Department of Human and Animal Physiology, Biological Faculty, Lomonosov Moscow State University, Moscow, Russia.
Department of Physiology, Russian National Research Medical University, Moscow, Russia.
Brain Behav. 2018 Aug;8(8):e01058. doi: 10.1002/brb3.1058. Epub 2018 Jul 6.
The 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.
Using 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.
Ryanodine (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 . 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.
We 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.
本研究旨在确定作为肌浆网钙通道受体激动剂的ryanodine 引起小鼠运动神经末梢微小终板电位 (MEPPs) 幅度增加的机制。
使用细胞内微电极记录 MEPPs 和诱发的终板电位 (EPPs)，研究了小鼠膈肌运动神经末梢运动突触中自发性和诱发的乙酰胆碱释放的变化。
ryanodine（0.1 μM）使 MEPPs 和 EPPs 的幅度均增加到相似程度（与对照相比增加了 130%）。降钙素基因相关肽 (CGRP) 的受体被截断肽 CGRP 阻断后，ryanodine 的作用被阻止。内源性 CGRP 储存在运动神经末梢的大致密核心囊泡中，可能作为共递质释放。用 vesamicol 抑制囊泡乙酰胆碱转运体或用 H-89 阻断蛋白激酶 A 的活性，可完全阻止 ryanodine 诱导的 MEPPs 幅度增加，表明内源性 CGRP 是在ryanodine 受体激活后释放的。CGRP 受体的激活反过来又可以增加乙酰胆碱进入突触囊泡的装载，从而增加量子大小。内源性 CGRP 活性的这个新特征类似于最近描述的外源性 CGRP 在小鼠运动突触中的作用。Ca/钙调蛋白依赖性激酶 II (CaMKII) 抑制剂 KN-62 或 KN-93 可阻止 ryanodine 作用。CaMKII 的抑制不能阻止外源性 CGRP 引起的 MEPPs 幅度增加。
我们提出，突触前 CaMKII 的活性对于 ryanodine 刺激内源性 CGRP 从运动神经末梢释放是必需的，但是 CaMKII 不参与 CGRP 受体激活后紧随其后的量子大小增加的信号转导。
