates ryanodine receptors (RyRs) of calcium stores, thus elevating intracellular concentration of calcium. So, the aim of this study was to check the possible functional coupling between activation of L-type Ca2+ channels, subsequent release of stored calcium through RyRs and evoked mediator secretion. The cut in vitro neuromuscular preparation of mouse left hemidiaphragm and intracellular microelectrode recordings were used in order to analyse rhythmically (50Hz) evoked endplate potentials (EPPs) and spontaneous miniature endplate potentials. Statistical analysis was performed using a Mann-Whitney test; all data are presented as mean±S.E.M. Pharmacological disinhibition of L-type Ca2+ channels (directly – by their agonist S(-) BAY K 8644 (1 microM), or indirectly – by blocker of Ca2+-activated K+ channels -iberiotoxin (100 nM)) was found to greatly potentiate EPP amplitudes (due to increase of quantal content of each single EPP in the train up to 41±8% (p<0.05, n=25) or 34±10%(p<0.05, n=25), respectively) in the train during rhythmic synaptic activity – up to 10 mV. Furthermore, these drugs additionally augment the facilitation phase up to 10-12% in the beginning of EPPs train. This upregulation of mediator secretion was completely prevented by preapplied nifedipine (10 microM) or verapamil (5 microM) – blockers of L-type Ca2+ channels (they weren’t able to affect amplitude or quantal content of EPPs by themselves). Blockade of intraterminal RyRs with ryanodine (2 microM) diminished the facilitation in EPPs train during rhythmic activity: from 110±1.5 in control to 103,3±1.7 (p<0.05, n=32). Moreover, the described effects of S(-) BAY K 8644 and iberiotoxin were totally prevented by primarily application of ryanodine. The obtained data indicate that potentiation of mediator release after involving of L-type Ca2+ channels in activity is not due to increased Ca2+ influx through these channels itself, but is mediated by activation of presynaptic RyRs and mobilization of stored calcium. So, activation of RyRs is a necessary step to facilitate rhythmically evoked transmitter release in mouse motor synapses after increased external Ca2+ influx through L-type Ca2+ channels.