Breakdown of phospholipids and the elevated nitric oxide are involved in M3 muscarinic regulation of acetylcholine secretion in the frog motor synapse

https://doi.org/10.1016/j.bbrc.2020.01.112Get rights and content

Highlights

  • Muscarine attenuates ACh release from frog motor terminals via activation of PLC.

  • PI3K activity is crucial for ACh secretion recovery after muscarine removal.

  • Muscarine-induced decrease of ACh release is linked to upregulation of NO signaling.

  • CB1 receptors do not contribute significantly to in the depressant effect of muscarine.

  • M3 receptors may modulate ACh secretion via PLC, PI3K and NO-dependent manner.

Abstract

Previously, we found that muscarine downregulates the acetylcholine release at the frog neuromuscular junction acting via M3 muscarinic receptors. Here, the molecular mechanisms underlying the inhibitory effect of muscarine on the quantal secretion of acetylcholine were studied. Inhibition of phospholipase C (with U-73122) prevented the reduction of evoked neurotransmitter release induced by muscarine. Interruption of synthesis of phosphatidylinositol 3-phosphate by the inhibitor of phosphoinositide-3-kinase (wortmannin) did not affect the depressant action of muscarine but precluded the restoration of secretion after removal of muscarine from the bathing solution. The effect of muscarine was not significantly modified by the blockade of endocannabinoid receptors (with AM 281), but it was abolished by the inhibitor of nitric oxide synthase (L-NAME) as well as extracellular nitric oxide (NO) chelator (hemoglobin). Moreover, muscarine increased NO-sensitive dye fluorescence in junctional region, which was prevented by the M3 receptor antagonist 4-DAMP. The data obtained indicate that the attenuation of acetylcholine release mediated by muscarine is associated with a change in the activity of both lipid-metabolizing enzymes and NO synthases.

Graphical abstract

The scheme of ACh secretion regulation by signaling pathways triggered by M3 choline receptor activation(M3AChR - M3 muscarinic receptor, NOS - nitric oxide synthase, EC - endocannabinoids, CB1R–CB1 cannabinoid receptor, IP3 - inositol-3-phosphate, PLC - phospolipase C, PI3K - phosphoinositide-3-kinase).

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Introduction

Muscarinic cholinoreceptors are known to be involved into the modulation of acetylcholine (ACh) secretion in the vertebrate neuromuscular junctions [[1], [2], [3], [4], [5]]. However, the precise molecular pathways of muscarinic regulation are not established so far, and seem to be dependent on a number of experimental conditions such as animal species, age of animals, extracellular Ca2+ level, temperature etc. [1,[4], [5], [6], [7], [8]].

Newman et al. [9] have investigated molecular mechanisms of ACh release regulation mediated by activation of M3 receptors in lizard neuromuscular synapses. It was shown that the activation of phospholipase C (PLC) followed by the rise in the production of endogenous cannabinoids and the activation of presynaptic cannabinoid receptors type 1 (CB1) are the necessary links of this chain. Second essential factor is nitric oxide (NO) which production seems to be enhanced upon M3 receptor activation.

Earlier we have shown that in frog neuromuscular junction the process of ACh secretion is modulated by muscarinic cholinergic receptors of M1, M2/M4 and M3 subtypes [10]. Application of cholinomimetic muscarine caused a significant decrease in the ACh quantal release. Depressing effect of muscarine persisted in the presence of non-selective muscarinic antagonists atropine and gallamine (unpublished data), and selective inhibitors of M1, M2 and M4 cholinergic receptors, but was completely prevented by pre-incubation of nerve-muscle preparation with M3 blockers 4-DAMP and J-104129.

During the past few decades, drugs targeting the muscarinic acetylcholine receptor family, including M3 type receptors, are considered as potential therapeutic tools [11,12]. Therefore, molecular mechanisms underlying M3 receptor dependent signaling is of importance.

Here we studied the molecular mechanisms underlying the action of muscarine on the evoked ACh secretion using EPPs recorded under conditions of low Ca2+/high Mg2+ in bathing solution. This approach allows to analyze not only the EPP quantal content but also the timing of the release by the analysis of real synaptic delay distribution [13].

It was shown that unlike lizard neuromuscular endplates, in frog, the inhibition of ACh secretion in presence of muscarine is associated for the greater part with an increased activity of important lipid-metabolizing enzymes − PLC and phosphoinositide-3-kinases (PI3K) as well as with a change in the activity of NO-synthase (NOS). Optical experiments provided direct evidence that muscarine increases NO production in junctional region, which is associated with the activation of M3 cholinoreceptors.

Section snippets

Ethics statement

Experiments were performed in accordance with the guidelines for the use of laboratory animals of Kazan Medical University, which are in compliance with the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol met the requirements of the EU Directive 2010/63/EU for animal experiments and was approved by the Ethical Committee of Kazan State Medical University. The authors further attest that all efforts were made to minimize the number of animals used and their

Results

As it was reported previously [10], under conditions of reduced extracellular Ca2+, 10 μM muscarine reversibly reduced the quantal content of EPPs by 41.5 ± 3.9% (from 0.33 ± 0.04 in control to 0.18 ± 0.03, n = 12, P < 0.001) and synchronized the secretion process (Fig. 1 A, B, C). These muscarinic effects were prevented by the application of M3 blockers, 4-DAMP and J-104129. In this study, we explored possible pathways of realization of muscarinic regulation of ACh secretion triggered by M3

Discussion

The concept of autoregulation of neuromuscular transmission involving muscarinic receptors has been attracting the attention of researchers for several decades [[1], [2], [3], [4], [5],[18], [19], [20], [21], [22], [23]]. Recently, some evidences appeared that cholinoreceptors of M3 type are present in the vertebrate neuromuscular junctions and can be functionally significant [9,10,[21], [22], [23]]. A number of studies [9,21] reported the existence of the negative autoregulation in the lizard

Author contributions

I.K. developed the initial concept. I.K, A.T and A.P wrote the paper. A.T., G.Z., and A.P. performed the experiments and analyzed the data.

Declaration of competing interest

The authors declare that there is no conflict of interests.

Acknowledgments

This work was done with financial support from the government assignment for FRC Kazan Scientific Center of RAS. Part of work (by A.P. and G.Z.) was done in the Instituite of Neuroscience, Kazan State Medical University.

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