Nitric oxide donor, S-nitroso-N-acetyl-dl-penicillamine, inhibits acetylcholinesterase activity in muscle homogenates
To determine whether NO molecules can inhibit AChE, biochemical assays for AChE activity were performed in EDL homogenates using different concentrations of the NO donor, SNAP. In the presence of SNAP, a dose-dependent decrease in AChE activity was observed, culminating in complete AChE inhibition (Fig. 1). The estimated IC50 for SNAP was 230 μm. SNAP is not an equimolar NO donor. For example, using the NO-selective electrode, Hermann & Erxleben (2001) showed that 1 mm of SNAP produced approximately 8 μm of NO. Therefore, an IC50 of 230 μm corresponds to 1.8 μm of NO in solution.
Figure 1. The NO donor, SNAP, reduces AChE activity in a dose-dependent manner in rat EDL homogenates. AChE activity was estimated using Ellman's method (Ellman et al., 1961). Control values were obtained in the absence of SNAP. Experimental values are presented as % control. Using Hill's equation, the estimated IC50 for SNAP was 230 μm. Data are presented as mean ± SEM (n = 3 muscles). Note: log-linear axis.
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Exogenous nitric oxide increases the amplitude and duration of miniature endplate currents
The amplitude and duration of spontaneous endplate currents change rapidly after AChE inhibition (Katz & Miledi, 1973). Notably, the current amplitude increases almost linearly in proportion to AChE inhibition, whereas the decay time constant increases only when 80–90% of AChE activity is inhibited (Anglister et al., 1994). Therefore, alterations in current amplitude and decay time constant can be used to follow AChE inhibition in the synaptic cleft.
The ability of NO to affect the amplitude of endplate potentials (quantal content) in the neuromuscular junction has been reported previously (Thomas & Robitaille, 2001). Therefore, the changes in the parameters associated with mEPCs were analysed only to avoid interference from the putative effects of NO on both mEPC amplitude and quantal content.
Based on the inhibition curve of AChE by NO in vitro, for electrophysiological recordings, SNAP was used at concentrations ranging from 20 μm to 2 mm. In controls, the mean mEPC amplitude and decay time constant (τ) were 2.77 ± 0.16 nA and 1.03 ± 0.06 ms, respectively (n = 20, Fig. 2). At the lowest concentration (20 μm), SNAP had no effect on mEPC parameters. Increasing the SNAP concentration to 200 μm enhanced mEPC amplitude by 13% (3.14 ± 0.09 nA vs. control, unpaired t-test, t38 = 2.04, P = 0.048, Fig. 2B) and had no effect on the decay time constant (Fig. 2C). SNAP at a concentration of 2 mm resulted in a dramatic increase in both mEPC amplitude (47% increase, 4.08 ± 0.16 nA vs. control, unpaired t-test, t38 = 5.85, P < 0.00001, Fig. 2B) and decay time constant (89% increase, 1.95 ± 0.10 ms vs. control, unpaired t-test, t38 = 7.71, P < 0.000001, Fig. 2C). This effect was fully reversible when SNAP was removed from the bathing solution (data not shown).
Figure 2. SNAP increases the amplitude and decay time constant (τ) of mEPCs in a dose-dependent manner. (A) Representative individual mEPCs in a control (black line) and in the presence of 2 mm SNAP (gray line). (B) The average of 100 individual signals recorded at the same endplate. (C) A moderate enhancement of mEPC amplitude was observed at 200 μm of SNAP. Application of 2 mm SNAP dramatically increased the mEPC amplitude (n = 20 per concentration and control). (D) Decay time constant (τ) was significantly increased by 2 mM SNAP. Lower SNAP concentrations had no effect (n = 20 per concentration and control). Data are presented as mean ± SEM. Note: log-linear axis. Asterisks indicate a statistically significant difference (*P < 0.05 and **P < 0.0001, respectively) from the control value.
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To examine whether the effects of SNAP on mEPC parameters are associated with inhibition of AChE, mEPC measurements were performed in the presence of the traditional anti-AChE compound, paraoxon. As expected, application of 10 μm paraoxon dramatically increased mEPC amplitude (4.93 ± 0.10 nA vs. control, unpaired t-test, t42 = 11.89, P < 0.00001, Fig. 3A) and prolonged the decay time constant by 299% (4.10 ± 0.13 ms vs. control, unpaired t-test, t42 = 20.50, P < 0.00001, Fig. 3B). Once paraoxon has exerted its influence on the mEPC amplitude and decay time constant, no further significant increase in mEPC parameters was observed when 2 mm SNAP was applied thereafter (amplitude: paraoxon + SNAP 5.13 ± 0.20 nA, paraoxon 4.93 ± 0.10 nA, unpaired t-test, t38 = 1.02, P = 0.31; τ of decay: paraoxon + SNAP 3.67 ± 0.10 ms, paraoxon 4.10 ± 0.13 ms, unpaired t-test, t38 = 1.66, P = 0.105).
Figure 3. The effects of SNAP on mEPC amplitude and decay time constant (τ) are associated with inhibition of synaptic AChE by released NO molecules. Incubation with the AChE inhibitor, paraoxon (10 μm), dramatically increased both mEPC amplitude (A) and τ of decay (B). The application of SNAP (2 mM) after preincubation with paraoxon did not affect either mEPC amplitude or τ of decay. The NO scavenger, haemoglobin (Hb) (30 μm), alone had no effect on mEPC parameters, whereas Hb significantly attenuated the effects of SNAP. Data are presented as mean ± SEM, pooled from four to five animals (19–24 endplates). Asterisks indicate a statistically significant difference (*P < 0.05 and **P < 0.0001, respectively) from the control value.
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The question arises whether a dose-dependent increase in mEPC amplitude and a prolongation of the decay time, which are typical of AChE inhibition, are the result of action by NO molecules or of SNAP-mediated depression of AChE activity. To address this question, SNAP (2 mm) was added to haemoglobin (30 μm), an NO scavenger (Mukhtarov et al., 2000; Thomas & Robitaille, 2001). Haemoglobin per se did not influence mEPC amplitude and duration (Fig. 3). However, the effects of SNAP on mEPC amplitude and duration were less pronounced in the presence of haemoglobin. The mEPC amplitude increased by only 22% (haemoglobin + SNAP, 3.37 ± 0.18 nA; haemoglobin, 2.76 ± 0.15 nA, unpaired t-test, t41 = 2.65, P = 0.011, Fig. 3A) and the decay time constant increased by only 24% (haemoglobin + SNAP, 1.39 ± 0.06 ms; haemoglobin, 1.13 ± 0.06 ms, unpaired t-test, t41 = 3.18, P = 0.003, Fig. 3B).
Therefore, the effects of the NO donor on mEPC amplitude and decay time were similar to the effects typically seen with AChE inhibition. Further, the absence of these effects after preincubation with an anti-AChE and significant attenuation of these effects in the presence of an NO scavenger support the hypothesis that exogenous NO molecules can depress AChE activity in the mammalian neuromuscular junction.
Activation of N-methyl-d-aspartate receptors increases miniature endplate current size through endogenous nitric oxide production
To address whether NMDA receptor activation triggers sufficient endogenous NO production to depress AChE activity, 100 μm glutamate was added to the bathing solution. Glutamate enhanced mEPC amplitude, but the effect became significant only 30–40 min after the addition of glutamate. Incubation for 1 h with glutamate increased the mEPC size by 9% (3.53 ± 0.10 nA vs. control: 3.23 ± 0.07 nA, unpaired t-test, t60 = 2.41, P = 0.02, Fig. 4). There was no difference in the effects of glutamate when the NMDA receptor co-agonist, glycine, was added to the solution at a low concentration (100 μm, data not shown). However, a higher concentration of glycine (700 μm) significantly enhanced the effect of glutamate, leading to an increase in mEPC amplitude of 19% (3.87 ± 0.11 nA vs. control, unpaired t-test, t75 = 5.08, P < 0.000001, Fig. 4). Glycine addition at any concentration did not change the duration of mEPCs. A further increase in glutamate concentration (up to 500 μm, in the presence of 700 μm glycine) did not amplify the effects on mEPC amplitude (data not shown). Finally, 700 μm glycine alone did not alter either the mEPC amplitude or duration (Fig. 4). A representative example of changes in mEPC amplitude during the combined application of glutamate and glycine is shown in Fig. 4 (insert).
Figure 4. Effects of glutamate (Glu) and glycine (Gly) on mEPC amplitude are associated with inhibition of synaptic AChE. (A) Representative example illustrating the time-course of mEPC amplitude enhancement during combined application of Glu and Gly in one typical experiment (each point represents the mean amplitude from ~200 mEPCs). The insert shows the average of 100 mEPCs recorded in the same endplate in a control (black line) and after incubation for 1 h with 100 μm Glu and 700 μm Gly (gray line). (B) Incubation for 1 h with 100 μm Glu resulted in a 9% increase in mEPC amplitude. The combined action of Glu (100 μm) and Gly (700 μm) caused even greater enhancement of mEPC amplitude (19% increase). The application of amino acids after pretreatment with the AChE inhibitor, paraoxon (10 μm), did not effect mEPC amplitude. Data are presented as mean ± SEM, pooled from four to seven animals (21–40 endplates). Asterisks indicate a statistically significant difference (*P < 0.05 and **P < 0.0001, respectively) from the control value.
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To test the hypothesis that the change in mEPC amplitude may result from a decrease in AChE activity, amino acids were applied after complete AChE inhibition by paraoxon. With paraoxon, the mean mEPC amplitude was 4.93 ± 0.10 nA (n = 24) and did not change significantly with application of glutamate and glycine (5.07 ± 0.14 nA vs. paraoxon, unpaired t-test, t47 = 0.85, P = 0.40, Fig. 4). In addition, AChE activity in EDL homogenates was not altered by application of glutamate or glycine, applied together or individually, in concentrations used for electrophysiological recordings (data not shown).
Therefore, it is likely that the amino acids, glutamate and glycine, act on mEPC amplitude through partial and indirect depression of AChE activity. Based on the results of this study, other potential mechanisms for this activity (e.g. an increase in quantum size or changes in acetylcholine receptor affinity) are less likely.
To investigate the hypothesis that glutamate-mediated NO production leads to depression of AChE activity via NMDA receptor activation, mEPCs were recorded in muscles preincubated with either the competitive NMDA receptor antagonist, AP-5, or the NO synthase inhibitor, L-NAME. Preincubation with AP-5 (25 μm) alone did not change mEPC amplitude (AP-5, 2.86 ± 0.11 nA; control, 3.09 ± 0.10 nA; unpaired t-test, t47 = 1.56, P = 0.12). After pretreatment with AP-5, the effect of glutamate and glycine on mEPC amplitude was eliminated (glutamate + glycine + AP-5, 2.82 ± 0.10 nA; AP-5, 2.86 ± 0.11 nA; unpaired t-test, t46 = 0.23, P = 0.82). The incubation of muscles with the NO synthase inhibitor, L-NAME (100 μm), resulted in a small but statistically significant decrease (8% decrease) in mEPC amplitude (2.96 ± 0.08 nA, control: 3.23 ± 0.07 nA, unpaired t-test, t72 = 2.51, P = 0.014, Fig. 5). The application of amino acids after incubation with L-NAME did not increase mEPC amplitude (glutamate + glycine + L-NAME, 2.79 ± 0.08 nA; L-NAME, 2.96 ± 0.08 nA; unpaired t-test, t57 = 1.51, P = 0.14, Fig. 5).
Figure 5. Effect of amino acids [glutamate (Glu) and glycine (Gly)] on mEPC amplitude is mediated via NO synthase activity. The combined action of Glu (100 μm) and Gly (700 μm) resulted in a 19% increase in mEPC amplitude. The NO synthase inhibitor, L-NAME (100 μm), slightly decreased mEPC amplitude (8% decrease) and fully prevented the effects of Glu and Gly. Data are presented as mean ± SEM, pooled from four to six animals (21–37 endplates). Asterisks indicate a statistically significant difference (*P < 0.05 and **P < 0.0001, respectively) from the control value.
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In additional experiments we attempted to attenuate the effect of amino acids on mEPC amplitude using the membrane-non-permeable NO scavenger haemoglobin (30 μm). Indeed, we did not observe any significant increase in mEPC amplitude after combined application of haemoglobin and amino acids (haemoglobin, 2.79 ± 0.11 nA; glutamate + glycine + haemoglobin, 2.99 ± 0.18 nA; unpaired t-test, t51 = 1.02, P = 0.31).
These results suggest that NMDA receptor activation triggers a long-term Ca2+-dependent pathway, which leads to enhancement of NO production and partial depression of AChE activity.
Increase in miniature endplate current amplitude with N-methyl-d-aspartate receptor activation is associated with phosphatase activity, rather than de novo protein synthesis
Two of the mechanisms most likely to explain the delayed effect of glutamate and glycine on the mEPC time-course are: (i) NMDA receptor activation enhances the synthesis of new proteins (Skinner et al., 2008) and, perhaps, enhances synthesis of NO synthase and (ii) Ca2+ entry through NMDA receptors initiates a Ca2+-dependent pathway that regulates NO synthase by changing its level of phosphorylation (Rameau et al., 2004).
To investigate whether NMDA receptor activation enhances the de novo protein synthesis and/or enhances synthesis of NO synthase, the selective protein synthesis inhibitor, cycloheximide, was applied. Incubation for 180 min with cycloheximide (350 μm) increased mEPC amplitude (cycloheximide, 3.59 ± 0.09 nA; control, 3.23 ± 0.07 nA, unpaired t-test, t69 = 3.21, P = 0.002). After incubation for the same time (180 min), the effect of glutamate and glycine on mEPC amplitude was preserved in the cycloheximide-containing bathing solution (cycloheximide + glutamate + glycine, 4.05 ± 0.14 nA; cycloheximide, 3.59 ± 0.09 nA, unpaired t-test, t56 = 2.78, P = 0.007, Fig. 6). Therefore, it is likely that de novo protein synthesis is not involved in the enhancement of NO production caused by glutamate.
Figure 6. The inhibition of PP1 and PP2A phosphatases, but not inhibition of protein synthesis, prevents the increase in mEPC amplitude induced by glutamate (Glu) and glycine (Gly). Preincubation with the protein synthesis inhibitor, cycloheximide (CHX) (350 μm), did not prevent the increase in mEPC amplitude in the presence of Glu (100 μm) and Gly (700 μm). Application of the PP1 and PP2A phosphatase inhibitor, OA (1 μm), completely prevented the increase in mEPC amplitude associated with Glu and Gly. Data are presented as mean ± SEM pooled from four to seven animals (21–40 endplates). Asterisks indicate a statistically significant difference (*P < 0.05 and **P < 0.0001, respectively) from the control value.
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Rameau et al. (2004) showed that delayed enhancement (40–60 min) of NO production after NMDA receptor activation can be mediated by dephosphorylation of NO synthase by the serine–threonine protein phosphatases 1 and 2A (PP1 and PP2A). The possibility that PP1 and PP2A are involved in the action of glutamate on mEPC amplitude was investigated using OA (1 μm), an inhibitor of PP1 and PP2A. The mean amplitude of mEPCs after pretreatment with OA was increased 14% compared with control (3.68 ± 0.11 nA, unpaired t-test, t68 = 3.62, P = 0.0006). Furthermore, OA fully prevented the action of glutamate and glycine on mEPCs (OA, 3.68 ± 0.11 nA; OA + glutamate + glycine, 3.43 ± 0.10 nA; unpaired t-test, t58 = 1.65, P = 0.104, Fig. 6). Therefore, the activation of NMDA receptors by exogenous glutamate induces inhibition of AChE activity via a pathway involving NO synthase dephosphorylation by a calcium-dependent mechanism.
Activation of N-methyl-d-aspartate receptors by endogenous glutamate results in an increase in miniature endplate current amplitude during high-frequency nerve stimulation
To investigate whether the changes in mEPC parameters can occur during rhythmic high-frequency stimulation, an analysis was conducted of the amplitude and temporal parameters of mEPCs recorded during interstimulus intervals. High-frequency (10 Hz) stimulation was chosen, as this frequency allowed for recording of a sufficient number of mEPCs during the interstimulus intervals. As mEPCs differ significantly between endplates, the parameters of mEPCs recorded at the same endplate at rest and during a high-frequency train of stimulation were compared, in both the absence and presence of glycine. In glycine-free solution, the mEPC amplitude and duration did not change before and during 10 Hz stimulation (before, 2.86 ± 0.16 nA; during, 2.84 ± 0.18 nA; paired t-test, t17 = 0.36, P = 0.72, Fig. 7). However, in the presence of glycine (700 μm), mEPC amplitude increased by 108.7 ± 2.9% during high-frequency stimulation (before vs. during, paired t-test, t16 = 2.33, P = 0.03, Fig. 7).
Figure 7. High-frequency nerve stimulation (10 Hz) increases mEPC amplitude, mediated by endogenous glutamate-induced NMDA receptor activation, with a subsequent elevation in NO production. (A) The average of 100 mEPCs recorded at the same endplate prior to stimulation onset (black line) and during 10 Hz stimulation (gray line) after a 30 min preincubation with 700 μm glycine (Gly). (B) In untreated endplates, mEPC amplitude was unchanged during 5 min of 10 Hz stimulation. Addition of Gly (700 μm) to the bathing solution resulted in a relative increase (9% increase) in mEPC amplitude recorded during interstimulus intervals. Preincubation with either the NMDA receptor blocker, AP-5 (25 μm), or the NO synthase inhibitor, L-NAME (100 μm), eliminated the effects associated with addition of Gly. Control values were recorded prior to stimulus onset. Data are expressed as % control, mean ± SEM, in the same 16–20 endplates (from four animals). Asterisk indicates a statistically significant difference from the control value (paired t-test, *P < 0.05).
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The mEPC duration did not change. Immediately following cessation of the stimulation, the mEPC amplitude tended to decline and was not statistically different from baseline at 5 min after cessation (data not shown).
When nerve–muscle preparations were preincubated with either AP-5 (25 μm) or L-NAME (100 μm), the mEPC amplitude did not change during 10 Hz stimulation performed in a glycine-containing bathing solution. Therefore, it is likely that, during normal synaptic functioning, the increase in synaptic signals due to NO-mediated partial AChE inhibition occurs via NMDA activation by glutamate and glycine. In contrast to the slowly developing effects of exogenous amino acids, the effect of endogenous glutamate appeared immediately after the onset of high-frequency nerve stimulation.