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Keywords:

  • N-methyl-d-aspartate receptor;
  • muscle endplate;
  • nitric oxide;
  • non-vesicular transmitter

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

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 Ca2+ from the bathing solution prevented the glutamate-induced decrease of the H-effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG-nitro-l-arginine methyl ester (l-NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H-effect. The results suggest that glutamate, acting on post-synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non-quantal secretion of ACh from motor nerve terminals. Non-quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non-quantal transmitter following muscle denervation triggers the early post-denervation depolarization of muscle fibres.

Abbreviations used
ACh

acetylcholine

AP5

dl-2-amino-5-phosphopentanoic acid

d-NAME

NG-nitro-d-arginine methyl ester

EPP

endplate potentials

l-NAME

NG-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

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 α-amino-3-hydroxy-5-methylisooxazole-4-propionate (AMPA)/kainate subtypes of ionotropic receptors for glutamate have also been found at the motor nerve terminals in developing neuromuscular synapses of Xenopus laevis tadpoles (Fu et al. 1995; Liou et al. 1996; Fu and Liu 1997; Fu et al. 1998) and NMDA receptors have been identified at the post-synaptic membrane in neuromuscular junction of adult rats (Berger et al. 1995; Urazaev et al. 1995, 1998; Grozdanovic and Gossrau 1998). In developing neuromuscular synapses of Xenopus, glutamate increases the amplitude and frequency of miniature endplate currents and potentials (Fu et al. 1995; Liou et al. 1996; Fu and Liu 1997; Fu et al. 1998). Recently we have shown (Urazaev et al. 1995, 1998) that glutamate released from nerve endings probably activates NMDA-receptor mediated Ca2+ entry into the sarcoplasm followed by activation of nitric oxide synthase and production of nitric oxide (NO). Non-quantal ACh, acting through M1-cholinergic receptors (Urazaev et al. 2000), activates synthesis of NO to serve as a trophic message from motoneurones that keeps the Cl transport inactive in the innervated sarcolemma (Urazaev et al. 1999). These data implicate glutamate as a possible modulator of neurotransmission at the vertebrate neuromuscular junction.

We report here that glutamate modulates the non-quantal release of ACh at rat neuromuscular junction (Vyskočil and Illes 1977, 1978; Minic et al. 2002) in a multi-step process that includes: the activation of post-synaptic NMDA receptors, stimulation of NO synthesis and its release from muscle whereupon it acts to inhibit secretion of non-quantal ACh from pre-synaptic motor nerve terminals.

Materials and animals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

Compounds used and their sources were as follows: glutamic acid, NMDA; NMDA receptor antagonists, dl-2-amino-5-phosphopentanoic acid, 7-chlorokynurenic acid; an NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME), and inactive enantiomer of l-NAME, NG-nitro-d-arginine methyl ester (d-NAME); a scavenger of extracellular NO, haemoglobin; a nicotinic antagonist (+)-tubocurarine, cGMP and dibutyryl cGMP were purchased from Sigma, St Louis, MO, USA; a NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801) and guanylyl cyclase inhibitor, 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were purchased from Tocris Cookson Inc., Ballwin, MO, USA. Armin (diethoxy-p-nitrophenyl phosphate), an inhibitor of acetylcholinesterase, was manufactured in the Institute of Organic Chemistry, Moscow, Russia. All other chemicals were purchased from Sigma.

Male Wistar rats (150–200 g body weight) were used for all experiments. The animals were kept in sawdust-lined plastic cages in a well-ventilated room. A standard diet and water were available at all times. All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the protocol of the experiments was approved by the Animal Care and Use Committee of Kazan State Medical University.

Tissue preparation and bathing solution

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

Diaphragms with a 10–15 mm nerve stump were removed rapidly from animals killed by cervical dislocation and decapitation under ether anaesthesia. The diaphragms were cut into strips, placed in transparent plastic dishes with a Sylgard 184 silicone elastomer coating (Dow Corning Co, Midland, MI, USA) and superfused with a standard oxygenated (95% O2 + 5% CO2) Ringer–Krebs solution containing, in mmol/L, NaCl 120.0, KCl 5.0, CaCl2 2.0, MgCl2 1.0, NaHCO3 11.0, NaH2PO4 1.0, d-glucose 11.0; pH 7.2–7.4. Drugs were added to the Ringer–Krebs solution at the time of experimentation. All experiments were performed at 20°C, the temperature at which the H-effect is maximal (Lupa et al. 1986). Nominally Ca2+-free Ringer–Krebs solution was prepared by replacing 2 mmol/L CaCl2 with 3 mmol/L NaCl.

Electrophysiology and data analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

Microelectrodes filled with 2.5 mol/L KCl and tip resistances between 5 and 10 MΩ were used for recording muscle fibre potentials. The endplate areas are localized along the intramuscular branches of the phrenic nerve and in our experiments we identified their location by the presence of miniature endplate potentials (mEPPs), prior to the treatment of muscle with an anti-cholinesterase agent. The effect of glutamate on modulation of release of ACh from motor nerve terminals and the sensitivity of the post-synaptic membrane to ACh was monitored by analysis of the electrophysiological events associated with spontaneous, evoked quantal, and non-quantal secretion of ACh.

The effect of experimental protocols on post-synaptic membrane receptor properties was estimated by analysis of the amplitude and time constant of the rise and decay of mEPPs generated from the spontaneous quantal secretion of ACh. Changes in pre-synaptic terminal properties of spontaneous quantal secretion of ACh were estimated by analysis of mean frequencies of the mEPP generation and distribution of the intervals between two subsequent mEPPs (inter-impulse intervals).

Evoked quantal secretion of ACh was evaluated by the recording of endplate potentials (EPP). Phrenic nerves were electrically stimulated by supra-threshold stimuli with 0.1 ms duration at 0.3 Hz to generate action potentials and the subsequent quantal release of ACh from nerve endings. Muscle contractions were blocked by decreasing the quantal content of EPPs using a low-Ca2+ (1.3 mmol/L) and high-Mg2+ (7 mmol/L) solution (Fatt and Katz 1952) or by transverse cutting of muscle fibres (Barstad and Lilleheil 1968). Quantal content of the EPP was calculated by dividing its mean amplitude by the mean amplitude of mEPPs using the correction for non-linear summation of quanta (Del Castillo and Katz 1954; Martin 1966).

Muscles were treated with the irreversible cholinesterase inhibitor, armin (10 μmol/L) for 30 min and then rinsed several times for 15 min with normal saline. Endplate membrane potential measurements were begun 20 min post anti-cholinesterase treatment in the presence of various drugs, unless otherwise stated. Non-quantal release of ACh, which causes depolarization of muscle fibres at the endplate zone, was quantified statistically by measuring membrane potentials in 20 or more fibres during a 5–10-min period before, and another 20 or more fibres 8–15 min after, the addition of (+)-tubocurarine (10 μmol/L) to the medium. The differences between the mean resting membrane potentials under these two conditions (H-effect) are generally considered to be due to the non-quantal release of ACh (Katz and Miledi 1977; Vyskočil and Illes 1977, 1978; for details see Vyskočil et al. 1983; Galkin et al. 2001). In each group, four to eight muscles from several mice were used. In these experiments, 10 μmol/L of choline chloride was added to Ringer–Krebs solution to delay the post-denervation decrease of non-quantal ACh secretion (Nikolsky et al. 1991).

Effect of glutamate on spontaneous quantal release of ACh

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

The amplitude and frequency of control mEPPs were 1.2 ± 0.1 mV and 1.00 ± 0.09 s−1, respectively (n = 6800 from 17 animals) and were unchanged by addition of glutamate (10 μmol/L to 1 mmol/L) to the medium (1.2 ± 0.2 mV and 0.97 ± 0.08 s−1, respectively, p > 0.5, n = 2800). Similarly, mean inter-impulse intervals (1.078 ± 0.072 s, n = 2000 from five muscles) and histograms of their distribution did not change following glutamate treatment. The time constant for control mEPP rise time was 0.27 ± 0.02 ms (n = 2800) and for decay was 1.34 ± 0.11 ms (n = 2800). In muscles treated with 100 μm/L glutamate, these values were 0.27 ± 0.02 ms and 1.35 ± 0.14 ms, respectively, and were not significantly different from the controls (p > 0.05, n = 2800).

Since activation of NMDA receptors requires the binding of both glutamate and glycine to independent sites on the receptor (Johnson and Ascher 1987; for review see Danysz and Parsons 1998), we tested whether addition of 700 μmol/L glycine in the Ringer–Krebs solution would promote an effect of glutamate on spontaneous quantal secretion of ACh. The amplitude and frequency of mEPPs in glycine-treated preparations were 1.3 ± 0.1 mV and 1.12 ± 0.27 s−1, respectively, and the rise time and time constant of potential decay were 0.35 ± 0.03 ms and 1.71 ± 0.14 ms (n = 2000 from five animals), not significantly different from control data without glycine. Addition of glutamate (10 μmol/L to 1 mmol/L) to the glycine-containing solution did not change amplitude and temporal parameters of mEPP. In muscles treated with 100 μmol/L glutamate in the presence of 700 μmol/L glycine, the amplitude and frequency of mEPP were 1.3 ± 0.1 mV and 1.24 ± 0.31 s−1, respectively, and the rise time and time constant of potential decay were 0.36 ± 0.07 ms and 1.73 ± 0.17 ms, respectively (for each group of data: p > 0.05, n = 2000 from five animals). The data suggest that glutamate and glycine do not alter either pre-synaptic mechanism related to spontaneous quantal release of ACh or post-synaptic sensitivity to ACh.

Effect of glutamate on evoked quantal ACh secretion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

In the presence of normal concentrations of Ca2+ and Mg2+ (2 mmol/L and 1 mmol/L, respectively) the quantal content of the EPP and its depolarizing amplitude was sufficient to generate action potentials in the muscle membrane and initiate muscle contraction. The EPP amplitude in control experiments was 12.7 ± 1.7 mV (n = 1600 from four animals) and in glutamate-treated preparations 11.8 ± 1.8 mV (p > 0.05; n = 1600). The contraction was blocked by transverse cut of muscle fibres.

In low-Ca2+ and high-Mg2+ solution, the quantal content and amplitude of EPPs were reduced to 5.2 ± 1.1 and 2.9 ± 0.4 mV, respectively (n = 2000 from five animals) making EPPs insufficient to generate action potentials and contraction. Glutamate (100 μmol/L) in the presence or absence of glycine (700 μmol/L) did not change the quantal content of the EPP: 5.2 ± 1.2 without and 6.0 ± 0.8 with glycine (p > 0.05, n = 2000) nor did it change the amplitude and temporal characteristics of these signals.

Effect of glutamate on the H-effect

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

The resting membrane potential in endplate areas of muscle fibres, treated with acetylcholinesterase inhibitor, was − 72.7 ± 0.3 mV (n = 108 from four animals). The inhibition of post-synaptic nicotinic receptors by (+)-tubocurarine in anti-acetylcholinesterase-treated muscles caused the endplate membrane to hyperpolarize to − 77.8 ± 0.3 mV (p < 0.001; n = 108) giving an H-effect of 5.1 ± 0.3 mV (n = 108; Fig. 1, □). The bath application of glutamate reduced the H-effect in a concentration-dependent manner (Fig. 1, filled squares).

image

Figure 1. The dose–response relationship between glutamate and the non-quantal acetylcholine release. Non-quantal acetylcholine release was estimated by recording of the amplitude of the endplate hyperpolarization (H-effect) following inhibition of nicotinic cholinoreceptors in muscles with inhibited acetylcholinesterase. The amplitude of the H-effect was 5.1 mV (□). The filled squares represent the effect of different concentrations of glutamate on the amplitude of H-effect. When 700 µmol/L glycine was added to the solution, the amplitude of H-effect (○) decreased compared with the control. Glutamate, in the presence of glycine, was approximately 5 × more effective in reducing the H-effect as indicated by the reduction in the concentration required (25.4 µmol/L vs. 4.8 µmol/L) for a half maximal decrease of the H-effect (●). These data suggest that glutamate in the presence or absence of glycine inhibits the non-quantal secretion of acetylcholine in the neuromuscular junction of rat. Data are presented as mean ± 1 SEM pooled from four to five animals (90–110 fibres).

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In the presence of glycine, the amplitude of the H-effect was approximately 2 mV less than in control experiments (Fig. 1, open circle). The decrease in the H-effect seen in these experiments could result from a potentiation by glycine of the effect of glutamate endogenously released in the synaptic cleft (Meister et al. 1993; Berger et al. 1995; Urazaev et al. 1998). When exogenous glutamate was added together with glycine there was a concentration-dependent decrease in the H-effect. The comparison of the effects of exogenous glutamate in the absence of glycine and in glycine-containing medium demonstrates that glycine potentiates the inhibitory effect of glutamate on the amplitude of the H-effect. The degree of potentiation by glycine is reflected in the concentration of glutamate required to decrease the H-effect by one half (EC50). In control experiments, the EC50 for glutamate was 25.4 ± 1.8 μmol/L (n = 4) and in the presence of glycine, 4.8 ± 0.2 μmol/L (n = 4; p < 0.001).

Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

A unique feature of NMDA receptors, compared with other subtypes of glutamate receptors, is the requirement for both glutamate and the co-agonist glycine for the efficient gating of the ion channel (Kleckner and Dingledine 1988). Thus, an increase in the glutamate-induced suppression of the H-effect by glycine implicates NMDA receptors on the muscle membrane (Berger et al. 1995; Urazaev et al. 1995, 1998; Grozdanovic and Gossrau 1998) as the most probable target for the action of glutamate seen in our experiments. This hypothesis was tested using NMDA receptor antagonists to alter glutamate and glycine inhibition of the H-effect. In the following experiments 100 μmol/L glutamate, in the presence or absence of 700 μmol/L glycine, was used since a maximum inhibition of the H-effect was observed in either circumstance (Fig. 1).

The decrease in the H-effect caused by glutamate, with or without glycine, was completely prevented by dl-2-amino-5-phosphopentanoic acid (AP5), a potent competitive NMDA receptor inhibitor, by MK801, a selective non-competitive antagonist of the NMDA receptor-associated ion channel and by 7-chlorokynurenic acid, a potent competitive NMDA receptor blocker acting at the glycine site (Figs 2a and b). In muscle strips exposed to a Ringer–Krebs solution medium containing any of the NMDA receptor antagonists alone, no change in the amplitude of the H-effect was observed when compared with controls.

image

Figure 2. The effects of NMDA receptors inhibition and Ca2+-free solution on the glutamate-induced decrease of the H-effect. As shown in this figure, 100 µmol/L NMDA or glutamate, significantly decreased the amplitude of the H-effect in experiments performed in both control Ringer–Krebs solution (a) and in the solution containing 700 µmol/L glycine added to augment the action of glutamate (b). The inhibition of NMDA receptors by AP5 (500 µmol/L), MK801 (200 nmol/L), 7-chlorokynurenic acid (20 µmol/L; 7ClKA), and nominally 0 Ca2+ Ringer–Krebs solution completely prevented the decrease of the H-effect by glutamate in both control and glycine-containing solutions. The data provide evidence that glutamate-induced inhibition of secretion of non-quantal acetylcholine is mediated through the activation of NMDA receptors. Data are presented as mean ± 1 SEM pooled from four to five animals (90–110 fibres).

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As expected, NMDA had the same action on the amplitude of the H-effect as glutamate. The amplitudes of the H-effect after exposure of the muscle to either glutamate or NMDA (100 μmol/L) were the same regardless of the presence or absence of glycine in saline (Fig. 2).

Role of Ca2+ on the reduction of the H-effect by glutamate

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

Activation of the NMDA receptor opens a cation-selective ion channel to permit an influx of Na+ and Ca2+ into the cytoplasm. As demonstrated in the experiments described in the previous section, MK801 removed the glutamate-induced decrease of the H-effect in the presence of normal extracellular Ca2+, raising the possibility that Ca2+ entering muscle via NMDA receptor channels may influence the H-effect.

A decrease in extracellular Ca2+ concentration to virtual zero by NaCl substitution completely prevented the glutamate-induced reduction in the H-effect (Fig. 2). The amplitude of the H-effect in Ca2+-free saline was the same with or without glutamate. In the presence of glycine, glutamate also failed to decrease the H-effect in Ca2+-free solution. Furthermore, the amplitude of the H-effect in these latter experiments was the same as seen in experiments with Ca2+-containing solutions without glutamate but significantly higher than the H-effect with saline containing normal Ca2+ and glycine.

Role of NO in the reduction of the H-effect by glutamate

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

Non-quantal release of ACh in the rat neuromuscular junction has been shown to be a self-regulating process, which includes the activation of muscle M1 cholinoreceptors with subsequent opening of voltage-dependent Ca2+ channels and NO production in muscle cytoplasm (Urazaev et al. 1997, 2000; Mukhtarov et al. 2000). Additionally, glutamate has been implicated as a synaptic agent controlling the active chloride transport and membrane potential in muscle membrane via activation of post-synaptic NMDA receptors with the subsequent synthesis of NO in sarcoplasm (Urazaev et al. 1995, 1998, 1999). Since the glutamate-induced decrease of the H-effect, as described above, is realized through activation of NMDA receptors, we hypothesized that the action of glutamate might also involve NO synthesis in and release from muscle cytoplasm.

L- and d-NAME

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

As illustrated in Fig. 3(a), the bath application of the NO synthase inhibitor l-NAME, but not its inactive enantiomer d-NAME, caused a considerable increase in the H-effect. This is consistent with the results reported in Mukhtarov et al. (2000). Glutamate failed to decrease the amplitude of the H-effect in l-NAME-treated fibres, whereas in d-NAME-treated muscles glutamate caused a decrease in the H-effect, action similar to that seen in control conditions.

image

Figure 3. The effect of NO synthase inhibition on the glutamate-induced decrease of the H-effect. The decrease of the H-effect by 100 µmol/L glutamate, in the absence (a) or presence (b) of 700 µmol/L glycine, was completely prevented by pre-incubation of muscle with l-NAME (100 µmol/L), an inhibitor of NO synthase, or haemoglobin (10 µmol/L; Hb), a scavenger of extracellular NO. Bath-application of l-NAME alone increased the amplitude of the H-effect compared with control conditions, suggesting that there is a background production of NO in muscle that partially inhibits non-quantal release of acetylcholine. As expected, d-NAME, an inactive enantiomer of l-NAME, did not prevent the action of glutamate on H-effect. These data suggest that glutamate decreases non-quantal secretion of acetylcholine via activation of synthesis of NO in muscle fibres. Data are presented as mean values ± 1 SEM pooled from four to five animals (90–110 fibres).

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The effects of l- and d-NAME, as described above, were unchanged by glycine (Fig. 3b). The bath application of l-NAME, but not d-NAME, alone increased the amplitude of the H-effect regardless of the presence of glycine. These results indicate that glutamate decreased the H-effect through the activation of NO synthase.

Haemoglobin

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

To determine whether NO diffuses through the sarcolemma to enter the nerve terminal to inhibit the H-effect, muscle preparations were pre-exposed to reduced haemoglobin, a known scavenger of extracellular NO. Haemoglobin completely prevented the glutamate-induced decrease of the H-effect regardless of the presence of glycine (Figs 3a and b). Haemoglobin, with or without glutamate, significantly increased the H-effect by 20% as compared with controls (Fig. 3), suggesting that the H-effect in control circumstances is influenced by endogenous glutamate and NO released into the synaptic cleft. These results provide further evidence that the H-effect is regulated to a large extent by the retrograde action of NO released from muscle fibres following their treatment with glutamate.

Guanylyl cyclase inhibition and exogenous cGMP

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

Since the main target for NO is soluble guanylyl cyclase (Murad 1994), the specific inhibitor ODQ (Garthwaite et al. 1995) was tested on the glutamate-induced reduction of the H-effect. ODQ itself increased the H-effect from 5.1 ± 0.3 mV to 7.5 ± 0.4 mV (n = 100; p < 0.05). When muscles were pre-treated with 1 μmol/L ODQ for 30 min, application of glutamate (100 μmol/L) was ineffective on the H-effect, which did not significantly differ from values in ODQ itself (7.4 ± 0.4 mV, n = 100, p > 0.05). In addition, the glycine potentiation of the inhibition of the H-effect described earlier was completely prevented (7.6 ± 0.4 mV, n = 100, p > 0.05) in the presence of 1 μmol/L M ODQ, 100 μmol/L glutamate and 700 μmol/L glycine.

Application of permeable dibutyryl cGMP (1 μmol/L) decreased the H-effect by 30%, from the control value of 5.1 mV to 3.6 ± 0.2 mV (n = 100, p < 0.05), whereas the impermeable cGMP (1 μmol/L) was ineffective (H-effect = 4.8 ± 0.3 mV, n = 105, p > 0.05).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References

A variety of studies of the past decade have strongly suggested that glutamate may be a signalling molecule in the mammalian neuromuscular junction. For example, glutamate-like immunoreactivity has been found in cytoplasm of cholinergic neurones and their nerve terminals where it is associated with synaptic vesicles and is co-released with ACh (Vyas and Bradford 1987; Israel et al. 1993; Meister et al. 1993; Waerhaug and Ottersen 1993). In addition, the NMDA subtype of glutamate receptors has been found on the post-synaptic membranes of the neuromuscular junction (Berger et al. 1995; Urazaev et al. 1995, 1998) in association with NO synthase (Grozdanovic and Gossrau 1998).

Activation of muscle NMDA receptors stimulates the synthesis of NO in sarcoplasm to participate in maintenance of the membrane potential through the negative control of furosemide-sensitive Na+,K+,Cl-symport activity which, by limiting intracellular Cl and its outwardly directed electrochemical gradient, tends to maintain a high resting muscle membrane potential (Urazaev et al. 1999). As a negative feedback regulation of membrane potential, the release of NO from the sarcoplasm has also been shown to depress the non-quantal secretion of ACh from motor nerve terminals giving drop of the H-effect (Mukhtarov et al. 2000). It is therefore possible that synaptic glutamate acting on NMDA receptors to activate NO synthase modulates the release of ACh from motor terminals to closely regulate muscle post-synaptic membrane potential.

Non-quantal release of ACh also contributes to the elimination of polyneuronal innervation during post-natal development of rat muscles (Vyskočil and Vrbova 1993) and may maintain increased endplate nicotinic receptors desensitization to shorten the post-synaptic response to quantal ACh (Giniatullin et al. 1993). Loss of non-quantal ACh release appears to be the earliest change in the nerve terminal following nerve section (Stanley and Drachman 1986; Nikolsky et al. 1994) and may be relevant to the denervation changes in muscle properties. These changes include the development of the early denervation depolarization of muscle (Albuquerque et al. 1971) as activation of sarcolemma Na+,K+,2Cl-transport occurs in the absence of nitric oxide synthase activity (Urazaev et al. 1987, 1999). During axonal regeneration the non-quantal release of ACh is restored 3–6 days before the appearance of detectable quantal release of mediator (Nikolsky et al. 1994), suggesting a possible role of non-quantal ACh in the re-establishment or maintenance of a functional post-synaptic neuromuscular receptor membrane.

In the study reported here glutamate was shown not to affect either spontaneous or evoked quantal release of ACh from the pre-synaptic terminal regardless of presence or absence of glycine in the bathing solution. This conclusion is reached on the basis of a lack of effect of glutamate on changes in the frequency and distribution of interstimuli intervals of mEPPs or on the amplitude and time course of EPPs and on their quantal content. In addition, glutamate also had no effect on amplitude or time course of mEPPs, implying that the sensitivity of post-synaptic nicotinic receptors to ACh and the kinetics of endplate ionic channels were not changed by glutamate.

On the other hand, glutamate was found to be highly effective in reducing the non-quantal release of ACh in a concentration-dependent manner, as indicated by the decrease in the H-effect. The EC50 for the glutamate-induced reduction in the H-effect was 25.4 μmol/L and was shifted dramatically to the left by glycine to 4.8 μmol/L. It is important to note that in the presence of glycine alone, without added glutamate, a decrease in the H-effect by 2 mV was observed, suggesting the presence of a small amount of endogenously released glutamate into the synaptic cleft sufficient to reduce the H-effect when potentiated by added glycine. Alternatively, a direct action of glycine on NMDA receptors cannot be excluded given the evidence that functional NMDA receptors can be activated by glycine, even in the absence of glutamate (Paudice et al. 1998; but see Dingledine et al. 1999 for the opposite view).

NMDA receptor antagonists reversed the glutamate- and glycine-induced reduction of the H-effect, strongly suggesting that the action of glutamate is mediated through post-synaptic NMDA receptors confirming earlier observations on diaphragms (Berger et al. 1995; Urazaev et al. 1995, 1998; Grozdanovic and Gossrau 1998). Glutamate also failed to decrease the H-effect in nominally zero Ca2+ solution, further indicating that glutamate probably induces Ca2+ entry through the NMDA receptor to implicate a role for Ca2+ in the regulation of the H-effect and by association, non-quantal release of ACh. One can hypothesize that the steady state 3 mV depolarization of the post-synaptic membrane caused by glutamate-induced Ca2+ entry through endplate NMDA channels (Urazaev et al. 1998) might be sufficient to induce NO synthesis in muscle cytoplasm in contrast to brief episodes of channel openings during excitatory post-synaptic currents at synapses in CNS.

In skeletal muscle, post-synaptic NMDA receptors and NO synthase, an enzyme that generates NO molecules, are co-localized (Grozdanovic and Gossrau 1998) and NO synthesis appears to be stimulated upon an increase in intracellular Ca2+ as a consequence of NMDA receptor activation by glutamate or NMDA (Urazaev et al. 1995, 1998). In the present experiments, NO synthase inhibition by l-NAME or application of the soluble guanylyl cyclase inhibitor, ODQ prevented a decrease in the H-effect by glutamate and glycine. The specificity of l-NAME-induced inhibition of NO synthase was addressed in our previous investigation (Mukhtarov et al. 2000) and was further confirmed in the in the experiments with the bath application of inactive enantiomer of l-NAME, d-NAME described in this investigation.

The hypothesis that muscle-produced NO has a role in regulation of non-quantal secretion of ACh from the pre-synaptic nerve terminal seems to be supported by experiments with haemoglobin. The glutamate-induced decrease in the H-effect was removed by the presence of haemoglobin, which binds and inactivates extracellular NO and also may deplete intracellular NO content (Urazaev et al. 1995, 1997; Mukhtarov et al. 2000).

In summary, the results of this investigation reveal a possible role of synaptic glutamate as a physiological modulator of non-quantal secretion of ACh at the neuromuscular junction through a NO cascade (Mukhtarov et al. 2000) together with muscle activity, metabotropic pre-synaptic ACh autoreceptors (Zemková and Vyskočil 1989), P1 purinergic receptors (Galkin et al. 2001) and several other factors including Na+ pump activity and Mg2+ (Vyskočil and Illes 1977; Zemkováet al. 1989; Vyskočil and Vrbova 1993). The action of glutamate appears to initiate the activation of post-synaptic NMDA receptors, allowing Ca2+ influx into the muscle, the subsequent activation of NO synthase followed by the production and release of NO. The NO diffuses from the muscle to inhibit the non-quantal release of ACh from the pre-synaptic nerve terminal. This in turn modulates resting membrane potential and activity of ion transporters on the post-synaptic side of the junction. This sequence of events is an example of feedback machinery at which two neurotransmitters, glutamate and ACh, can effectively participate in the regulation of the post-synaptic membrane potential and the sensitivity of its receptors.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials and animals
  5. Tissue preparation and bathing solution
  6. Electrophysiology and data analysis
  7. Statistics
  8. Results
  9. Effect of glutamate on spontaneous quantal release of ACh
  10. Effect of glutamate on evoked quantal ACh secretion
  11. Effect of glutamate on the H-effect
  12. Effect of agonists and antagonists of NMDA receptors on the glutamate-evoked depression of the H-effect
  13. Role of Ca2+ on the reduction of the H-effect by glutamate
  14. Role of NO in the reduction of the H-effect by glutamate
  15. L- and d-NAME
  16. Haemoglobin
  17. Guanylyl cyclase inhibition and exogenous cGMP
  18. Discussion
  19. Acknowledgements
  20. References
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