Effect of N‐acetylaspartylglutamate (NAAG) on non‐quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

Abstract

N-Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of nonquantal secretion of acetylcholine (ACh) from motor nerve terminals. Non-quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H-effect) caused by inhibition of nicotinic receptors by (+)-tubocurarine and acetylcholinesterase by armin (diethoxyp-nitrophenyl phosphate). Application of exogenous NAAG decreased the H-effect in a dose-dependent manner. The reduction of the H-effect by NAAG was completely removed when N-acetyl-b-aspartylglutamate (bNAAG) or 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane-associated ectoenzyme that hydrolyzes NAAG to glutamate and N-acetylaspartate. Bath application of glutamate decreased the H-effect similarly to the action of NAAG but N-acetylaspartate was without effect.Inhibition of NMDA receptors by DL-2-amino-5-phosphopentanoic acid, (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), and 7-chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by N G -nitro-L-arginine methyl ester and 3-bromo-7-nitroindazole completely prevented the decrease of the H-effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath-applied NAAG, can modulate non-quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.

Non-Cholinergic Signaling Pathways at Vertebrate Neuromuscular Junctions

“…Experiments on rat NMJ showed that NAAG is able to depress non-quantal ACh release [90]. The mechanism of neuropeptide action is realized through its extracellular hydrolysis by the GCP II with the formation of glutamate molecules, which, as was shown earlier [89], activate glutamate postsynaptic NMDA receptors and thereby trigger the NO-mediated mechanism of reducing the intensity of the non-quantal ACh release [104].…”

“…In the later stages of amphibians development, namely in tadpoles and adult frogs metabotropic glutamate receptors were found [80,[84][85][86], which, apparently, are localized postsynaptically [80,85]. In contrast to the amphibian NMJ, in the endplate of mammals to date were found only ionotropic glutamate NMDA and AMPA receptors, and all the experimental data show exclusively postsynaptic localization of these proteins [87][88][89][90][91][92].…”

“…Thus, it is shown that glutamate facilitates the quantal release of ACh at early stages of establishment and maturation of the NMJ in frog [72,82,94], whereas in adult animals, on the contrary, the amino acid inhibits the quantal release of ACh [80,[84][85][86]. At the same time any effect of glutamate on the quantal release of ACh in the NMJ of mammals was not established [89], however, the inhibition of non-quantal ACh release was revealed [89,90]. And since this type of the mediator is able to perform trophic function [17], in this case, glutamate may be considered as a regulator of neurotrophic control of the properties of the postsynaptic membrane.…”

“…And since this type of the mediator is able to perform trophic function [17], in this case, glutamate may be considered as a regulator of neurotrophic control of the properties of the postsynaptic membrane. Due to the fact that the activation of glutamate receptors both in amphibians [85], and mammals [88][89][90] may be accompanied with increased synthesis of nitric oxide molecules (NO), then it should be assumed that the amino acid is able to participate in a wide range of physiological functions, since the contribution of NO-mediated signaling was revealed in metabolism and contraction of muscle fibers [95,96].…”

Effect of dipeptide N-acetylaspartylglutamate on denervation-induced changes in the volume of rat skeletal muscle fibers

“…Activation of Na + -, K + -, Cl --cotransport is one of the key stage in the development of denervationinduced alterations in MF, including the increase in MF volume [1,6]. It was previously found that the intensity of this co-transport is controlled by the inhibitory action of the motoneuron realized via activation of glutamate receptors located on the sarcolemma [2,6].Motor nerve terminals in rats contain dipeptide N-acetylaspartylglutamate (NAAG) [3] participating in the synaptic transmission as an agonist of ionotropic glutamate NMDA-and metabotropic mGlu3-receptors [7,8] or as a glutamate precursor produced during hydrolysis catalyzed by glutamate carboxypeptidase II [4,5].Our aim was to study the possible role of NAAG in the neurotrophic control of MF volume. …”