Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H-effect) following blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine and cholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Glutamate was shown to inhibit non-quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate-induced decrease of the H-effect was enhanced by glycine. Glycine alone also lowered the H-effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N-methyl-D-aspartate (NMDA) receptors with (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), DL-2-amino-5-phosphopentanoic acid (AP5) and 7-chlorokynurenic acid or the elimination of Ca It is well known that glutamate serves as a neurotransmitter at invertebrate neuromuscular junctions (Lunt and Olsen 1988) whereas acetylcholine (ACh) serves that role in the vertebrate. However, investigations during the last decade indicate a role for glutamate as a signalling molecule at the neuromuscular junction of vertebrates (see for review Grozdanovic and Baumgarten 1999). In support of this notion, glutamate transporter mRNA has been shown to be present in the cytoplasm of rat spinal motoneurones along with a glutamate-like immunoreactivity (Meister et al. 1993). Glutamate has been found in nerve terminals of rat motoneurones in association with synaptic vesicles (Waerhaug and Ottersen 1993) and evidence that it is co-released with ACh in cholinergic nerve terminals has been documented (Vyas and Bradford 1987;Israel et al. 1993;Meister et al. 1993). In addition, N-methyl-D-aspartate (NMDA) and Received August 15, 2002; revised manuscript received November 20, 2002; accepted December 19, 2002. Address correspondence and reprint requests to Dr Albert K. Urazaev, Department of Physiology, The Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA. E-mail: urazaeva@mail.ecu.eduAbbreviations used: ACh, acetylcholine; AP5, DL-2-amino-5-phosphopentanoic acid; D-NAME, N G -nitro-D-arginine methyl ester; EPP, endplate potentials; L-NAME, N G -nitro-L-arginine methyl ester; mEPP, miniature endplate potentials; MK801, (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine; NO, nitric oxide; ODQ, 1H [1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one.

High-frequency stimulation (100 Hz) of isolated giant axons of the small squid Alloteuthis subulata and the large squid Loligo forbesi caused the periaxonal Schwann cell resting potential (Em = -40 mV) to hyperpolarize up to 11 mV in direct proportion to train duration and action potential amplitude. In both species, the Schwann cell also hyperpolarized up to 17 mV with the application of L-glutamate (10(-9) to 10(-6) M), in a dose-dependent manner. By contrast, in the presence of 10(-8) M d-tubocurarine (d-TC) to block the cholinergic component of the Schwann cell response, Schwann cells depolarized 8-9 mV during electrical stimulation of the axon or application of L-glutamate. In the presence of 10(-5) M 2-amino-4-phosphonobutyrate (2-APB), the hyperpolarization to glutamate and to axon stimulation was blocked, whereas the cholinergic (carbachol-induced) hyperpolarization was unaffected. In experiments with Alloteuthis, L-aspartate (10(-7) M) also caused a Schwann cell hyperpolarization, but this was not blocked by 2-APB. In tests with glutamate receptor agonists and antagonists, quisqualate (10(-5) M) produced a hyperpolarization blocked by 10(-4) M L-glutamic acid diethylester (GDEE), which also blocked the response to axonal stimulation. Kainic acid (10(-4) M) also caused a hyperpolarization, but n-methyl-D-aspartate (NMDA; 10(-4) M), ibotenate (10(-5) M), alpha-amino-3-hydroxy-5-methyl-isoxazole proprionate (AMPA; (10(-4) M), and isethionate (10(-5) M) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

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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.

1. Resting membrane potential and the current-voltage relation were measured in crayfish giant axons bathed in various potassium solutions with and without ouabain. 2. Ouabain caused a depolarization of the membrane at each [K]o used but did not affect membrane resistance. 3. The ouabain-sensitive transport current was least (3 microamperemeter/cm2) in 0 mM [K]o and greatest (7 microamperemeter/cm2) in 16.2 and 21.6 mM [K]o. 4. The assumption was made an some indirect evidence presented that axons equilibrated in various potassium solutions maintain constant internal sodium and potassium concentrations for up to 3 h. 5. On the basis of this assumption, the apparent ratio of coupled Na : K transport was calculated. It was found to be least (-1.3/1) in 0 mM [K]o and to approach infinity in 16.2 and 21.6 mM [K]o. 6. The data indicate that the apparent variability of the Na : K exchange ratio likely represents an intrinsic property of the exchange mechanism and is less likely to be explained by a fixed-ratio coupled Na : K transport operating in parallel with electro-neutral Na : Na or K : K exchange.