Involvement of Nitric Oxide in Nicotinic Receptor-Mediated Myopathy1

  1. Manar D. El-Dada and
  2. Maryka Quik
  1. Department of Pharmacology, McGill University, Montreal, Quebec, Canada
     
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    Abstract

    Previous studies have shown that nicotinic cholinergic agonists induce muscle cell degeneration. Although an involvement of calcium is well documented, subsequent intracellular steps have not been identified. The present experiments test whether nitric oxide (NO) may play such a role. Both the irreversible nitric oxide synthase inhibitorl-5N-iminoethyl ornithine andl-nitroarginine methyl ester, a reversible inhibitor, protected the muscle cells from the myopathic effects of nicotine. These results may suggest that nicotinic receptor stimulation produces an increase in NO that results in muscle cell degeneration. In line with this interpretation, exposure of the muscle cultures to the NO donor sodium nitroprusside resulted in a dose-dependent decline in myotube branch points. Neither l-5N-iminoethyl ornithine nor nitroprusside altered the binding of the nicotinic receptor agonist 125I-α-bungarotoxin to muscle cells in culture, which indicates that the effect of these agents was not mediated through an interaction at the nicotinic receptor recognition site. The results with agents that inhibit guanylate cyclase or modify extracellular levels of cGMP suggest an involvement of this cyclic nucleotide in the nicotinic receptor-mediated myopathy. To conclude, the present results suggest that nicotinic receptor activation causes skeletal muscle degeneration through an increase in NO production and a possible involvement of cGMP.

    NO is a short-lived, ubiquitous molecule that serves as a mediator of vasodilation (Moncada et al., 1991), platelet aggregation (Radomski et al., 1990a; Lowenstein et al., 1994) and cellular toxicity (Dawson et al., 1991; Xie et al., 1992; Schulzet al., 1995; Dawson, 1995; Przedborski et al., 1996; Schulz et al., 1996). NO also mediates transmission in the CNS and the peripheral nervous system (Dawson et al., 1992; Snyder, 1992; Garthwaite and Boulton, 1995), where it has been implicated in long-term potentiation and depression (Chapman et al., 1992; Lin and Bennett, 1994; Dinerman et al., 1994; O’Dell et al., 1994). NO is synthesized froml-arginine by the enzyme NOS. Three distinct isoforms of NOS have been identified to date. These include two constitutive, Ca++/calmodulin-dependent gene products, of which one is neuronal NOS (nNOS), a cytosolic enzyme that is highly concentrated in brain, neurons of the myenteric plexus, kidney macula densa (Huanget al., 1993) and skeletal muscle (Kobzik et al., 1994). The second constitutive isoform, endothelial NOS (eNOS), is a membrane-bound enzyme (Robinson et al., 1995) present in the endothelium of blood vessels, in the epithelium of many tissues including the bronchial tree, in the pyramidal cells of the hippocampus (O’Dell et al., 1994) and in skeletal muscles (Kobziket al., 1995). The third NOS isoform, the inducible NOS (iNOS), is a cytosolic, Ca++-independent enzyme. iNOS is transcriptionally regulated and is activated by certain cytokines and endotoxin lipopolysaccharides; this form is present in nearly all nucleated cells, including macrophages, Kupffer cells, endothelial cells, fibroblasts, vascular smooth muscle cells and mesangial cells (Radomski et al., 1990b; MacMicking et al., 1995).

    ACh released at the neuromuscular junction mediates its effects by interacting with the muscle-type nicotinic receptor, one of the best-characterized ligand-gated ion channel receptors. In addition to its classical role in neurotransmission, ACh has been shown to affect a variety of growth-related functions ranging from developmental to degenerative processes (Ariens et al., 1969; Fenichelet al., 1972; Leonard and Salpeter, 1979, 1982; Mattson, 1989; Quik, 1995). In analogy to the neurotoxic actions of excitatory amino acids at their receptors (Meldrum and Garthwaite, 1990; Choi, 1990; Dawson et al., 1991), nicotinic ACh receptor activation may also result in damage to the endplate region of skeletal muscle. Inhibition of acetylcholinesterase, which increases ACh levels at the neuromuscular junction, produces an extensive muscle necrosis (Fenichel et al., 1972; Engel et al., 1973;Freeman et al., 1976). The severity of the myopathy was decreased by prior nerve sectioning, by depletion of ACh within the nerve terminal by the ACh transport inhibitor hemicholinium and by application of the nicotinic receptor blockerd-tubocurarine. These results suggest that the response is mediated by ACh through an interaction at the receptor (Ariens et al., 1969; Fenichel et al., 1972; Hudson et al., 1978). In vitro studies have shown that application of the cholinergic agonist carbachol resulted in a calcium-dependent muscle damage that was prevented by nicotinic receptor blockade (Leonard and Salpeter, 1979, 1982). These studies provide evidence for a myopathic/degenerative role mediated by excess ACh released at the neuromuscular junction.

    Interestingly, excitatory amino acids such as glutamate exert, at their receptors, neurotoxic effects that are mediated by an initial flux of calcium into the cell (Meldrum and Garthwaite, 1990; Garthwaite and Boulton, 1995). Accumulating evidence now suggests that glutamate receptor-activated neurotoxicity is mediated by NO (Dawson et al., 1991; Reif, 1993; Zhang et al., 1994; Dawson, 1995; Schulz et al., 1995). These observations raised the question of whether NO may be involved in ACh-induced muscle cell degeneration. nNOS is present in skeletal muscle (Nakane et al., 1993; Kobzik et al., 1994, 1995). Studies also show that NO modulates muscle contractility and relaxation (Kobziket al., 1994, 1995) and is involved in activity-dependent synaptic suppression during development at the neuromuscular synapses (Wang et al., 1995). NO also directly modulates mitochondrial function, the mitochondrial electron transport chain (Schweizer and Richter, 1994) and oxygen consumption in intact skeletal muscle (King et al., 1994).

    The present experiments were done to determine whether NO is involved in agonist-induced myopathy at the neuromuscular junction. For this purpose, neonatal muscle cells in culture were exposed to nicotine in the absence or presence of NOS inhibitors, as well as drugs that release NO. The results are the first to suggest that NO is a second messenger that may mediate the degenerative effects of nicotinic receptor activation in skeletal muscle.

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    Materials and Methods

    Materials.

    The following drugs and chemicals were used in this study: Cytosine arabinose (cytosar), d-tubocurarine, BSA, l-NAME, d-NAME, 8-bromo cGMP and dibutyryl cGMP (Sigma Chemical Co., St. Louis, MO), SNP (Fisher, Montreal, Quebec), NIO (Cayman, Ann Arbor, MI), LY83583 (Research Biochemicals International, Natick, MA) nicotine hydrogen (+)-tartrate (BDH Ltd., Poole, England), trypsin, medium 199, minimal essential medium, horse serum, penicillin and streptomycin (Gibco/BRL, Grand Island, NY) and125I-α-bungarotoxin, 10–20 μCi/μg, (New England Nuclear, Boston, MA).

    Muscle cell culture.

    Rat myotube cultures were prepared, under sterile conditions, from 1 to 2-day-old Sprague-Dawley rats (12–16 pups) as previously described (Braun et al., 1989) with some modifications. Minced muscle from the pectoralis and hind limb was washed in phosphate-buffered saline containing 0.5 mM Mg++. The muscle tissue was dissociated for 45 min in 10 ml 0.25% trypsin, during which time the cells were gently mixed with a magnetic stirrer. Ten milliliters of 0.25% trypsin was then added, and the cells were allowed to dissociate for a further 30 min. An equal volume of Hanks’ balanced salt solution (magnesium- and calcium-free) was added to the cell suspension, which was centrifuged for 10 min at 1000 × g. The supernatant was discarded, and the pellet was resuspended in culture medium that consisted of 65% minimal essential medium, 25% medium 199, 10% horse serum, penicillin (50 U/ml) and streptomycin (50 μg/ml).

    For the experiments involving morphological studies, cells were plated onto 35-mm collagen-coated dishes (Nunc) at a density of 1.1 to 1.3 million cells/dish. After 3 days in culture, the medium was replaced with one containing 10 μM cytosar to limit the proliferation of fibroblasts. Cytosar was removed after 1 or 2 days, and the cells were maintained in regular culture medium to which various concentrations of the drugs were added.

    For the 125I-α-BGT binding studies, cells were plated onto collagen-coated, 24-well multiwell plates at a density of 0.30 to 0.35 million cells/well. Cultures were incubated in a humidified atmosphere of 5% CO2/95% air. Cells were not maintained for more than 8 to 10 days after plating because, with increasing myotube development, enhanced contraction caused cells to lift from the culture plates.

    Assessment of myotube branch formation.

    Numerical analysis of myotube branching was done at various times after plating, using phase-contrast microscopy. Previous studies (Quik et al., 1992) have shown that determination of the number of myotube fusion or branch points provided a good index of muscle cell viability; declines in the number of branch points as a result of nicotine exposure were associated with a corresponding reduction in muscle fiber length and nicotinic receptor binding. To count the number of myotube branch points, we counted a series of fields along the diameter of the plate at 100× magnification for each culture plate. This included 12 or 15 fields per plate, depending on the particular experiment; the same number of fields was counted in any one experiment to ensure an appropriate comparison of control and treated groups. The area of each field was 2.27 mm; thus the areas counted for 12 fields and for 15 fields were 27 and 34 mm, respectively. The number of myotubes ranged from 3 to 30 per field, depending on the condition to which the cells were subjected. Each culture condition was tested in quadruplicate; that is, four separate culture dishes were counted per experimental condition. The data are presented as either percent control or the average number of branch points, when the control number of branch points between platings was very similar.

    125I-α-BGT binding to neonatal muscle cells in culture.

    Radiolabeled α-BGT binding to neonatal muscle cells was done as described (Quik et al., 1992). Before the binding assay, the cells were washed twice with 2 ml of DMEM containing 1 mg/ml BSA. Cells in culture were preincubated for 60 min at 37°C in DMEM containing 10 mg/ml BSA in the presence or absence of the indicated drugs. This was followed by a 90-min incubation at 37°C in the presence of 125I-α-BGT (1.8 nM). Binding was terminated by removal of the medium followed by four 1-ml washes with DMEM containing 0.1% BSA. The cells were then resuspended in 0.5 ml 1.0 N NaOH, and the radioactivity was determined using a gamma counter. Nonspecific binding was defined as the binding in the presence of 10−4 M d-tubocurarine, a concentration that results in a maximal inhibition of 125I-α-BGT binding.

    Statistics.

    Statistical analyses were done on INSTAT software using two-way analysis of variance (ANOVA).

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    Results

    Effects of nicotine on myotube morphology.

    A 24 to 48-h exposure to nicotine was established as the appropriate condition under which to examine drug-induced effects on myotubes. Figure1 shows an example of nicotine-induced muscle cell degeneration; 3 × 10−5 M nicotine (fig. 1B) markedly decreased cell size and the number of branch points in the differentiated myotubes as compared with the control untreated cells (fig. 1A). Muscle cell degeneration was characterized by regression of myotubes with subsequent detachment of the myotube branches from each other and/or the culture dish.

    Figure 1
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    Figure 1

    Phase-contrast photomicrographs of neonatal muscle cells in culture under control conditions and 2 days after nicotine exposure. Cells were plated and maintained in culture for 3 days, after which time the medium was changed to one containing 10 μM cytosar. One day later, cytosar was removed and nicotine or buffer added. A) Control cells. B) Cells exposed to 3 × 10−5 M nicotine. Note the decline in the number of branch points and muscle fiber length after nicotine treatment compared with control. Magnification 100×.

    Effect of nicotinic antagonists on the nicotine-induced myopathy.

    To determine whether the nicotine-induced muscle degeneration was due to a specific interaction at nicotinic receptors, we examined the effect of the nicotinic receptor antagonistd-tubocurarine. Muscle cultures were preincubated with 10−4 M d-tubocurarine for 2 h before nicotine addition. Twenty-four hours later, muscle cell viability (branching) was assessed using phase-contrast microscopy. Figure2 illustrates that nicotine resulted in a dose-dependent decline in the number of muscle branch points that was statistically significantly different from control at nicotine concentrations of 10−6 M, 10−5 M, 3 × 10−5 M and 10−4 M (*P < .05, **P < .01). The data in figure 2 depict the effect of nicotine on branch point formation after a 1-day exposure period; the highest concentration of nicotine used was 10−4 M because previous work has shown that a maximal effect was observed with 3 × 10−5 M (Quik et al., 1992). To observe a greater reduction in the number of branch points, a longer exposure time is required.

    Figure 2
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    Figure 2

    The effect of the nicotinic receptor antagonistd-tubocurarine on the nicotine-induced decrease in myotube branching. Muscle cells were incubated in the absence of presence ofd-tubocurarine (10−4 M); nicotine/buffer was added 2 h later. Changes in cell morphology were assessed after 24 h. Nicotine decreased the number of branch points in a dose-dependent manner; this was prevented by d-tubocurarine. The results are the mean ± S.E.M. of five culture dishes and are representative of two separate experiments. Significance of difference from control in the absence of nicotine: *P < .05, **P < .01.

    The nicotinic antagonist d-tubocurarine (10−4M) completely prevented the degenerative effects of nicotine on the muscle cells in cultures, which indicates that the effect of nicotine was receptor-mediated.

    Effects of NOS inhibitors on nicotine-induced muscle cytotoxicity.

    To test whether NO is involved in the nicotine-induced effects on the muscle cultures, we added the irreversible NOS inhibitor NIO to the muscle cultures in the presence or absence of nicotine. The degree of branching was assessed after 24 h. NIO, at either 3 × 10−5 M or 10−4 M, did not significantly affect myotube branching when compared with the control untreated condition (fig.3). As previously shown, nicotine at 3 × 10−5 and 10−4 M induced a decrease in the myotube number of branch points. On the other hand, when cultures were exposed to both nicotine and NIO (3 × 10−5 M and 10−4 M), the nicotine-induced myopathy was partially prevented (fig. 3); we observed almost complete protection of the nicotine (3 × 10−4 M)-induced effect with 10−4 M NIO. The results with 3 × 10−4 M NIO were similar to those observed with 10−4 M NIO. No protection was observed with the lower concentration of 10−5 M.

    Figure 3
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    Figure 3

    Effect of the irreversible nitric oxide inhibitor NIO on the nicotine-induced decrease in muscle branch points. Muscle cells were incubated with different concentrations of nicotine in the absence or presence of 3 × 10−5 M NIO and 10−4 M NIO. Note the partial prevention of the degenerative effects of nicotine by different NIO concentrations. The results are the mean ± S.E.M. of five culture dishes and are representative of four separate experiments. Significance of difference from nicotine in the absence of NIO: *P < .05, **P < .01.

    The results of the experiments with NIO prompted us to test the effects of the reversible NOS inhibitor l-NAME. When cultures were treated with both l-NAME and nicotine, there was a significant reversal of the nicotine-induced myopathy at all concentrations of l-NAME tested. On the other hand,d-NAME, the biologically inactive enantiomer, had no effect (fig. 4). It should be noted that the culture medium in which the muscle cells were grown contains l-arginine (0.5 mM); for this reason, relatively high concentrations of the reversible NOS inhibitor l-NAME were required. Lower concentrations ofl-NAME (<1.0 mM) were tested but had no effect; this is probably due to a competition between the inhibitor and thel-arginine in the culture medium. Experiments were not done with culture medium lacking l-arginine, because this amino acid is required for normal cell growth and maintenance. Collectively, the results with NOS inhibitors suggest a role for NO in nicotine-induced degeneration of muscle cells.

    Figure 4
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    Figure 4

    The effect of the reversible NOS inhibitorl-NAME or the biologically inactive enantiomerd-NAME on the nicotine-induced decrease in muscle branch points. Muscle cells in culture were exposed to the indicated concentrations of l-NAME, d-NAME or buffer. Nicotine ( 3 × 10−5M) or buffer was subsequently added. The number of myotube branch points were counted 24 h later. Note the reversal of the nicotine-induced decrease in the number of branch points at all l-NAME, but not d-NAME, concentrations. The results are the mean ± S.E.M. of four culture dishes and are representative of four and of two experiments forl-NAME and d-NAME, respectively. Significance of difference between control and nicotine-treated: aP < .001. Significance of difference between nicotine alone and nicotine in the presence of various concentrations of l-NAME:bP < .001. Significance of difference between control and either nicotine-treated only or nicotine-treated in the presence of various concentrations of d-NAME: cP < .01.

    Effect of the NO donor SNP on the nicotine-induced myopathy.

    Because the NOS inhibitors protect against the effects of nicotine, it is conceivable that NO donors result in degenerative effects on the muscle cultures and/or increase the myopathic effects of nicotine. To test this hypothesis, we exposed the muscle cultures to the spontaneous NO donor SNP in the absence or presence of 10−5 M and 3 × 10−5 M nicotine. SNP was also added 4 to 6 h after the initial exposure to drugs. SNP resulted in a dose-dependent decrease in the number of branch points (fig. 5B; fig.6) after a 24-h exposure as compared with control (fig.5A). SNP was not tested at higher concentrations because 10−5 M is considered a fairly high concentration; however, more marked reduction in the number of branch points was observed when the cultures were counted at 48 h rather than 24 h. Muscle cultures were then exposed to increasing concentrations of SNP in combination with nicotine (table 1). Nicotine (10−5 M and 3 × 10−5 M) resulted in a 35 ± 12% and a 22 ± 6% decrease, respectively, in the number of branch points as compared with the control condition (100 ± 5%). When muscle cultures were exposed to either 10−5 M or 3 × 10−5 M nicotine and 3 × 10−6 M SNP in combination, the decrease in muscle branch points was similar to that observed with nicotine alone. These results suggest that SNP mimics the degenerative effects of nicotine, possibly by acting through a common pathway, or, alternatively, through a distinct pathway that results in common morphological manifestations.

    Figure 5
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    Figure 5

    Phase-contrast photomicrographs of the effect of the nitric oxide donor SNP on muscle cell morphology. A) Control cells. B) Cells after a 1-day exposure to 10−5M SNP. Note the reduction in branching after SNP treatment compared with control.

    Figure 6
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    Figure 6

    Effect of SNP on muscle cell branching. Muscle cells were incubated for 2 days in the absence or presence of various concentrations of SNP. Note the dose-dependent decrease in the number of branch points after SNP treatment. The results are the mean ± S.E.M. of five culture dishes and are representative of three separate experiments. Significance of difference between control and SNP-treated: *P < .01; **P < 0.001.

    View this table:
    Table 1

    Effect of SNP and nicotine on the number of myotube branch points

    Effect of drugs that modify NO levels on 125I-α-BGT binding to muscle cells.

    To investigate the possibility that the NOS inhibitor NIO and the NO donor SNP exert their effect through a direct interaction at the nicotinic receptor, we determined the effect of these drugs on 125I-α-BGT binding to neonatal muscle cultures. Table 2 shows that neither NIO nor SNP altered specific binding of 125I-α-BGT to the nicotinic receptors.

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    Table 2

    Effect of SNP and NIO on 125I-α-BGT binding to muscle cells in culture

    Effect of cGMP analog and a guanylate cyclase inhibitor on nicotinic receptor-mediated muscle cell degeneration.

    Because NO mediates many of its actions through cGMP, experiments were done to determine whether the nicotinic receptor-mediated effect could be reversed by a guanylate cyclase inhibitor. LY83583 was selected because previous studies had suggested that this agent was fairly selective in inhibiting guanylate cyclase (Mulsch et al., 1989). The results given in table 3 show that exposure of the cells to the inhibitor completely prevented the degenerative effects induced by exposure of the cells to 3 × 10−5 M nicotine. The guanylate cyclase inhibitor alone had no effect on muscle cell morphology.

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    Table 3

    Effect of a guanylate cyclase inhibitor and cGMP analogs on the nicotine-induced decline in the number of myotube branch points

    Experiments were subsequently done to determine whether cyclic GMP analogs might mimic the effect of nicotine. Surprising, both cGMP analogs, 8-bromo cGMP and dibutyryl cGMP, also reversed the nicotine-induced muscle cell degeneration. As is evident from the results depicted in table 3, exposure of the cultures to cGMP analogs protected against the nicotinic receptor-mediated degenerative effects, whereas the cGMP analogs on their own had no significant effect.

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    Discussion

    The present results show that exposure of neonatal muscle cultures to nicotine resulted in a dose-dependent decline in myotube branching, which was blocked by the nicotinic receptor antagonistd-tubocurarine. On its own, the nicotinic receptor blockerd-tubocurarine (10−4 M) affected neither the degree of myotube branching nor the morphology of the muscle cell. These results are in line with previous studies showing that the administration of acetylcholinesterase inhibitors, which result in an increased level of ACh, produce extensive muscle necrosis through a nicotinic receptor-mediated mechanism.

    Experimental evidence indicates that activation of the nicotinic receptor at the neuromuscular junction leads to a small calcium influx (Decker and Dani, 1990), approximately 2% of the current carried through the receptor being attributable to calcium under physiological conditions. Although this is not large, there may be a higher local calcium accumulation, particularly because voltage-gated calcium channels may also be activated and could contribute to a larger calcium influx than that mediated by the nicotinic receptor alone. This calcium entry may play a role in the rapid regulation of synaptic function, as well as in long-term processes such as agonist-induced myopathy (Leonard and Salpeter, 1979, 1982). Subsequent molecular pathways mediating this myopathy have not been identified. However, evidence has shown that NO is involved in calcium-mediated cellular toxicity in other systems and that NOS is present in skeletal muscle and is a physiological modulator of skeletal muscle function (Kobzik et al., 1994). Moreover, nNOS is also concentrated at the synaptic junctions of the motor end plates in skeletal muscles (Brenman et al., 1995), colocalizing with both α1-syntrophin, a dystrophin-associated protein, and nicotinic receptors (Brenmanet al., 1996). Recently, Brenman et al. (1996)have demonstrated that nNOS is associated with α1-syntrophin and that this association is lost in Duchenne muscular dystrophy, which suggests a possible link between nNOS and nicotinic receptors at the neuromuscular junction in muscle pathology.

    The present work indicates that NO may also be involved in nicotinic receptor-mediated myopathy. Evidence for this stems from experiments showing that the reversible NOS inhibitor l-NAME and NIO, an irreversible inhibitor of NOS, both resulted in a dose-dependent inhibition of the myopathic effects of nicotine. Thus, inhibition of NOS partially protected muscle cells against the myopathy. These results suggest that nicotinic receptor activation leads to myopathyvia the production of NO. This interpretation is further supported by the observation that the NO donor SNP also resulted in a dose-dependent myotube degeneration similar to the nicotinic receptor-mediated effect.

    The results of the binding experiments indicate that neither the NO donor SNP nor an NOS inhibitor such as NIO exerts its effects through an interaction at the nicotinic receptor recognition site. However, it is still possible that these agents affect nicotinic receptor channel function. If they interact at the channel, then they are likely to act as channel blockers. In this case, they might be expected to produce results similar to those of a competitive antagonist such asd-tubocurarine. However, d-tubocurarine alone had no effect on muscle cell morphology, whereas SNP induced muscle cell degeneration. Thus SNP, at least, is probably not acting as a channel blocker. If the NOS inhibitors were to act as channel blockers (although this has not previously been shown), then they would be expected to protect against myopathy, which they did. On the other hand, the NOS inhibitors may also exert their effect on channel function through alterations in NO, because NO has been shown to act as a regulator of muscle activity (Kobzik et al., 1994). Experiments are in progress to assess these possibilities by determining the effect on nicotinic receptor-mediated function and assessing the effect of nicotine on NOS activity in cells in culture.

    It is consistent with our results demonstrating a myopathic effect of nicotine through NO that the increased production of this second messenger has been linked to diverse pathophysiological conditions in other systems, including septic- and cytokine-induced circulatory shock (Moncada et al., 1991) and vascular disorders. In addition, NO has been implicated in ischemic brain damage (Nowicki et al., 1991; Reif, 1993; Zhang et al., 1994; Dawson, 1995; Schulz et al., 1995) due to an overstimulation of CNS glutamate receptors. More recent studies have also shown that inhibitors of nNOS play a neuroprotective role against NMDA-kainic acid- and AMPA-induced excitotoxicity (Schulz et al., 1995) and that MPTP-induced Parkinsonism in mice was prevented by the nNOS inhibitor 7-nitroindazole (Pzrzedborski et al., 1996). Neurotoxicity was also attenuated in neuronal NOS knockout mice (Schulzet al., 1996).

    Activation of guanylate cyclase, which leads to an increase in cGMP levels, is one of the major pathways through which NO exerts its physiological effects (Radomski et al., 1990a; Moncadaet al., 1991; Schmidt et al., 1993; Lowensteinet al., 1994). NO activates the soluble guanylate cyclase by binding to the heme moiety of the enzyme. This results in an increased cGMP concentration, which is associated with a host of functional effects, including the modulation of skeletal muscle activity (Kobziket al., 1994, 1995) and NMDA receptor-mediated excitoxicity (Choi, 1990; Nowicki et al., 1991; Snyder, 1992; Southam and Garthwaite, 1993), two findings that are of immediate relevance to the present study. In addition, nicotinic ACh receptor activation has been shown to increase cGMP levels in skeletal muscle (Nestler et al., 1978), and Briggs (1992) has demonstrated that cGMP analogs and NO generators potentiated nicotinic transmission in rat superior cervical sympathetic ganglion. The present results show that exposure of the cells to a guanylate cyclase inhibitor also protects against the effect on nicotinic receptor-mediated function. These data might imply that the effects of nicotinic receptor activation in the present system are mediated through activation of guanylate cyclase; however, the observation that a similar protective effect was observed with the cGMP analogs suggests that any effects mediated by cGMP are complex in nature. A possible conclusion is that the second messenger cGMP is involved but that the exact nature of its involvement remains to be elucidated.

    A wide variety of neuromuscular disorders are characterized by muscle cell injury and degeneration, which may be associated with inflammation (Engel et al., 1994). The induction of iNOS, a high-output source of NO, is relevant to the pathophysiology of muscle disorders. iNOS is activated by infiltrating lymphocytes and macrophages in inflammatory associated myopathies (Engel et al., 1994;Dalakas, 1994). Further evidence for the role of iNOS in myopathy stems from the beneficial effects of anti-inflammatory glucocorticoids in treating muscle tissue pathologies (Kaplan et al., 1990). Glucocorticoid inhibition of iNOS expression has been confirmed in many cellular systems, such as macrophages, endothelial cells (Radomskiet al., 1990a, 1990b) and muscle tissue (Moncada et al., 1991). This observation may explain the effectiveness of glucocorticoid therapy in protecting muscle tissue integrity against inflammatory processes.

    To conclude, the present results show that NOS inhibitors prevented the degenerative effects of nicotine on muscle cells, whereas SNP, a NO donor, resulted in muscle cell degeneration. These findings suggest that overstimulation of the nicotinic receptor may result in pathophysiological processes through an activation of NOS and subsequent production of NO, which in turn activates other cellular processes, leading ultimately to muscle cell injury and death.

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    Footnotes

    • Send reprint requests to: Dr. M. Quik, Parkinson’s Institute, 1170 Morse Ave, Sunnyvale, CA 94089.

    • ↵1 The authors gratefully acknowledge support from the Medical Research Council, Canada, and the Muscular Dystrophy Association, United States.

    • Abbreviations:
      α-BGT
      α-bungarotoxin
      BSA
      bovine serum albumin
      cytosar
      cytosine arabinoside
      DMEM
      Dulbecco’s modified Eagle’s medium
      d-NAME
      d-nitroarginine methyl ester
      l-NAME
      l-nitroarginine methyl ester
      NIO
      l-5N-iminoethyl ornithine
      NO
      nitric oxide
      NOS
      nitric oxide synthase
      SNP
      sodium nitroprusside
      • Received October 3, 1996.
      • Accepted February 4, 1997.
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