Abstract
ObjectiveThe aim of this study was to identify the mechanism responsible for an increase in miniature endplate potentials (MEPPs) amplitude, induced by ryanodine as an agonist of ryanodine receptors in mouse motor nerve terminals.MethodsUsing intracellular microelectrode recordings of MEPPs and evoked endplate potentials (EPPs), the changes in spontaneous and evoked acetylcholine release in motor synapses of mouse diaphragm neuromuscular preparations were studied.ResultsRyanodine (0.1 μM) increased both the amplitudes of MEPPs and EPPs to a similar extent (up to 130% compared to control). The ryanodine effect was prevented by blockage of receptors of calcitonin gene‐related peptide (CGRP) by a truncated peptide CGRP 8‐37. Endogenous CGRP is stored in large dense‐core vesicles in motor nerve terminals and may be released as a co‐transmitter. The ryanodine‐induced increase in MEPPs amplitude may be fully prevented by inhibition of vesicular acetylcholine transporter by vesamicol or by blocking the activity of protein kinase A with H‐89, suggesting that endogenous CGRP is released in response to the activation of ryanodine receptors. Activation of CGRP receptors can, in turn, upregulate the loading of acetylcholine into synaptic vesicles, which will increase the quantal size. This new feature of endogenous CGRP activity looks similar to recently described action of exogenous CGRP in motor synapses of mice. The ryanodine effect was prevented by inhibitors of Ca/Calmodulin‐dependent kinase II (CaMKII) KN‐62 or KN‐93. Inhibition of CaMKII did not prevent the increase in MEPPs amplitude, which was caused by exogenous CGRP.ConclusionsWe propose that the activity of presynaptic CaMKII is necessary for the ryanodine‐stimulated release of endogenous CGRP from motor nerve terminals, but CaMKII does not participate in signaling downstream the activation of CGRP‐receptors followed by quantal size increase.
Highlights
We have found that ryanodine-stimulated calcium release from intracellular calcium stores initiates a calcium-d ependent increase in miniature endplate potentials (MEPPs) amplitude and ACh quantal size in mouse neuromuscular junctions (NMJs) (Skiteva, Lapteva, & Balezina, 2012)
We found that the ryanodine-induced increase in MEPP amplitude and ACh quantal size is associated with the release of endogenous calcitonin gene- related peptide (CGRP)
We found that none of these drugs affected MEPP amplitude during their application for 90 min (Figure 5a,b,d,e)
Summary
We have found that ryanodine-stimulated calcium release from intracellular calcium stores initiates a calcium-d ependent increase in MEPP amplitude and ACh quantal size in mouse neuromuscular junctions (NMJs) (Skiteva, Lapteva, & Balezina, 2012). Both the mechanism of this ryanodine effect and possible ryanodine influence on the multiquantal evoked ACh release remain unknown. The aim of this study was to test the hypothesis that ryanodine can trigger calcium-dependent release of CGRP from motor nerve terminals of mice and further presynaptic action of endogenous peptide may underlie the increase in MEPP amplitude and ACh quantal size. We found for the first time that endogenous CGRP, which was released into synaptic cleft of motor synapses, may act presynaptically stimulating the loading of ACh into synaptic vesicles
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