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. 2024 Aug;49(8):2021-2037.
doi: 10.1007/s11064-024-04153-5. Epub 2024 May 30.

Mechanism of Purinergic Regulation of Neurotransmission in Mouse Neuromuscular Junction: The Role of Redox Signaling and Lipid Rafts

Arthur R Giniatullin  1 , Kamilla A Mukhutdinova  2   3 , Alexey M Petrov  4   5   6
Affiliations

Mechanism of Purinergic Regulation of Neurotransmission in Mouse Neuromuscular Junction: The Role of Redox Signaling and Lipid Rafts

Arthur R Giniatullin et al. Neurochem Res. 2024 Aug.

Abstract

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic Gi protein-coupled P2Y13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P2Y13 receptors (MRS 2211), Ca2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca2+ with subsequent elevation of ROS levels. The elevation of [Ca2+]in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of Gi protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P2Y13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P2Y13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P2Y13 receptor/Gi protein/Ca2+/protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner.

Keywords: ATP; Acetylcholine; Lipid raft; Neuromuscular junction; Purinoceptor; Reactive oxygen species.

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