Prejunctional effects of the nicotinic ACh receptor agonist dimethylphenylpiperazinium at the rat neuromuscular junction

doi:10.1111/j.1469-7793.1998.451bh.x

Journal List > J Physiol > v.511(Pt 2); Sep 1, 1998

J Physiol. 1998 September 1; 511(Pt 2): 451–460.

doi: 10.1111/j.1469-7793.1998.451bh.x.

PMCID: PMC2231127

Prejunctional effects of the nicotinic ACh receptor agonist dimethylphenylpiperazinium at the rat neuromuscular junction

Shila Singh and Chris Prior

Department of Physiology and Pharmacology, University of Strathclyde, 204 George Street, Glasgow G1 1XW, UK

Corresponding author C. Prior: Department of Physiology and Pharmacology, University of Strathclyde, 204 George Street, Glasgow G1 1XW, UK. Email: c.b.prior/at/strath.ac.uk

Received March 31, 1998; Accepted June 1, 1998.

This article has been cited by other articles in PMC.

Abstract

We have studied the effects of the nicotinic acetylcholine (ACh) receptor agonist dimethylphenylpiperazinium (DMPP) on the evoked release of ACh from motor terminals in the rat isolated hemidiaphragm using an electrophysiological approach.
DMPP (1–4 μM) had no effect on the rate of spontaneous quantal ACh release but increased the number of quanta of ACh released per impulse during 50 Hz stimulation. The DMPP-induced increase in evoked ACh release was dependent on the frequency of stimulation, being absent when it was reduced to 0.5 Hz, but was not Ca²⁺ dependent, being unaffected at 50 Hz by a 4-fold decrease in the extracellular Ca²⁺ concentration.
The facilitation of evoked ACh release at 50 Hz by 2 μM DMPP was abolished by 10 μM of the calmodulin antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride) and, in the presence of W7, 2 μM DMPP depressed evoked ACh release at 0.5 Hz. The ability of the nicotinic ACh receptor antagonist vecuronium (1 μM) to depress evoked ACh release at 50 Hz was also abolished by 10 μM W7.
The present findings demonstrate, using an electrophysiological technique, that DMPP can produce changes in the evoked ACh release from rat motor nerve terminals that are consistent with the existence of facilitatory nicotinic ACh receptors on the motor nerve endings. Further, they indicate a role for calmodulin-dependent systems in this facilitatory effect of the compound.

Most electrophysiological models of the prejunctional effects of acetylcholine (ACh) at the neuromuscular junction rely on the ability of nicotinic ACh receptor antagonists to alter evoked ACh release. The effect seen depends upon the receptor specificity of the antagonist that is used. Antagonists selective for the muscle subclass (e.g. vecuronium) produce a Ca²⁺-independent decrease in evoked ACh release at high frequencies of motor nerve stimulation (Tian et al. 1992, 1994). Conversely, antagonists selective for neuronal subclasses (e.g. hexamethonium) produce a Ca²⁺-dependent increase in ACh release at low frequencies of nerve stimulation (Wilson & Thomsen, 1992; Tian et al. 1997). The complex effects of nicotinic ACh receptor antagonists on ACh release have been interpreted in terms of multiple facilitatory and inhibitory subclasses of prejunctional nicotinic ACh autoreceptors (Tian et al. 1994, 1997). However, to date, no validation of the model has been made with respect to the prejunctional actions of nicotinic ACh receptor agonists. Further, nothing is known of the intracellular mechanisms that underlie the effects mediated by these autoreceptors. Thus, the present study was conducted to determine the effects of a nicotinic ACh receptor agonist on evoked ACh release from motor terminals and to elucidate the intracellular mechanism involved. The agonist studied was 1,1-dimethyl-4-phenylpiperazinium (DMPP) because it increases the evoked overflow of [³H]ACh from rat motor terminals (Wessler et al. 1986, 1987) and we also wished to reconcile data from the two diverse techniques.

DMPP is primarily regarded as an agonist at neuronal subclasses of nicotinic ACh receptors (Boyd, 1997). However, it is also active at the muscle subclass (Cooper et al. 1996; Yost & Winegar, 1997) and produces a depolarization block of twitches in rat skeletal muscle (Ling, 1959). Therefore, to give an insight into the receptor subclass mediating any prejunctional effect of DMPP we studied the Ca²⁺ and frequency dependency of any effects of the compound on ACh release. In addition, to identify the intracellular mechanisms underlying the prejunctional effects of DMPP, we studied the calmodulin (CaM) dependency of its action on ACh release. This system was chosen for study because it has previously been shown that enhanced CaM activity can alter the mobilization and release of neurotransmitter (Llinás et al. 1985; Lin et al. 1990).

We show that DMPP produces a concentration-dependent increase in evoked (50 Hz) ACh release from motor terminals in the rat hemidiaphragm. This effect is absent at 0.5 Hz and is not dependent on the extracellular Ca²⁺ concentration ([Ca²⁺]_o). These dependencies are consistent with an effect on prejunctional muscle-type nicotinic ACh receptors. Thus, the study strengthens the hypothesis that there exists, on rat motor terminals, muscle-type nicotinic ACh receptors, activation of which leads to an enhancement of its release during periods of sustained motor terminal activity. Further, the ability of W7 to attenuate the prejunctional actions of DMPP and vecuronium suggests a role for calmodulin in the facilitatory effects of ACh hypothesized to be mediated by prejunctional positive-feedback muscle-type nicotinic ACh receptors (Tian et al. 1994, 1997).

METHODS

Experimental preparation

Male Sprague-Dawley rats (150–200 g) were killed, in accordance with UK Home Office guidelines, by dislocation of the cervical vertebrae followed by immediate exsanguination. Left and right hemidiaphragm muscles, along with their associated phrenic nerves were rapidly removed from the animal and placed in freshly oxygenated (95% O₂ with 5% CO₂) physiological saline of the following composition (mM): NaCl, 118; KCl, 5; KH₂PO₄, 1.2; MgSO₄, 1; NaHCO₃, 25; CaCl₂, 1.8; and glucose, 11 (pH 7.2-7.4). The two hemidiaphragms were separated and each placed in a 5 ml Sylgard-lined tissue bath perfused with physiological solution at 32°C at a rate of 10–12 ml min⁻¹. Muscle preparations were either left intact for potential recording or, for current recording, immobilized by cutting (Barstad & Lilliheil, 1968). This method involves severing the muscle fibres transversely leaving approximately 2 mm on either side of the main intramuscular branches of the nerve. The cut muscle preparation was used to obviate the use of unwanted pharmacological treatments to immobilize the tissues during recording. For recording evoked responses, the phrenic nerve was stimulated via a pair of silver wire ring electrodes with pulses of 0.05-0.1 ms duration and supramaximal voltage (typically 10–20 V). These pulses were generated by a Grass S88 stimulator linked to a Grass SIU5 stimulus isolation unit. Two nerve stimulation protocols were used: 50 Hz for 2 s and continuous 0.5 Hz stimulation for 3 min, each to evoke approximately 100 individual endplate currents (EPCs).

Potential recording technique

The ability of DMPP (1 and 10 μM) to activate postjunctional nicotinic ACh receptors was determined by recording the effect of the compound on resting membrane potential recorded at the motor endplates of whole, undamaged muscle fibres. Single intracellular microelectrodes filled with 3 M KCl (resistances, 5–10 MΩ) were used to monitor continuously resting membrane potential during the course of a 5–10 min bath application of DMPP (1 or 10 μM) at a rate of 10–12 ml min⁻¹. Potential records were monitored using the voltage follower on a voltage-clamp amplifier (TEV 2000, Dagan Corp., Minneapolis, MN, USA). Signals from the voltage follower were digitized at 10 Hz (Lab-PC+ laboratory interface, National Instruments, Newbury, UK) and the resultant digital record displayed DC-coupled at low gain on a computer screen using an in-house acquisition and display program. Membrane potential was recorded before application of the drug, at the peak effect of the drug and following complete recovery from the effects of the drug.

Current recording technique

EPCs and miniature endplate currents (MEPCs) were recorded from motor endplate voltages clamped at −55 mV using a two-microelectrode technique described in detail elsewhere (Dionne & Stevens, 1975; Prior et al. 1993). Microelectrodes (2–5 MΩ) were made from borosilicate capillary glass and filled with either 3 M KCl (voltage recording) or with 2 M potassium citrate (current passing). Accurate placement of the microelectrodes at the motor endplate was determined from the rate of rise of spontaneous MEPCs (less than 0.5 ms being regarded as acceptable). Adjusting the gain and bandwidth of the voltage-clamp amplifier (Axoclamp 2A, Axon Instruments) set optimum voltage-clamp performance. Both EPCs and MEPCs were recorded with the same voltage clamp settings. Only those current signals where the peak voltage escape was less than 1% of the signal driving force (taken as the holding potential, −55 mV) were considered for analysis. All current signals were recorded on FM tape (Racal Store 4DS, DC - 5 kHz) for subsequent off-line analysis.

Experiments were performed at a range of DMPP concentrations (1–4 μM) with a fixed [Ca²⁺]_o of 1.8 mM and at a range of [Ca²⁺]_o (0.23-1.8 mM) with a fixed DMPP concentration of 2 μM. The concentrations of DMPP used were chosen to match those previously shown to have an effect on [³H]ACh release overflow studies (Wessler et al. 1986, 1987). In each fibre, EPCs were elicited at 50 or 0.5 Hz. For high frequency EPCs, a 1 min recording of MEPCs was followed by a single train of EPCs at 50 Hz for 2 s. For low frequency EPCs, MEPCs and EPCs at 0.5 Hz were recorded simultaneously over a 3 min period. After recording the MEPCs and EPCs, a solution containing DMPP was perfused through the tissue bath for 5 min. Finally, MEPC and EPC records were taken, from the same endplate, in the presence of the DMPP.

To determine the role of CaM in the nicotinic ACh receptor-mediated regulation of ACh release we studied the ability of the CaM inhibitor W7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulphonamide) to alter the prejunctional effects of DMPP and vecuronium. MEPCs and EPCs (0.5 or 50 Hz) were recorded before and after a 5 min exposure to either DMPP (2 μM) or vecuronium (1 μM) in the continued presence of 10 μM W7. We also determined the effects of W7 alone on evoked ACh release. For all W7 experiments [Ca²⁺]_o was 1.8 mM.

In each muscle one or two fibres were sampled, with a minimum of 90 min washing in DMPP-free physiological solution between the two recordings. Each day 1 mM stock solutions of DMPP and W7 (in distilled water) and vecuronium (in 10 mM citric acid) were freshly prepared from powder. When not in use, these solutions were kept at 4°C. DMPP and W7 were purchased from Sigma Chemical Co. and Tocris Cookson (Bristol, UK), respectively. Vecuronium bromide was a gift from Organon Laboratories Ltd (Newhouse, Lanarkshire, UK).

Data analysis and statistical testing

EPCs and MEPCs were analysed off-line using a suite of purpose designed analysis programs (Dempster, 1988, 1993). Following digitization at 25 kHz (Lab-PC+ laboratory interface) signals were stored on the hard disk of a microcomputer (Viglen 486DX) for subsequent analysis. EPCs were analysed individually for amplitude and time course as described elsewhere (Prior et al. 1993). Because of an initial rundown of EPC amplitude at the start of the 50 Hz stimulation period only the last eighty signals, where EPC amplitude was constant, were considered for analysis. For MEPCs we have determined that the best estimates of peak amplitude and time course are obtained from averaged current records (Tian et al. 1994, 1997). Therefore, for each experimental condition 40–200 individual MEPCs were averaged, following alignment to the midpoint of their rising phases, and the single average signal was subsequently analysed in the same manner as the individual EPCs. Quantal ACh release was calculated from the amplitudes of MEPCs and/or EPCs using binomial (Miyamoto, 1975; McLachlan, 1978; Glavinovic, 1979) or Poisson (Martin, 1955, 1966) statistical analysis. For each condition studied, six to ten individual experiments were performed and results for these were averaged to give the presented means and standard error of the mean (s.e.m.). Statistical testing was performed using Student's two-tailed paired t test with P < 0.05 being taken to indicate significance.

RESULTS

Postjunctional effects of DMPP

To determine if DMPP, at the concentrations studied, had any postjunctional action, we studied its effects on the resting membrane potential and on the time constant of decay of MEPCs and EPCs. DMPP (1–10 μM) produced a concentration-dependent depolarization of the motor endplate. With 10 μM DMPP, the depolarization was from 69.3 ± 3.2 to 57.9 ± 3.5 mV (n = 6, P < 0.05) and this was reversed by washout, the membrane potential returning to 69.1 ± 2.7 mV. Peak depolarization was reached within 2–3 min of drug application and, even at 10 μM, showed no spontaneous reversal throughout the 5–10 min exposure period.

The MEPC and EPC decay time constants were unaffected by DMPP up to 2 μM. However, the highest concentration of DMPP used in the ACh release studies (4 μM) produced small (around 14%), but statistically significant, reductions in the time constants of decay of both MEPCs and EPCs. The EPC decay time constant was reduced from 0.95 ± 0.13 to 0.82 ± 0.11 ms while the MEPC decay time constant was reduced from 0.75 ± 0.09 to 0.63 ± 0.06 ms (for both EPC and MEPC data, n = 7; P < 0.05). These DMPP-induced reductions probably reflect the appearance of a small degree of endplate ion channel block. However, given that the reductions in time constants of decay were small and similar for both MEPCs and EPCs, we consider it unlikely that the presence of this endplate ion channel block would have a significant impact on the quantal analysis of ACh release (see also Tian et al. 1997).

DMPP (1–4 μM) significantly depressed MEPC amplitude (Fig. 1) and over the range studied there was a slight concentration dependence to this effect (Fig. 2). However, a fixed concentration of DMPP (2 μM) produced an approximate 20% depression in MEPC amplitude regardless of [Ca²⁺]_o. We consider that the depression of MEPC amplitude by DMPP represents a degree of fast desensitization of the postjunctional nicotinic ACh receptors.

Figure 1

Representative effects of 2 μM DMPP on MEPCs and EPCs recorded from the rat hemidiaphragm preparation

Figure 2

Effects of DMPP on MEPC amplitudes in the rat hemidiaphragm preparation

Prejunctional effects of DMPP

DMPP (1–4 μM) had no effect on the frequency of occurrence of MEPCs. Thus, in the presence of 4 μM DMPP, MEPC frequency was 2.5 ± 0.6 Hz, which was not significantly different from the control value of 2.8 ± 0.8 Hz (n = 7). This indicates a lack of an effect on spontaneous quantal ACh release. In contrast to its effects on MEPC amplitude, the effects of DMPP on the amplitude of EPCs elicited at 50 Hz were less clear. In most cases EPC amplitude was unaffected by DMPP (Figs 1 and 3). However, in one instance (2.0 μM DMPP with a [Ca²⁺]_o of 0.9 mM) there was a slight (11%), but statistically significant, DMPP-induced increase in EPC amplitude (Fig. 3B). In no instance did DMPP decrease EPC amplitude. The differential effect of DMPP on MEPC and EPC amplitudes can be attributed to an effect of the compound on the per-impulse quantal release of ACh - the EPC quantal content. Calculation of this parameter from EPC and MEPC amplitudes clearly showed that DMPP produced a concentration-dependent (Fig. 4A), but [Ca²⁺]_o-independent (Fig. 4B), increase in ACh release.

Figure 3

Effects of DMPP on EPC amplitudes (at 50 Hz) in the rat hemidiaphragm preparation

Figure 4

Effect of DMPP on EPC quantal content (at 50 Hz) in the rat isolated hemidiaphragm muscle preparation

We also investigated the effects of 2.0 μM DMPP on EPCs elicited at 0.5 Hz (in a [Ca²⁺]_o of 1.8 mM). As at 50 Hz, there was a significant depression of MEPC amplitude (18.8 ± 4.4%, n = 8, P < 0.05) and no change in their frequency of occurrence. However, there was no DMPP-induced increase in quantal content: control EPC quantal content, 40.4 ± 2.7; EPC quantal content in 2 μM DMPP, 35.2 ± 3.1 (n = 8). Thus the facilitatory effect of DMPP on evoked ACh release is only seen with high frequency stimulation.

We have no immediate evidence that the DMPP-induced mechanism by which MEPC amplitudes are depressed also applies to the larger evoked EPC signal. It could be argued that it is improbable that the pre- and postjunctional effects of DMPP would be so evenly matched as to make it appear that in most cases the drug had no effect on EPC amplitude. Therefore, we decided to determine the effects of DMPP on quantal ACh release under conditions where estimates could be made independent of the knowledge of MEPC and EPC amplitudes. To do this, we looked at the effects of DMPP on EPC quantal content (at 50 Hz) at very low values (< 1 quantum per impulse) where alternative statistical processes apply. EPC quantal content was reduced to around 0.5 by reducing [Ca²⁺]_o to 0.23 mM and, under these ‘low-probability’ conditions, quantal release follows a Poisson distribution (del Castillo & Katz, 1954a,b) and the mean EPC quantal content can be determined from the proportion of ‘zero quanta’ EPCs - i.e. those instances where nerve stimulation fails to release any quanta of ACh (Martin, 1955, 1966). In the presence of a [Ca²⁺]_o of 0.23 mM, DMPP (2 μM) produced a similar depression in MEPC amplitude as seen with higher [Ca²⁺]_o (Table 1). However, unlike at higher [Ca²⁺]_o, under very low ACh release conditions DMPP (2 μM) markedly increased mean EPC amplitude (Table 1). Hence, DMPP increased EPC quantal content and this was true irrespective of whether this was calculated from the ratio of MEPC and EPC amplitudes or from the incidence of EPC failures. Thus, we believe that the lack of an effect of DMPP on EPC amplitudes at higher EPC quantal contents is a consequence of coincidentally similar degrees of postjunctional decrease (fast desensitization) and prejunctional increase (increased ACh release).

Table 1

Effect of 2 μm DMPP on EPCs (at 50 Hz) and MEPCs recorded from rat hemidiaphragm preparations under conditions of low quantal ACh release

Effect of the calmodulin inhibitor W7 on the prejunctional effects of DMPP

To determine the role of CaM-dependent systems in the DMPP-induced modulation of evoked ACh release we examined the effects of DMPP (2 μM) on the amplitudes of EPCs and MEPCs under circumstances where CaM activity was eliminated with 10 μM of its specific inhibitor W7. At this concentration, W7 itself exhibited a frequency-dependent effect on evoked ACh release. At a low frequency of stimulation (0.5 Hz), evoked ACh release was unaffected by W7: control EPC quantal content, 33.1 ± 4.7; EPC quantal content in 10 μM W7, 31.5 ± 5.0 (n = 8). However, at a high frequency of stimulation (50 Hz), W7 produced an approximate 15% reduction in evoked ACh release: control EPC quantal content, 32.4 ± 3.2; EPC quantal content in 10 μM W7, 28.9 ± 3.5 (n = 11, P < 0.05). The CaM inhibitor had no effect on the rate of spontaneous ACh release: control MEPC frequency, 1.8 ± 0.2 Hz; MEPC frequency in 10 μM W7, 1.8 ± 0.2 Hz (n = 11, P < 0.05).

As in its absence, in the continued presence of 10 μM W7, DMPP (2 μM) had no effect on the frequency of occurrence of MEPCs and produced a small, statistically significant, reduction in MEPC amplitude (Fig. 5A, +W7). However, the presence of 10 μM W7 markedly altered the effect of DMPP on the amplitude of EPCs recorded at 50 Hz. In the absence of W7, DMPP (2 μM) had no depressant effect on EPC amplitude (Fig. 5B, -W7). However, in the presence of 10 μM W7, DMPP (2 μM) produced a marked decrease in EPC amplitude at 50 Hz (Fig. 5B, +W7) which was proportionally greater than its effect on MEPC amplitude under the same experimental conditions (cf. Fig. 5A, +W7). This indicates that, in the presence of 10 μM W7, there is a DMPP-induced depression of EPC quantal content (Fig. 5C, +W7).

Figure 5

Effect of W7 on the DMPP-induced changes in MEPCs and EPCs (at 50 Hz) in the rat hemidiaphragm muscle

We also studied the effects of 2 μM DMPP on EPCs evoked at 0.5 Hz in the continued presence of 10 μM W7. At 0.5 Hz and in the absence of W7, 2 μM DMPP equally depressed EPC and MEPC amplitudes (Fig. 6A and B, -W7) and thus had no effect on EPC quantal content (Fig. 6C, -W7). However, in the presence of 10 μM W7, 2 μM DMPP produced a proportionally greater depression of EPC amplitudes (Fig. 6B, +W7) than MEPC amplitudes (Fig. 6A, +W7). Thus, in the presence of 10 μM W7 and at a low frequency of stimulation, 2 μM DMPP reduced EPC quantal content (Fig. 6C, +W7).

Figure 6

Effect of W7 on the DMPP-induced changes in MEPCs and EPCs (at 0.5 Hz) in the rat hemidiaphragm muscle

Effect of W7 on the prejunctional effects of vecuronium

The ability of 10 μM W7, when applied alone, to decrease EPC quantal content at 50 Hz suggests that the CaM regulation of evoked ACh release is part of a facilitatory positive-feedback modulatory system involving endogenous ACh. To investigate this further, we studied the effects of 10 μM W7 on the prejunctional actions of vecuronium, an antagonist of the muscle subclass of nicotinic ACh receptors. Vecuronium (1 μM) had no effect on the frequency of occurrence of MEPCs, irrespective of the presence or absence of W7 (data not shown). However, the compound depressed MEPC amplitude by around 30%, in both the presence and absence of W7 (Fig. 7A). At 50 Hz and in the absence of W7, vecuronium (1 μM) depressed EPC amplitude (Fig. 7B, -W7). The decrease was proportionally greater than that for seen for MEPCs under the same conditions (cf. Fig. 7A, -W7). This indicates that vecuronium reduces EPC quantal content in the absence of W7 (Fig. 7C, -W7). This is consistent with previously published data for the compound (Tian et al. 1994). In the presence of 10 μM W7, vecuronium (1 μM) produced a decrease in EPC amplitude at 50 Hz (Fig. 7B, +W7) which was similar to that seen for MEPCs under the same conditions (cf. Fig. 7A, +W7). This indicates that vecuronium (1 μM) has no effect on EPC quantal content in the presence of 10 μM W7 (Fig. 7C, +W7).

Figure 7

Effect of W7 on the vecuronium-induced changes in MEPCs and EPCs (at 50 Hz) in the rat hemidiaphragm muscle

DISCUSSION

Effects of DMPP on MEPCs

DMPP-induced changes in ACh release were evident because of a differential effect of the compound on EPC and MEPC amplitudes. Thus, it is important to consider how DMPP decreases MEPC amplitudes and to determine whether anything other than a concomitant increase in ACh release could account for the lack of an effect on EPC amplitudes.

The most likely reason for the DMPP-induced reduction in MEPC amplitudes is fast desensitization of the endplate ACh receptors. This has been used to account for similar cholinergic agonist-induced depression of MEPC amplitudes in isolated mouse (Pennefather & Quastel, 1982) and cat (Wray, 1981) muscles. However, despite the possibility of some postjunctional ACh receptor inactivation, a large proportion of the ACh receptors must remain operational since, even at 10 μM, DMPP caused a sustained membrane depolarization. The fact that this depolarization was maintained suggests that slow desensitization has no importance in the context of the present studies. If fast desensitization of a fraction of the endplate ACh receptors underlies the effects of DMPP on MEPCs then it would be expected that EPC amplitudes, whatever the level of evoked ACh release, would be similarly affected. Therefore, the differential effect of DMPP on MEPC and EPC amplitudes does reflect changes in ACh release.

It is also possible that the DMPP-induced decrease in MEPC amplitude is due to open ion channel block. The main indicator of this is a decrease in MEPC and EPC time constants of decay (Adams, 1976; Beam, 1976; Ruff, 1977), and this was detected with 4 μM DMPP. Since ion channel block can also reduce the amplitude of currents (Prior et al. 1990), it is important to consider its potential effects on quantal analysis. However, we believe that endplate ion channel block does not underlie the apparent change in EPC quantal content for two reasons. Firstly, effects of channel block on current amplitudes are only detected in association with large decreases in the decay time constants of the signals (Prior et al. 1990), something not seen here. Secondly, driving function analysis has shown that, under the conditions employed here, small amounts of endplate ion channel block equally affect MEPC and EPC amplitudes and so do not affect the analysis of ACh release (Tian et al. 1997). Finally, the accumulation of DMPP-blocked endplate channels during repetitive 50 Hz stimulation cannot underlie the differential effects of the compound on EPC and MEPC amplitudes since this would be expected to depress EPCs more than MEPCs and hence produce an apparent decrease in evoked ACh release.

Is DMPP a prejunctional agonist or antagonist?

We argue that the DMPP-induced depression of MEPC amplitude is due to fast desensitization of a proportion of the postjunctional ACh receptors. This raises the possibility that the facilitatory effect of DMPP on evoked ACh release could be due to a desensitization-induced antagonist activity on prejunctional negative-feedback ACh receptors rather than an agonist activity on prejunctional positive-feedback ACh receptors. Facilitatory effects of nicotinic ACh receptor antagonists on ACh release have been reported for compounds with selectivity for neuronal receptor subclasses such as hexamethonium (Wilson & Thomsen, 1992; Tian et al. 1997). However, several pieces of evidence lead us to believe that DMPP is not acting prejunctionally as an antagonist. Firstly, in studies of evoked [³H]ACh release from the rat hemidiaphragm it has been shown that the effects of DMPP on [³H]ACh release are concentration and time dependent (Wessler et al. 1986, 1987). With a short exposure period or at a low concentration, DMPP augments evoked [³H]ACh release from rat hemidiaphragm and nicotinic ACh receptor antagonists block this effect. However, a longer exposure to a high concentration produces a depression of evoked [³H]ACh release that can be attributed to DMPP-induced nicotinic ACh receptor desensitization. From a comparison of our exposure protocols with those of Wessler et al. (1986, 1987), we believe that neither the duration of exposure nor the concentrations of DMPP used were sufficient to produce marked nicotinic ACh receptor desensitization.

Further evidence for a prejunctional agonistic activity of DMPP is that the facilitatory effect of DMPP on ACh release is frequency but not Ca²⁺ dependent. This profile matches more closely that seen for the depression of ACh release by muscle-type antagonists such as vecuronium (Tian et al. 1994) than that seen for the augmentation of ACh release by neuronal-type antagonists such as hexamethonium (Tian et al. 1997). Thus the effect of DMPP on ACh release is more readily explained as an agonist action on prejunctional facilitatory nicotinic ACh receptors of the muscle type. Can this notion be supported by the known receptor subtype specificity of DMPP? DMPP is regarded as a classical agonist of the neuronal subclasses since it is typically more potent at these receptors than at the muscle subclass (Ling, 1959). However, the compound shows marked variability in its effects on the neuronal subclasses (Boyd, 1997) and does have activity at muscle-type nicotinic ACh receptors (Cooper et al. 1996; Yost & Vinegar, 1997). Further, the fact that DMPP augments ACh release over the same range of concentrations that produces a sustained endplate depolarization would indicate the potential for agonistic activity at prejunctional facilitatory muscle-type nicotinic ACh receptors. Finally, the fact that the depressant effects of vecuronium and the facilitatory effect of DMPP are both CaM dependent would argue for an agonistic activity of the latter.

The fact that DMPP had no effect on ACh release at 0.5 Hz would initially imply that the compound does not act on the negative-feedback ACh autoreceptor system associated with low frequencies of nerve stimulation (Tian et al. 1997). However, nicotinic ACh agonists can depress quantal ACh release from motor terminals (van der Kloot, 1993; van der Kloot et al. 1997) and we show that, in the presence of W7, DMPP did reduce ACh release at 0.5 Hz. We suggest that this is due to an unmasked agonist action of DMPP on the negative-feedback system that is hypothesized to involve prejunctional neuronal-type nicotinic ACh receptors (Wilson & Thomsen, 1992; Tian et al. 1997). Supporting this, the DMPP-induced depression of ACh release from frog motor terminals is mediated by neuronal-type nicotinic ACh receptors (van der Kloot, 1993) and possibly involves activation of prejunctional N-type Ca²⁺ channels (van der Kloot et al. 1997).

The role of calmodulin in the facilitatory effects of DMPP

Our present data implicate CaM in the facilitatory effect of DMPP on ACh release evoked by high frequency stimulation. Further, the observation that W7 abolishes the depressant effect of vecuronium on ACh release at 50 Hz suggests an autofacilitatory role for this system during normal neuromuscular function. The lack of an effect of either vecuronium or W7 on evoked ACh release at low frequencies of stimulation is consistent with the hypothesis that the main purpose of this CaM-dependent system is to enhance mobilization of ACh for release during periods of sustained demand. How might this high-frequency-associated CaM-dependent mobilization phenomena function? One clue to a possible mechanism comes from the work of Llinás et al. (1985) and Lin et al. (1990) who have shown that the phosphorylation state of synapsin I affects quantal release from the squid giant synapse. Release is depressed by dephosphorylated synapsin I and enhanced by CaM-activated protein kinase II (PK-II). Synapsin I anchors synaptic vesicles to the cytoskeletal matrix by binding to both F-actin (Bahler & Greengard, 1987) and the synaptic vesicle membrane PK-II (Benfenati et al. 1992). Phosphorylation of synapsin I by PK-II inhibits its binding to PK-II. Thus, the level of CaM activity controls the anchoring of synaptic vesicles to the cytoskeletal matrix via synapsin I and PK-II. Therefore, anything that increases CaM activity has the potential to enhance synaptic vesicle mobilization and hence evoked ACh release.

How can activation of prejunctional nicotinic ACh receptors enhance CaM activity? One possibility is that there is a rise in the intraterminal Ca²⁺ concentration ([Ca²⁺]_i) either directly as a result of Ca²⁺ conductance by the nicotinic ACh receptors or indirectly as a consequence of nicotinic ACh receptor-mediated changes in nerve terminal Ca²⁺ fluxes. It is unlikely that the facilitatory effect of DMPP on evoked ACh release involves a generalized rise in [Ca²⁺]_i since, unlike DMPP, the Ca²⁺ ionophore A-23187 (Pressman & Fahim, 1982) greatly increases in the frequency of MEPCs at the neuromuscular junction. However, this does not preclude the possibility that a localized rise in [Ca²⁺]_i could selectively alter CaM activity in a critical region of the nerve terminal and hence underlie the facilitatory effects of DMPP on ACh release.

In conclusion, DMPP increases ACh release from rat motor nerve terminals at high frequencies of nerve stimulation. The effect is consistent with a prejunctional agonist-like action of the compound on muscle-type nicotinic ACh autoreceptors. The mechanism by which activation of a non-selective cation channel on the motor terminal leads to increased ACh release appears to be CaM dependent and could possibly involve an inhibition of the anchoring of synaptic vesicles to the cytoskeletal matrix by synapsin I. Overall, our data offer considerable support for the existence on motor terminals of a physiologically relevant positive-feedback modulatory system for ACh release.

Acknowledgments

The authors gratefully acknowledge the support and advice offered by Professor Ian G. Marshall and Dr J. Dempster during the course of these studies. S. S. is supported by a postgraduate scholarship from the Overseas Research Student Award Scheme.

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