Purine P2Y receptors in ATP-mediated regulation of non-quantal acetylcholine release from motor nerve endings of rat diaphragm

Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 lM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABA A receptor blocker, but was attenuated following application of the GABA B receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABA B receptor (GABA B R1 and GABA B R2) in the neuromuscular junction. These findings suggest that metabotropic GABA B receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release. Keywords: GABA receptors, phospholipase C, quantal and non-quantal acetylcholine release. A compound called c-aminobutyric acid (GABA), which is synthesized by the enzyme L-glutamate decarboxylase (GAD; EC 4.1.1.15), is the basic inhibitory neurotransmitter in the synapses of the central nervous system (CNS). It plays a key role in the development, maturation, and functioning of the adult brain by modulating neuronal activity (Watanabe et al. 2002;Obata 2013). The synaptic action of GABA occurs via the activation of ionotropic GABA A receptors and metabotropic GABA B receptors (Bowery et al. 2002;Olsen and Sieghart 2008). The physiological role of GABA in the CNS is studied largely because GABAergic signaling dysfunction underlies the development of a broad spectrum of neurological and psychiatric diseases, including anxiety, sleep disorder, muscle rigidity, Parkinson's disease, Huntington's disease, epilepsy, and schizophrenia (Obata 2013; Smith-Hicks 2013).

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“…; Malomouzh et al . ). The bath application of GABA decreased the amplitude of the H‐effect in a dose‐dependent manner (Fig.…”

Section: Resultsmentioning

confidence: 97%

Metabotropic GABA_B receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction

Malomouzh

Петров

Нуруллин

et al. 2015

Journal of Neurochemistry

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“…The selective blockade of M 1 receptors by pirenzepine did not cause any measurable changes in NEO-induced fade, unless presynaptic adenosine A 2A receptors were blocked [34]. The absence of the effect of pirenzepine alone on NEO-induced fade shows that the activation of M 1 receptors located in skeletal muscle [48,49] is highly unlikely in the presence of NEO. Anticholinesterase-induced fade has been proposed to be modulated by ACh causing post-synaptic nicotinic receptor desensitization, but such desensitization was not observed when the function of muscle-type α1-containing receptors was evaluated during fade produced by tetanic stimulation [50].…”

Section: Discussionmentioning

confidence: 99%

Tetanic Facilitation of Neuromuscular Transmission by Adenosine A<sub>2A</sub> and Muscarinic M<sub>1</sub> Receptors is Dependent on the Uptake of Choline via High-Affinity Transporters

Castellão-Santana¹,

Abiko²,

Ambiel³

et al. 2018

Pharmacology

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Background/Aims: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A_2A and muscarinic M₁ receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. Methods: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500–750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. Results: Activation of A_2A and M₁ receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 μmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 μmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 μmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 μmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 μmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 μmol/L) was also prevented by HC-3 (4 μmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 μmol/L). Conclusion: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A_2A and M₁ receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.

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“…P2X7 receptor subunits were found on presynaptic motor nerve terminals of mouse, but there is no evidence for P2X1, P2X2, P2X3, P2X4, P2X5 or P2X6 receptor subunits [49]. According to a number of electrophysiological studies metabotropic P2Y receptors are also localized on the motor nerve endings of both amphibian and mammals NMJ [44,[50][51][52]. However, P2Y receptors were found on the postsynaptic membrane of skeletal muscle fiber.…”

Section: Purinergic Signalingmentioning

confidence: 98%

“…Adenosine also inhibits quantal release of ACh, acting through P1 receptors and its mechanism of action does not affect the operation of calcium channels [50,51]. As for the influence of purines on the non-quantal release of ACh it is established that its intensity remains unchanged in the presence of adenosine, but it decreases via activation of P2Y receptors by the ATP molecules and this mechanism is not coupled to presynaptic voltage-dependent Ca 2+ channels [52,63].…”

Section: Purinergic Signalingmentioning

confidence: 99%

Non-Cholinergic Signaling Pathways at Vertebrate Neuromuscular Junctions

Malomouzh¹

2012

Skeletal Muscle - From Myogenesis to Clinical Relations

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Purine P2Y receptors in ATP-mediated regulation of non-quantal acetylcholine release from motor nerve endings of rat diaphragm

Cited by 18 publications

References 52 publications

Metabotropic GABA_B receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction

Metabotropic GABA_B receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction

Tetanic Facilitation of Neuromuscular Transmission by Adenosine A<sub>2A</sub> and Muscarinic M<sub>1</sub> Receptors is Dependent on the Uptake of Choline via High-Affinity Transporters

Non-Cholinergic Signaling Pathways at Vertebrate Neuromuscular Junctions

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Purine P2Y receptors in ATP-mediated regulation of non-quantal acetylcholine release from motor nerve endings of rat diaphragm

Cited by 18 publications

References 52 publications

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction

Tetanic Facilitation of Neuromuscular Transmission by Adenosine A<sub>2A</sub> and Muscarinic M<sub>1</sub> Receptors is Dependent on the Uptake of Choline via High-Affinity Transporters

Non-Cholinergic Signaling Pathways at Vertebrate Neuromuscular Junctions

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Metabotropic GABA_B receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction

Metabotropic GABA_B receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction