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J Physiol Volume 559, Number 2, 423-432, September 1, 2004 DOI: 10.1113/jphysiol.2004.064469
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Nitric oxide, cAMP and the biphasic muscarinic modulation of ACh release at the lizard neuromuscular junction

Austin R. Graves, Katherine A. Lewin and Clark A. Lindgren

Department of Biology, Grinnell College, Grinnell, IA 50112, USA


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we characterized the pharmacology and physiology of the automodulation of ACh release at the lizard neuromuscular junction (NMJ). The activation of muscarinic ACh receptors generated a biphasic modulation of synaptic transmission. Muscarine-induced activation of M3 receptors (0–12 min) decreased release, whereas M1 activation (> 12 min) enhanced release. Both phases of the biphasic effect are dependent on nitric oxide. However, cAMP acting via protein kinase A is also necessary for the M1 effect. In summary, we present a novel biphasic role for muscarine and implicate M3 receptors in the inhibition and M1 receptors in the enhancement of transmitter releaseat the cholinergic lizard NMJ.

(Received 16 March 2004; accepted after revision 30 June 2004; first published online 2 July 2004)
Corresponding author C. A. Lindgren: Department of Biology Grinnell College 1116 8th Avenue Grinnell, IA 50112, USA. Email: lindgren{at}grinnell.edu


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Though it is generally accepted that muscarinic acetylcholine receptor (mAChR) activation modulates the release of ACh from motor nerve terminals, there exists disagreement as to the precise effects of muscarinic agonists. Ganguly & Das (1979) suggested that oxotremorine increases ACh release in rats. However, several more recent studies have shown that muscarine and oxotremorine inhibit ACh release from nerve terminals at the neuromuscular junctions (NMJs) of mammals and amphibians (Duncan & Publicover, 1979; Michaelson et al. 1979; Standaert, 1982; Wali et al. 1988; Felder, 1995; Slutsky et al. 1999, 2001; Minic et al. 2002). It has been suggested that this depression is mediated by the activation of mAChRs, possibly located on the closely associated glial cells (Georgiou et al. 1994; Lindgren et al. 1997; Bourque & Robitaille, 1998; Robitaille, 1998; Slutsky et al. 1999; Prothero et al. 2000). Furthermore, Slutsky et al. (1999) demonstrated that muscarine enhanced release in the presence of methoctramine, a selective M2 antagonist, but inhibited release in the presence of pirenzepine, a selective M1 antagonist, suggesting that M2 receptors mediate inhibition, while the M1 receptor subtype mediates enhancement. There is also evidence that the M3 subtype controls synaptic depression in the central nervous system (Hsu et al. 1995).

In addition to controversy over the precise effects of muscarinic agonists and the receptor subtypes that are involved, relatively little is known about the mechanism(s) by which this modulation occurs. The presence of nitric oxide synthase (NOS) at the vertebrate NMJ suggests that nitric oxide (NO) may be the signal molecule involved in the feedback depression (Jahromi et al. 1992; Lindgren & Laird, 1994; Prast et al. 1998; Descarries et al. 1998; Castonguay et al. 2001). However, the relationship of NO to these muscarinic effects has not been systematically tested.

This study endeavored to clarify the nature of muscarine's effects on ACh release at the lizard NMJ. We discovered a temporally biphasic modulation of synaptic transmission, wherein muscarine – acting via M3 receptors – first decreased release (0–12 min), then enhanced ACh release (> 12 min) by activating M1 receptors. Both phases of the biphasic effect are dependent on NO, while cAMP-dependent protein kinase A (PKA) is necessary only for the M1 effect. In summary, we propose a novel biphasic automodulation of ACh release that involves the M1 and M3 subtypes of the mAChR and requires the synthesis and extracellular diffusion of nitric oxide. Some of the results reported here have appeared in preliminary form (Lindgren & Young, 2002; Lindgren et al. 2003).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Lizard preparation and solutions

The ceratomandibularis muscle of the lizard, demonstrated as a good model system for studying various features of synaptic transmission (Lindgren & Moore, 1989), was isolated from American chameleons (Anolis carolinensis; Trans-Mississippi Biological Supply). All of the experiments were approved by the Animal Welfare Committee/Institutional Review Board of Grinnell College. Prior to being pithed, the lizards were placed at 7–10°C for 8–10 min to slow them down and facilitate the quick and accurate ablation of the forebrain. The ceratomandibularis muscle (with its associated nerve) was isolated and pinned down in a Sylgard-coated chamber containing fresh Ringer solution composed of 158 mM NaCl, 2 mM KCl, 2 mM MgCl2, 5 mM Hepes, 2 mM CaCl2, and 2 g l–1 of dextrose (pH adjusted to 7.4 using NaOH). Fresh Ringer solution was made daily from stock solutions. During the dissection, the bath solution was changed every 5 min to increase the longevity of the preparation. Evoked end-plate potentials (EPPs) were reduced below the action potential threshold of the muscle by applying 10 µM tubocurarine chloride (Sigma-Aldrich). All chemicals were purchased from Sigma-aldrich, St Louis, MO, USA.

Applications of all drugs were conducted in the same manner: the muscle was bathed in the given concentration of the drug dissolved in fresh Ringer solution. With the exception of the NO donor DEA-NO, which we prepared immediately before application (at 100 µM), aliquots were prepared ahead of time, frozen, and then diluted to the following concentrations at the beginning of the experiment: 5 µM muscarine, 10 µM pirenzepine, 1 µM 4-DAMP, 1 mM L-NAME, 40 µM carboxy-PTIO, 200 µM 8-bromo cAMP, and 20 µM Fragment 14–22 PKA inhibitor. All chemicals were purchased from Sigma-Aldrich, St Louis, MO, USA.

Electrophysiology and data analysis

We elicited EPPs by stimulating the motor nerve axon with a continuous train of depolarizing square pulses of 1–10 V, for 0.04 ms, at 0.5 Hz (Grass S88 Stimulator). d-Tubocurarine chloride (10 µM; Sigma-Aldrich) was applied to reduce EPP amplitudes below threshold for eliciting an action potential, and subsequent contraction, in the muscle. EPPs were measured using standard intracellular recording techniques and glass microelectrodes (filled with 3 M KCl, resistance ~15 M{Omega}). Membrane potentials were amplified with a Dagan 8700 Amplifier and collected with a MacLab (AD Instruments) data acquisition system. EPP amplitudes were measured after averaging 16 individual EPPs. Each trial (n) represents the mean EPP amplitude recorded at six to eight different locations (i.e. NMJs) in a single preparation. Resting membrane potentials were between –70 and –90 mV. We evaluated statistical significance of data using the Student's t test, taking P < 0.05 as significant. The same procedure was used to measure spontaneous miniature end-plate potentials (MEPPs), except in this case d-tubocurarine chloride was not applied and the motor nerve was not stimulated.

Immunofluorescence

Muscles were fixed in 3% paraformaldehyde for 10 min, rinsed for 30 min with Ringer solution, permeablized for 30 min at 37°C in 0.3% Triton X-100, pre-incubated for 15 min at room temperature in blocking solution (0.01% Triton X-100, 4% non-fat dry milk in Ringer solution), and incubated in primary antibody (10 µg ml–1 Ringer solution) for 5 h at room temperature and then for 10 h at 5°C. Muscles were rinsed for 1 h in blocking solution, incubated with FITC-conjugated goat anti-rabbit secondary antibody (5 µg ml–1 Ringer solution) for 2 h at 37°C, rinsed in blocking solution for 30 min, immersed in Slowfade Antifade buffer for 10 min, and mounted on slides with Slowfade Antifade solution and 20% glycerol in Ringer solution. Immunofluorescence was observed using either an Olympus BX-51 fluorescence microscope equipped with a Q-Imaging digital video camera or a laser scanning confocal microscope (Noran Odyssey attached to a Zeiss Axiovert).

To visualize the perisynaptic Schwann cells (PSCs), some muscles were incubated for 10 min at room temperature with 0.5 or 1.0 µM POPO-3 iodide nucleic acid stain immediately following permeablization. To visualize nerve terminals, other muscles were incubated for 10 min at room temperature with tetramethylrhodamine {alpha}-bungarotoxin nicotinic ACh receptor (nAChR) stain (0.1 mg ml–1) prior to permeablization, prior to incubation with Slowfade Anti-fade buffer, or following the NADPH-diaphorase reaction.

NADPH-diaphorase (NADPH-d) histochemistry

Muscles were fixed for 10 min in 3% paraformaldehyde, rinsed for 30 min in Ringer solution (pH 8.0), permeablized for 30 min in 0.3% Triton X-100 in Ringer solution (pH 8.0), and incubated for 75 min at 37°C in NADPH-d reaction solution (1 mg ml–1 ß-NADPH, 1 mg ml–1 Nitro Blue Tetrazolium in Ringer solution; pH 8.0). In some cases, muscles were incubated with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, as a control. In these experiments, 1 mM L-NAME was included in 0.3% Triton X-100 in Ringer solution (pH 8.0), muscles were incubated with 1 mM L-NAME in Ringer solution (pH 8.0) for 60 min prior to the NADPH-d reaction, and 1 mM L-NAME was included in the NADPH-d reaction solution. As an additional control, ß-NADPH was omitted from the reaction solution in one experiment.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Biphasic effects of muscarine: immediate depression and delayed facilitation

Muscarinic ACh agonists modulate synaptic transmission at the lizard neuromuscular junction (NMJ) in a temporally biphasic manner. End-plate potential (EPP) amplitude following bath application of muscarine first decreased (36.9 ± 3.9% change from control, 0–12 min, n = 7, P < 0.03) then increased (110.8 ± 3.5% increase from control, beyond 12 min, n = 11, P < 0.02, Fig. 1A–C). This biphasic change reflects two partially superimposed effects, an immediate depression and a delayed facilitation. Oxotremorine-M, another muscarinic agonist, demonstrated qualitatively similar effects at the lizard NMJ (data not shown), but muscarine was the primary agonist used in our study as it neither has postsynaptic effects at cholinergic NMJs nor is it preferentially selective for any subtype of muscarinic ACh receptor (mAChR; Slutsky et al. 2001).



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  		Figure 1.  Biphasic effect of muscarine
A, 5 µM muscarine yields two distinct effects at the lizard NMJ. Between 0 and 12 min after muscarine addition, the agonist depresses EPPs. Beyond 12 min after application, muscarine enhances EPPs. The ‘IM’ bar depicts the average effect of immediate 5 µM muscarine, the ‘DM’ bar depicts the effects of delayed 5 µM muscarine, and the ‘R’ bar depicts Rinse. Error bars represent standard error of the mean. P values were calculated by a Student's paired t test. *Statistical significance relative to control. B, trace graph depicting typical EPPs recorded before (Control), after immediate and delayed application of 5 µM muscarine, and after rinsing with control saline (Rinse). C, summary of the time courses of the biphasic effect. A best fit curve is shown; n = 1. Application of 5 µM muscarine was at time = 0.

 
Muscarine's effects are presynaptic

To determine whether the biphasic modulation of EPP amplitude was due to a change in the amount or ACh released (i.e. a presynaptic effect) or due to a change in the sensitivity of the nicotinic ACh receptors (nAChR) in the muscle membrane (i.e. a postsynaptic effect) spontaneous miniature end-plate potentials (mEPPs) were recorded at neuromuscular junctions both before and during exposure to muscarine. The mean mEPP amplitude never varied by a statistically significant amount during the course of three experiments. The result from one such experiment is shown in Fig. 2. Although in this experiment we observed a gradual increase in mEPP amplitude, when the amplitudes were clustered into three time intervals (0–4 min before application of muscarine, 6–10 min after and 14–18 min after the application of muscarine) none of the mean values from each cluster were significantly different from either of the other two. Furthermore, the overall change was far too small to account for the magnitude of change of evoked EPP amplitudes observed following the application of muscarine (see Fig. 1). Thus, the biphasic effect of muscarine appears to be due to a presynaptic change (i.e. a change in ACh release or quantal content) and not due to a postsynaptic change (i.e. a change in nAChR sensitivity). Therefore, throughout this paper we attribute muscarine-induced changes in evoked EPP amplitude to changes in ACh release.



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  		Figure 2.  Muscarine does not change miniature EPP amplitude
The effect of muscarine on spontaneous miniature end-plate potential (mEPP) amplitude was examined. Each data point represents the mean mEPP amplitude within a 30 s time interval bin. Data points correspond to the left axis. Each bar represents the mean mEPP amplitude over a 4 min interval (4 min prior to application of muscarine, 6–10 min after, and 14–18 min after application of muscarine). Bars correspond to the right axis, which is also mean mEPP amplitude (mV). Error bars denote standard error of the mean. There is no significant difference between the means of any of the intervals. Muscarine (5 µM) was applied at 0 min, as denoted by the black bar. The membrane potential ranged from –86 to –90 mV. No tubocurarine chloride was present in the Ringer solution.

 
Using the same data set we also determined the effect of muscarine on mEPP frequency. The mean mEPP frequency was significantly reduced – compared to control – following 6 min of muscarine application (P < 0.05). This decrease in frequency is consistent with the immediate (0–12 min) decrease in evoked EPP amplitude observed following the application of muscarine (see Fig. 1). Due to the large variability of mEPP frequency and the difficulty we had in maintaining a good long-term intracellular recording in a single cell, we were unable to conclusively determine whether there was an increase in mEPP frequency that might correspond with the delayed (> 12 min) increase in evoked EPP amplitude (Fig. 1). This warrants further investigation (see Discussion).

Pharmacological characteristics of biphasic muscarinic effect

As expected, the non-selective M1–M5 antagonist atropine (1–10 µM) was sufficient to block both the immediate depression and delayed enhancement normally induced by 5 µM muscarine (data not shown). To determine the specific subtype(s) of receptors that mediate the muscarine-induced depression and enhancement of EPPs at the lizard NMJ, selective muscarinic antagonists were applied. There is considerable evidence that the M1 receptor subtype controls enhancement of release at several cholinergic synapses (Caulfield, 1993; Slutsky et al. 1999). To determine whether this subtype is responsible for the delayed enhancement observed at the lizard NMJ, the selective M1 antagonist pirenzepine was applied. As shown in Fig. 3, pirenzipine (10 µM) blocks the delayed enhancement of EPPs by muscarine (1.06 ± 6.32% increase from control, n = 5, P < 0.02 relative to delayed muscarine). However, pirenzepine does not affect the immediate depression caused by muscarine (43.6 ± 4.2% decrease from control, n = 4). It is worth noting that by blocking the delayed enhancement of ACh release with pirenzipine it was possible to observe the time course of the immediate depression per se. The depression persisted for approximately 20 min. Thus, during the period of time from 10 to 20 min after the application of muscarine the two phases, depression and enhancement, overlap.



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  		Figure 3.  Pharmacology of the biphasic effects of muscarine
Effect of pirenzepine and 4-DAMP on both phases (immediate and delayed) of muscarine's effects. The bar labelled ‘M’ depicts the average effect of 5 µM muscarine, the ‘M + P’ bar depicts the average effect of muscarine and 10 µM pirenzepine, the ‘M + D’ bar depicts the average effect of muscarine and 1 µM 4-DAMP, and the ‘R’ bar depicts Rinse. Bars represent mean percentage change from control EPP amplitude. Error bars represent standard error of the mean. P values were calculated by a Student's paired t test. *Statistical significance relative to control. Pirenzepine knocks out the delayed facilitation, suggesting M1 receptors mediate enhancement. 4-DAMP knocks out the immediate depression induced by muscarine, suggesting M3 receptors control depression.

 
Rouse et al. (1998) demonstrated that the M2 receptor subtype is the most common presynaptic muscarinic receptor at the rat granule cell synapse and Slutsky et al. (1999) demonstrated that muscarine enhanced neurotransmitter release in the presence of methoctramine, a selective M2 antagonist, at the amphibian NMJ; thus, it seemed reasonable that M2 receptors may mediate the inhibitory effect of muscarine on ACh release at the lizard NMJ. To investigate the role of the M2 receptor, we applied the selective M2 antagonist methoctramine. Unexpectedly, methoctramine (750 nM–10 µM) irreversibly abolished EPPs. This may have been due to a secondary effect of methoctramine on a high-affinity GTPase activity of G proteins (Daeffler et al. 1999). Concentrations of methoctramine below 750 nM did not have any discernable effects on EPP amplitude. Since we could not obtain an alternative selective M2 antagonist, we abandoned the investigation of the M2 receptor in synaptic transmission at the lizard NMJ.

There is some evidence that the M3 receptor subtype mediates depression at cholinergic synapses (Hsu et al. 1995; D'Agostino et al. 2000). Thus, the selective M3 antagonist 4-DAMP was applied to investigate the role of M3 receptors in synaptic modulation at the lizard NMJ. As can be seen in Fig. 3, the immediate depression induced by muscarine was eliminated by 4-DAMP (1 µM), holding EPPs at control levels (0.82 ± 3.99% decrease from control, n = 4, P < 0.02 relative to immediate muscarine). In contrast, the muscarine-induced delayed enhancement was not affected by 4-DAMP (98.9 ± 4.2% increase from control, n = 4), as EPP values were not significantly different from muscarine per se.

Endogenous ACh activates muscarinic receptors

It has long been suggested that endogenous ACh binds to muscarinic receptors at the NMJ, thus tonically inhibiting ACh release (Katz & Miledi, 1977; Slutsky et al. 1999). When we applied atropine (1 µM) in the absence of exogenous muscarine, EPP amplitude increased relative to control (data not shown), a result complementary to that of Slutsky et al. (1999). To determine whether endogenous ACh activates either or both the M1 and M3 muscarinic receptor subtypes, we applied their respective antagonists, pirenzipine and 4-DAMP, in the absence of exogenous muscarine. (It should be pointed out that the nerve was continuously stimulated at 0.5 Hz to make these measurements of EPP amplitude.) The data presented in Fig. 4 suggests that both subtypes are activated by endogenous ACh. Selective inhibition of the M3 receptor subtype increases EPP amplitude in the absence of exogenous muscarine (9.92 ± 2.27% increase from control, n = 4, P < 0.05). In contrast to this, selective inhibition of the M1 receptor subtype decreases mean EPP amplitude (21.1 ± 4.4% decrease relative to control, n = 11, P < 0.04 relative to control, Fig. 4). Thus, it appears that endogenously released ACh – during low-frequency nerve stimulation (0.5 Hz) – has two simultaneous effects: inhibition and excitation. Our observation, along with those of others (Katz & Miledi, 1977; Slutsky et al. 1999), that atropine enhances EPP amplitude in the absence of exogenous muscarine, further suggests that endogenously released ACh has a stronger inhibitory effect, mediated via M3 receptors, than an enhancing effect, mediated via M1 receptors.



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  		Figure 4.  Activation of M1 and M3 receptors by endogenous ACh
Effect of pirenzepine and 4-DAMP on EPP amplitude in the absence of exogenous muscarine. The ‘Pir’ bar depicts the average change in EPP amplitude relative to control following application of 10 µM pirenzepine and the ‘4-DAMP’ bar depicts the average effect of 1 µM 4-DAMP. Error bars represent standard error of the mean. P values were calculated by a Student's paired t test. *Statistical significance relative to control. Presumably pirenzepine reduced EPP amplitude by blocking the tonic activation of M1 receptors and 4-DAMP increased EPP amplitude by blocking the activation of M3 receptors by endogenous ACh.

 
NO is necessary for both phases of muscarinic effect

To determine whether NO plays a role in the biphasic muscarinic effect, muscles were pretreated with the NOS inhibitor L-NAME or the extracellular NO chelator carboxy-PTIO, and the effects of muscarine were examined. Figure 5 shows that L-NAME abolished both phases of the muscarinic effect: immediate (0.8 ± 6.3% decrease from control, n = 5) and delayed (0.25 ± 6.28% decrease from control, n = 4). This suggests that NO production via NOS is necessary for both the muscarine-induced depression and enhancement of ACh release. In similar fashion, carboxy-PTIO blocks both the immediate (2.00 ± 7.7% decrease from control, n = 5) and delayed effect of muscarine (4.6 ± 8.8% increase from control, n = 4), implying that NO must diffuse through the extracellular space to mediate the effects of muscarine (Fig. 5). To rule out the possibility that L-NAME was acting through a non-specific effect not involving NO, the NO donor DEA-NO was applied to NMJs that had been treated with L-NAME and muscarine. As shown in Fig. 5, application of DEA-NO rescued both the immediate (37.3 ± 9.9% decrease from control, n = 3) and the delayed (108 ± 6.6% increase from control, n = 3) effects of muscarine in the presence of L-NAME (compare to an immediate decrease of 36.9 ± 3.9% and a delayed increase of 110.8 ± 3.5% when muscarine was applied without L-NAME; see Fig. 1).



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  		Figure 5.  Synthesis and extracellular diffusion of NO
Effects of L-NAME (an NO synthase inhibitor) and carboxy-PTIO (an extracellular NO chelator) on biphasic muscarine. The ‘M’ bar is 5 µM muscarine, the ‘L + M’ bar is 1 mM L-NAME and muscarine, the ‘C + M’ bar is 40 µM carboxy-PTIO and muscarine and the ‘L + M + NO’ bar is 1 mM L-NAME, 5 µM muscarine and 100 µM DEA-NO. Bars represent mean percentage change from control EPP amplitude. Error bars represent standard error of the mean. P values represent Student's t tests relative to control EPPs. * Statistical significance relative to control. L-NAME knocks out both phases of the muscarinic effect, suggesting the production of NO via NO synthase is integral to the biphasic effect. Carboxy-PTIO also knocks out both phases, thus suggesting the extracellular diffusion of NO is also integral. The ability of DEA-NO to rescue both phases of muscarine's effects when L-NAME is present further supports the unique role of NO in this process.

 
Nitric oxide synthase is located at the lizard NMJ

If NO is involved in the biphasic modulation of ACh release at the lizard NMJ, then its synthetic enzyme, Nitric Oxide Synthase (NOS) must also be present. We first used NADPH-diaphorase (NADPH-d) histochemistry to determine whether NOS is present at the lizard NMJ (Descarries et al. 1998). Figure 6A reveals extensive NADPH-d staining. Immunofluorescence was used to verify this result and determine whether the NOS was of the neuronal isoform (nNOS). Figure 6B shows that nNOS-like immunoreactivity (green) colocalizes with POPO-3 iodide nucleic acid stain (red). This suggests that nNOS is present and active in the PSCs and muscle cells. Figure 6B also reveals that nNOS-like immunoreactivity is located outside the PSCs, presumably in the nerve terminals and/or the muscle. The extensive distribution of nNOS further supports the conclusion that NO plays a critical role in modulating ACh release at the lizard NMJ.



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  		Figure 6.  Localization of nitric oxide synthase
NOS activity detected at the lizard neuromuscular junction. The scale bar applies to both images (A and B). A, NADPH-diaphorase staining is prevalent in the nerve terminal, PSCs and muscle cells, indicating that NOS is present in all three compartments of the lizard NMJ. B, the colocalization of nNOS immunoreactivity (green) and POPO-3 iodide nucleic acid stain (red) suggests the presence of nNOS in the PSCs and muscle cells. Images were visualized using brightfield and fluorescence microscopy.

 
Role of cAMP via PKA in the biphasic effect

Though NO is necessary for both phases of muscarine's effects, the following experiment was performed to determine whether NO alone could mimic either the inhibition or enhancement of release. Bath application of the NO donor DEA-NO (1–100 µM) had no effect on EPP amplitude (2.3 ± 2.8% decrease from control, n = 4, Fig. 7). Other NO donors, such as sodium nitroprusside, were also applied without effect (data not shown). Thus, NO is necessary but not sufficient to modulate both phases of the biphasic muscarinic effect.



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  		Figure 7.  Role of cAMP via PKA in the biphasic muscarinic effects
Effects of NO, cAMP and the PKA inhibitor Fragment 14–22. The ‘NO’ bar is 100 µM NO, the ‘cAMP’ bar is 40 µM 8-bromo cAMP, the ‘NO + cAMP’ bar is 100 µM NO with 40 µM cAMP, the ‘14–22 + M (imm)’ bar is 20 µM Fragment 14–22 PKA inhibitor with 5 µM immediate muscarine, and the ‘14–22 + M (del)’ bar is 20 µM Fragment 14–22 PKA inhibitor with 5 µM delayed muscarine. Both NO and cAMP are necessary but not sufficient to mimic the delayed muscarinic EPP amplitude increase. cAMP via PKA is necessary for the delayed but not immediate effects of muscarine. Bars represent mean percentage change from control EPP amplitude. Error bars represents standard error of the mean. P values represent Student's t tests relative to control EPPs. *Statistical significance relative to control.

 
As application of NO alone was insufficient to mimic the biphasic effect, we examined the potential role of cAMP. Bath application of the cAMP analogue 8-Br-cAMP (40 µM) by itself did not alter EPPs (0.88 ± 4.0% decrease from control, n = 3, Fig. 7). However, coapplication of 100 µM NO with 40 µM 8-Br-cAMP increased EPPs in a manner qualitatively similar to that of the delayed enhancement produced by muscarine (79.3 ± 14.1% increase relative to control, n = 4, P < 0.03, Fig. 7). Coapplication of cAMP and NO did not, on any timescale, inhibit release. Thus, NO and cAMP may be part of two independent pathways within the larger signal transduction pathway leading to the delayed increase of release.

Since cAMP usually exerts its cellular effect by activating protein kinase A (PKA; Wenzel et al. 2002; Wong et al. 2002; Zhao et al. 2004), we examined the effects of the PKA inhibitor Fragment 14–22 on both phases of muscarine's effect on ACh release. Figure 7 demonstrates that inhibition of PKA blocks the delayed increase of EPP amplitude (5.7 ± 3.3% increase from control, n = 4, P < 0.03 relative to delayed muscarine), but does not affect the immediate inhibition of ACh release by muscarine. This provides further support for a role of cAMP, via PKA, in the delayed enhancement of release by muscarine.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The main results of our study are: (1) muscarine has a biphasic effect on ACh release at the lizard neuromuscular junction (NMJ); from 0 to 12 min (immediate) it decreases release, while muscarine application beyond 12 min (delayed) increases release; (2) M3 receptors mediate the inhibition of release, while M1 receptors mediate the enhancement; (3) these receptors are activated by endogenous ACh, with the M3 receptor producing a stronger effect than the M1 receptor; (4) the synthesis and the extracellular diffusion of NO are necessary for both phases of muscarine's effects; (5) application of NO alone is insufficient to modulate ACh release; however, coapplication of NO and cAMP mimics the delayed effects of muscarine; (6) activation of protein kinase A is necessary for the delayed enhancement of ACh release induced by muscarine.

Muscarine elicits an immediate depression and delayed enhancement of ACh release

We present the first evidence that muscarine modulates ACh release in a temporally biphasic manner. Whether this phenomenon helps resolve the controversy regarding the precise effects of muscarinic agonists in all organisms or is merely an intriguing distinction of cholinergic transmission at the lizard NMJ remains to be seen. Our findings, however, offer a potential resolution between the disparate findings of Slutsky et al. (1999, 2001), who showed that muscarine decreased release in frogs, and (Ganguly & Das, 1979), who suggested that oxotremorine increased release in rats. Furthermore, our findings suggest that the lizard NMJ may be a good model system for studying the cellular mechanisms of other examples of biphasic synaptic modulation.

Muscarine exhibits two distinct effects at the lizard NMJ. Initially, ligand binding to presynaptic M3 receptors reduces ACh release. The activation of M1 receptors causes a delayed enhancement of ACh relase, which predominates after 12 min (Fig. 1). This novel biphasic role of muscarine is extremely intriguing. Clearly, the classical depiction of ACh release, wherein quantal release is regulated by presynaptic muscarinic autoreceptors (e.g. Parnas et al. 2000), is insufficient to fully explain synaptic transmission at all NMJs (e.g. lizard).

Muscarine's effects on miniature end-plate potentials (mEPPs)

Our observation that mEPP amplitude remained constant during the application of muscarine (Fig. 2) allowed us to conclude that the changes in evoked EPP amplitudes were due to changes in the number of neurotransmitter quanta released and not due to changes in the postsynaptic efficacy of a quantum of neurotransmitter (Del Castillo & Katz, 1954). Thus, the presynaptic nerve terminal must be the final target of both the M1- and M3-initiated changes in neuromuscular transmission. However, this does not rule out the involvement of the muscle and/or the perisynaptic Schwann cells as intermediates in the process.

The decrease in frequency of mEPPs that we observed fits well with the decrease in EPP amplitude that follows short (0–12 min) applications of muscarine. Unfortunately, the experimental approach we used – recording continuously from a single NMJ – made it difficult to determine convincingly what happens to mEPP frequency following longer (> 12 min) applications. MEPP frequency was highly variable and our recordings became unstable following long applications of muscarine (> 25 min), which is the time at which time the enhancement of EPP amplitude is maximal (see Fig. 1) and would be the most likely time to observe a corresponding increase in mEPP frequency. The application of membrane permeable forms of cAMP has been shown previously to increase mEPP frequency at both rat (Goldberg & Singer, 1969; Hirsh & Silinsky, 2002) and frog NMJ (Hirsh et al. 1990). Furthermore, this increase can be prevented by inhibiting protein kinase A (Hirsh et al. 1990; Hirsh & Silinsky, 2002). Since muscarine's delayed effect at the lizard NMJ involves cAMP acting via protein kinase A (Fig. 7), it would be very interesting to determine whether the mEPP frequency parallels that observed in the frog and rat. Further experiments are in progress to make this determination at the lizard NMJ.

M3 and M1 receptors mediate the biphasic inhibition and enhancement of release

Though it is widely accepted that ACh release is modulated by muscarinic receptors, there is disagreement as to the specific receptor subtype(s) involved. Our results are consistent with the general consensus that M1 receptors mediate an enhancement of ACh release at the vertebrate NMJ (Caulfield, 1993; Slutsky et al. 1999). Slutsky et al. (1999) suggested that the M2 subtype is responsible for the autoinhibition of release, while Hsu et al. (1995) implicated M3 receptors in the depression. Although we were unable to properly test for the involvement of the M2 subtype, our results do support the suggestion that the M3 receptor subtype mediates inhibition of release.

NO is necessary but not sufficient to mimic the effects of biphasic muscarine

Nitric oxide is a ubiquitous intercellular signalling molecule that has been shown to play numerous roles in neuronal signalling (Lindgren & Laird, 1994; Mothet et al. 1996; Mathes & Thompson, 1996; Descarries et al. 1998; Burette et al. 2002; D'Ascenzo et al. 2002). In a previous study, we have shown that the application of exogenous NO decreases ACh release at the frog NMJ (Lindgren & Laird, 1994). The present study concludes that neuronal NO synthase (nNOS) is present at the lizard NMJ and the synthesis and extracellular diffusion of NO is necessary to evoke both the immediate depression and delayed enhancement of ACh release by muscarine (Figs 5 and 6). However, it is not clear from our data what cellular component of the NMJ produces NO in response to muscarine. Since NO must be able to diffuse through the extracellular space for muscarine to modulate ACh release and since its target is most likely within the nerve terminal, we think it is most likely that the NO is produced in either the surrounding perisynaptic Schwann cells (PSCs) or muscle (i.e. not the nerve terminal).

Though NO clearly plays a critical role in the biphasic modulation of ACh release at the lizard NMJ, application of exogenous NO alone does not have a measurable effect on ACh release (Fig. 7). Thus, NO is necessary but not sufficient to modulate either the immediate depression or the delayed enhancement of ACh release at the lizard NMJ. Interestingly, we did find that application of NO enhanced release when coapplied with 8-Br-cAMP (Fig. 7), a membrane-permeable cAMP analogue. This suggests that cAMP and NO are messengers in two separate signal transduction pathways that – together – mediate the enhancement of delayed release. This is consistent with evidence that cAMP mediates an increase in ACh release at the mammalian NMJ (Goldberg & Singer, 1969) and frog sympathetic ganglia (Kuba et al. 1981). Furthermore, we show that the activation of PKA is necessary for the delayed enhancement. PKA inhibition abolished the effects of delayed muscarine but did not have an impact on the immediate depression of release (Fig. 7). Thus, we conclude that the delayed facilitation of release by muscarine is regulated by both NO and cAMP.

Since M1 receptor activation is not known to increase cAMP synthesis, we suspect that another messenger molecule – which has not yet been identified – is involved in the pathway initiated by activation of M1 receptors. Pinard et al. (2003) have presented a compelling mechanism of cholinergic modulation at the frog NMJ, wherein metabatropic glutamate receptors regulate ACh release. Though we do not have data localizing the PKA or the M1 receptor to any of the specific components at the NMJ (the nerve terminal, the PSCs, or the muscle), the possibility we favour is that the M1 receptor is located on the PSCs and its activation leads to the release of a signalling molecule (possibly glutamate) which then elevates cAMP levels and activates PKA in the nerve terminal. This intriguing hypothesis merits further scrutiny at the lizard NMJ to establish a clearer understanding of the mechanisms of muscarinic modulation of ACh release.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Bourque MJ & Robitaille R (1998). Endogenous peptidergic modulation of perisynaptic Schwann cells at the frog neuromuscular junction. J Physiol 512, 197–209.[Abstract/Free Full Text]

Burette A, Zabel U, Weinberg RJ, Schmidt HHW & Valtschanoff JG (2002). Synaptic localization of nitric oxide synthase and soluable guanylyl cyclase in the hippocampus. J Neurosci 22, 8961–8970.[Abstract/Free Full Text]

Castonguay A, Lévesque S & Robitaille R (2001). Glial cells as active partners in synaptic function. Prog Brain Res 132, 227–240.[Medline]

Caulfield MP (1993).Muscarinic receptors – characterization, coupling and function. J Pharmacol Exp Ther 58, 319–379.

Daeffler L, Chahdi A, Gies J-P & Landry Y (1999). Inhibition of GTPase activity of Gi proteins and decreased agonist affinity at M2 muscarinic acetylcholine receptors by spermine and methoctramine. Br J Pharm 127, 1021–1029.[CrossRef][Medline]

D'Agostino G, Bolognesi ML, Lucchelli A, Vicini D, Balestra B, Spelta V, Melchiorre C & Tonini M (2000). Prejunctional muscarinic inhibitory control of acetylcholine release in the human isolated detrusor: involvement of the M4 receptor subtype. Br J Pharmacol 129, 493–500.[CrossRef][Medline]

D'Ascenzo M, Martinotti G, Azzena GB & Grassi C (2002). cGMP/Protein Kinase G-dependent inhibition of the N-type Ca2+ channels induced by nitric oxide in human neuroblastoma IMR32 cells. J Neurosci 22, 7485–7492.[Abstract/Free Full Text]

Del Castillo J & Katz B (1954). Quantal components of the end-plate potential. J Physiol 124, 560–573.[Free Full Text]

Descarries LM, Cai S & Robitaille R (1998). Localization and characterization of nitric oxide synthase at the frog neuromuscular junction. J Neurocytol 27, 829–840.[CrossRef][Medline]

Duncan CJ & Publicover SJ (1979). Inhibitory effects of cholinergic agents on the release of transmitter at the frog neuromuscular junction. J Physiol 294, 91–103.[Abstract/Free Full Text]

Felder CC (1995). Muscarinic acetylcholine receptors: signal transduction through multiple effectors. FASEB J 9, 619–625.[Abstract]

Ganguly DK & Das M (1979). Effects of oxotremorine demonstrate presynaptic muscarinic and dopaminergic receptors on motor nerve terminals. Nature 278, 645–646.[CrossRef][Medline]

Georgiou J, Robitaille R, Trimble WS & Charlton MP (1994). Synaptic regulation of glial protein expression in vivo. Neuron 12, 443–455.[CrossRef][Medline]

Goldberg AL & Singer JJ (1969). Evidence for a role of Cyclic AMP neuromuscular transmission. Proc Natl Acad Sci 64, 134–141.[Abstract/Free Full Text]

Hirsh JK & Silinsky EM (2002). Inhibition of spontaneous acetylcholine secretion by 2-chloroadenosine as revealed by a protein kinase inhibitor at the mouse neuromuscular junction. Br J Pharm 135, 1897–1902.[CrossRef][Medline]

Hirsh JK, Silinsky EM & Solsona CS (1990). The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings. Br J Pharm 101, 311–318.[Medline]

Hsu KS, Huang CC & Gean PW (1995). Muscarinic depression of excitatory synaptic transmission mediated by the presynaptic M3 receptors in the rat neostriatum. Neurosci Lett 197, 141–144.[CrossRef][Medline]

Jahromi BS, Robitaille R & Charlton MP (1992). Transmitter release increases intracellular calcium in perisynaptic Schwann cells in situ. Neuron 8, 1069–1077.[CrossRef][Medline]

Katz B & Miledi R (1977). Suppression of transmitter release at the neuromuscular junction. Proc Roy Soc Lond 196, 465–469.

Kuba K, Kato E, Kumamoto E, Koketsu K & Hirai K (1981). Sustained potentiation of transmitter release by adrenaline and dibutyryl cyclic AMP in sympathetic ganglia. Nature 291, 654–656.[CrossRef][Medline]

Lindgren CA, Emery DG & Haydon PG (1997). Intracellular acidification reversibly reduces endocytosis at the neuromuscular junction. J Neurosci 17, 3074–3084.[Abstract/Free Full Text]

Lindgren CA, Graves AR & Lake B (2003). Nitric Oxide is necessary but not sufficient for the biphasic muscarinic modulation of synaptic transmission at the lizard neuromuscular junction. Soc Neurosci Abstr 29, 168.11.

Lindgren CA & Laird MV (1994). Nitroprusside inhibits neurotransmitter release at the frog neuromuscular junction. Neuro Report 5, 2205–2208.[CrossRef][Medline]

Lindgren CA & Moore JW (1989). Identification of ionic currents at presynaptic nerve endings of the lizard. J Physiol 414, 201–222.[Abstract/Free Full Text]

Lindgren CA & Young KM (2002). The inhibition of ACh release by muscarinic agonists at the neuromuscular junction requires nitric oxide synthesis. Soc Neurosci Abstr 28, 838.3.

Mathes C & Thompson SH (1996). The nitric oxide/cGMP pathway couples muscarinic receptors to the activation of Ca2+ influx. J Neurosci 16, 1702–1709.[Abstract/Free Full Text]

Michaelson DM, Avissar S, Kloog Y & Sokolovsky M (1979). Mechanism of acetylcholine release: possible involvement of presynaptic muscarinic receptors in regulation of acetylcholine release and protein phosphorylation. Proc Natl Acad Sci 76, 6336–6340.[Abstract/Free Full Text]

Minic J, Molgó J, Karlsson E & Krejci E (2002). Regulation of acetylcholine release by muscarinic receptors at the mouse neuromuscular junction depends on the activity of acetylcholinesterase. Eur J Neurosci 15, 439–448.[CrossRef][Medline]

Mothet JP, Fossier P, Tauc L & Baux G (1996). NO decreases evoked quantal ACh release at a synapse of Aplysia by a mechanism independent of Ca2+ influx and protein kinase G. J Physiol 493, 769–784.[Abstract/Free Full Text]

Parnas H, Segel L & Parnas I (2000). Autoreceptors, membrane potential and the regulation of transmitter release. Trends Neurosci 23, 60–68.[CrossRef][Medline]

Pinard A, Lévesque S, Vallée J & Robitaille R (2003). Glutamatergic modulation of synaptic plasticity at a PNS vertebrate cholinergic synapse. Eur J Neurosci 18, 3241–3250.[CrossRef][Medline]

Prast H, Tran MH, Fischer H & Philippu A (1998). Nitric oxide-induced release of acetylcholine in the nucleus accumbens: role of cyclic GMP, glutamate, and GABA. J Neurochem 71, 266–273.[Medline]

Prothero LS, Mathie A & Richards CD (2000). Purinergic and muscarinic receptor activation activates a common calcium entry pathway in rat neocortical neurons and glial cells. J Neuropharm 39, 1768–1778.[CrossRef]

Robitaille R (1998). Modulation of synaptic efficacy and synaptic depression by glial cells at the frog neuromuscular junction. Neuron 21, 847–855.[CrossRef][Medline]

Rouse ST, Gilmor ML & Levey AI (1998). Differential presynaptic amd postsynaptic expression of M1–M4 muscarinic acetylcholine recepors at the perforant pathway/granule cell synapse. J Neurosci 86, 221–232.[CrossRef]

Slutsky I, Parnas H & Parnas I (1999). Presynaptic effects of muscarine on ACh release at the frog neuromuscular junction. J Physiol 514, 769–782.[Abstract/Free Full Text]

Slutsky I, Silman I, Parnas I & Parnas H (2001). Presynaptic M2 muscarinic receptors are involved in controlling the kinetics of ACh release at the frog neuromuscular junction. J Physiol 536, 717–725.[Abstract/Free Full Text]

Standaert FG (1982). Release of transmitter at the neuromuscular junction. Br J Anaesth 54, 131–145.[Free Full Text]

Wali FA, Suer AH, McAteer E, Dark CH & Jones CJ (1988). Effects of atropine and glycopyrrolate on neuromuscular transmission in the rat phrenic nerve-diaphragm preparation. Gen Pharm 19, 285–290.[Medline]

Wenzel B, Elsner N & Heinrich R (2002). mAChRs in the grasshopper brain mediate excitation by activation of the AC/PKA and the PLC second-messenger pathways. J Neurophysiol 87, 876–888.[Abstract/Free Full Text]

Wong DL, Anderson LJ & Tai TC (2002). Cholinergic and peptidergic regulation of phenylethanolamine N-methyltransferase gene expression. Ann N Y Acad Sci 971, 19–26.[Medline]

Zhao Q, Tao J, Zhu Q, Jia PM, Dou AX, Li X, Cheng F, Waxman S, Chen GQ, Chen SJ, Lanotte M, Chen Z & Tong JH (2004). Rapid induction of cAMP/PKA pathway during retinoic acid-induced acute promyelocytic leukemia cell differentiation. Leukemia 18, 285–292.[CrossRef][Medline]


   Acknowledgements
 
We would like to thank Priya Malik for contributing data to Fig. 1C, Erin Conboy for her calcium and NO imaging expertise, Babs Lake for her preliminary work with NADPH histochemistry and nNOS immunofluorescence, Kendra Young for her preliminary electrophysiological investigations of muscarinic agonists, and Gerald Walther for his work concerning NO donors. We would also like to thank the Howard Hughes Medical Institute and Grinnell College for their financial support. This article is contribution no. 2 from the Barr Laboratory of Developmental Biology and Cellular Neuroscience at Grinnell College.





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