Verapamil alters the amplitude and time course of miniature endplate current

The effect of verapamil on neuromuscular transmission was examined by recording miniature endplate currents (mepcs) in voltage-clamped frog sartorius muscle fibres. In the presence of 100 microM verapamil, the amplitude and time constant of decay of the mepcs (tau D) were reduced to 68% and 55% of control respectively, and the normal voltage dependency of tau D was decreased. Part of the decrease in amplitude of the mepc was independent of changes in tau D because, on washout, recovery of tau D was more rapid than that of amplitude, and in some cells smaller concentrations of verapamil (1 and 10 microM) decreased amplitude without affecting tau D. Evidence of open and closed channel blockade by verapamil (5-20 microM) was obtained from ionophoretic end-plate current trains and it is proposed that these effects are mediated via an allosteric mechanism.     

Effects of hycanthone on the neuromuscular transmission

The effects of the antischistosomal drug, hycanthone, on the synaptic transmission at the frog neuromuscular junction were studied. The mean quantal content increased in the presence of 20 microM hycanthone. The amplitude of the miniature end-plate current was unaffected by 20 microM hycanthone, while 2 microM hycanthone decreased the ionophoretic ACh response (ACh induced current). The decay time constants of the evoked end-plate current and the miniature end-plate current were increased with 1-5 microM hycanthone, but were decreased at concentrations over 20 microM. Analysis of the ACh induced noise revealed that 1 microM hycanthone slightly increased the channel lifetime whereas the single channel conductance was not affected. It was concluded that the primary site of action of hycanthone is the 'transient state' or ACh bound but closed conformation of the ACh receptor ion channel, but this drug also has other sites of action (presynaptic nerve terminal and open conformation of ACh receptor-ion channel complex).     

Neuromuscular blocking action of two hemicholinium-3 analogs

Two hemicholinium-3 (HC-3) analogs (NAM-204 and NAM-224) in which the C = O moiety was reduced to -CH2 and 2- or 3-CH3 substitution on the piperidine ring were demonstrated to produce a different type of neuromuscular inhibition than that induced by HC-3. The neuromuscular blocking activity was tested in several different preparations and it was shown that these compounds are potent, short-acting, and was reversed by neostigmine but not by choline. Although these compounds possess weak anticholinesterase activity, significant alterations of blood pressure and heart rate are absent. Miniature end-plate potentials' amplitude were found to be diminished by these compounds with some alteration of frequency. In voltage-clamped frog sartorius muscle NAM-224 and NAM-204 decreased amplitude of miniature end-plate current (mepcs) and accelerated the time constant of mepc decay in a concentration-dependent manner, without altering the single exponential nature of mepc decay. NAM-204 and NAM-224 at high concentrations also produced a voltage- and concentration-dependent non-linearity in current/voltage relationships, especially at more negative membrane potentials. Hence, these pharmacological and electrophysiological studies indicate NAM-224 and NAM-204 have significant activity at both pre- and post-junctional sites and their post-junctional blocking activity results from blocking ACh receptor/ion channel complex.     

Inhibitory actions of vecuronium bromide on acetylcholine and glutamate responses at the frog and crayfish neuromuscular junction

Effects of vecuronium bromide, an analog of pancuronium, on the cholinergic and glutamatergic neuromuscular junction were investigated. Vecuronium depressed the postsynaptic response of the frog end-plate at lower concentrations than 10(-6) g/ml without affecting the presynaptic events. Vecuronium decreased the amplitude of the double ACh potential, but the second potential was more markedly reduced than the first. In analogy with d-tubocurarine, this suggests that vecuronium may act in part as an open channel blocker at the frog end-plate. Vecuronium depressed both the glutamate response and the excitatory junctional potential at the crayfish neuromuscular junction, although high concentrations were required. The drug increased the decay rate of extracellularly recorded excitatory junctional potentials at the crayfish neuromuscular junction. The reduction of the crayfish synaptic response caused by vecuronium can be explained by the open channel blocking action at this junctional site. The problem that cholinergic antagonists possess a property of channel blocking at the other transmitter system was discussed.     

Prion protein potentiates acetylcholine release at the neuromuscular junction.

Cellular prion protein (PrP(c)), the normal isoform of the pathogenic peptide (PrP(sc)) responsible of the transmissible spongiform encephalopaties (TSEs), is present in many neural tissues, including neuromuscular junctions (NMJ). To analyze if this protein could influence the synaptic transmission, we performed an electrophysiological approach to study the effect of cellular prion protein on a mammalian neuromuscular junction. The loose patch clamp (LPC) technique enables the study of the whole preparation including the pre- and the post-synaptic domains. In a mouse phrenic-diaphragm preparation, nanomolar concentrations of cellular prion protein were able to induce a very striking potentiation of the acetylcholine (ACh) release. The effect was mainly pre-synaptic with an increase of the amplitude of the miniature end-plate currents, probably calcium dependent. Moreover, an apparent facilitation of the synaptic transmission was noted. The results clearly indicate that cellular prion protein may play a key role in the function of the neuromuscular junction.     

Effects of lindane upon transmitter release and end-plate responsiveness in the neuromuscular junction of the frog

Studies were carried out to determine whether the insecticide, lindane, influenced calcium-mediated electrical activity or release of neurotransmitter in vitro. In neuroblastoma cells, lindane (1-100 microM) did not appreciably modify action potentials elicited in normal or sodium-free solutions nor did it have a significant effect upon either transient or sustained-type calcium channels. Exposure of cutaneous pectoris neuromuscular junctions of the frog to 100 microM lindane increased spontaneous release of transmitter. In neuromuscular junctions perfused with 0.5 mM Ca++/6 mM Mg++ Ringer (low quantal release conditions), lindane increased evoked release of transmitter. The absolute increase amounted to just less than one quanta and the extent of the increase was inversely proportional to the mean number of quanta released by the nerve terminal before exposure to lindane. No effect on release of transmitter could be demonstrated when neuromuscular junctions were perfused in normal Ringer. Exposure of neuromuscular junctions to 100 microM lindane also decreased the amplitudes of miniature end-plate potentials. Amplitudes of responses to iontophoretically-applied acetylcholine (ACh) were similarly affected, suggesting that the decrease in amplitudes of miniature end-plate potentials was a reflection of decreased sensitivity of the end-plate to ACh. At neuromuscular junctions of the cutaneous pectoris of the frog, lindane appeared to produce two major effects. Presynaptically, it produced changes in spontaneous and evoked release of transmitter that were consistent with a small increase in free intracellular concentration of Ca++. Postsynaptically, it reduced the sensitivity of the end-plate to ACh.     

Linalool modifies the nicotinic receptor-ion channel kinetics at the mouse neuromuscular junction.

Linalool is a monoterpene compound reported to be a major component of essential oils in various aromatic species. Several linalool-producing species are used in traditional medical systems. Among these is Aeolanthus suaveolens G. Dom (Labiatae) which is used as an anticonvulsant in the Brazilian Amazon. Psychopharmacological in vivo evaluation of linalool showed that this compound has dose-dependent marked sedative effects at the central nervous system (CNS), including hypnotic, anticonvulsant and hypothermic properties. It has been suggested that these neurochemical effects might be ascribed to the local anaesthetic activity of linalool. The present study reports an inhibitory effect of linalool on the acetylcholine (ACh) release and on the channel open time in the mouse neuromuscular junction. These findings could provide a rational basis to confirm the traditional medical use of linalool-producing plant species. Indeed, our data demonstrate some interactions in the modulation of the ACh release at the mouse neuromuscular junction, which are well correlated with the suggested molecular mechanisms. Linalool induced a reduction of the ACh-evoked release. The possibility that this effect could be ascribed to some interaction with pre-synaptic function is noteworthy. Moreover, the inhibitory effect induced on the kinetics of the miniature end-plate current decay demonstrates a local anaesthetic action, either on the voltage or on the receptor-activated channels.     

Effects of atypical antipsychotics on vertebrate neuromuscular transmission

A study was made of the effects of the atypical antipsychotics clozapine, olanzapine, sulpiride and risperidone on nicotinic synaptic transmission at the frog neuromuscular junction. At concentrations higher than 10 microM, these atypical antipsychotics partially reduced the amplitude of miniature end-plate currents (mEPCs) in a dose-dependent and reversible manner. Atypical antipsychotics were, however, less effective than typical neuroleptics of the phenothiazine family at inhibiting mEPCs. In addition to decreasing mEPC amplitude, the atypical antipsychotics reduced the half-decay time of mEPCs. In the case of clozapine, the reduction in mEPC amplitude and duration was not markedly voltage-dependent. Beside their post-synaptic effects, all atypical neuroleptics, except sulpiride, increased the frequency of mEPCs in a concentration-dependent manner, with the strongest effect seen with clozapine. Altogether, these results raise the possibility that atypical neuroleptics could derive some of their therapeutic effects not only from their well-known inhibitory action on dopaminergic receptors, but also from their pre- and post-synaptic modulation of nicotinic neurotransmission.     

The phosphatidylinositol 4-kinase inhibitor phenylarsine oxide blocks evoked neurotransmitter release by reducing calcium entry through N-type calcium channels

The effects of the phosphatidylinositol 4-kinase inhibitor, phenylarsine oxide (PAO), on acetylcholine (ACh) release and on prejunctional Ca(2+) currents were studied at the frog neuromuscular junction using electrophysiological recording techniques. Application of PAO (30 microM) increased both spontaneous ACh release reflected as miniature end-plate potential (mepp) frequencies and evoked ACh release reflected as end-plate potential (epp) amplitudes with a similar time course. Following the initial increase in epp amplitudes produced by PAO, epps slowly declined and were eventually abolished after approximately 20 min. However, mepp frequencies remained elevated over this time period. PAO (30 microM) also inhibited the perineural voltage change associated with Ca(2+) currents through N-type Ca(2+) channels (prejunctional Ca(2+) currents) at motor nerve endings. Addition of British anti-lewisite (BAL, 1 mM), an inactivator of PAO, partially reversed both the inhibition of epps and the inhibition of the prejunctional Ca(2+) current. The effects of PAO on N-type Ca(2+) channels were investigated more directly using the whole cell patch clamp technique on acutely dissociated sympathetic neurons. Application of PAO (30 - 40 microM) to these neurons decreased the voltage-activated calcium currents through N-type Ca(2+) channels, an effect that was partially reversible by BAL. In combination, these results suggest that inhibition of neurotransmitter release by PAO occurs as a consequence of the inhibition of Ca(2+) entry via N-type calcium channels. The relationship between the effects of PAO on N-type Ca(2+) channels in motor nerve endings and in neuronal soma is discussed.     

Mechanism of the blocking action of beta-eudesmol on the nicotinic acetylcholine receptor channel in mouse skeletal muscles.

beta-Eudesmol, an uncharged alcohol contained in Atractylodes lancea, blocks the neuromuscular junction. Atractylodes lancea is prescribed in a traditional Chinese medicine and plays a main role for "alleviation of pain in skeletal muscle". By using the cell-attached patch-clamp or conventional intracellular technique, the site of action of beta-eudesmol on the nicotinic acetylcholine (ACh) receptor (nAChR) channel in skeletal muscle of the adult mouse, was investigated and compared with that of different types of blockers of the nicotinic ACh receptor channel (bupivacaine, chlorpromazine and phencyclidine). beta-Eudesmol (200 microM) depressed completely the nerve-evoked twitch tension and reduced the amplitude and quantal size of endplate potentials but did not alter either the quantal content, resting membrane potential or action potential. beta-Eudesmol (100-200 microM) decreased the amplitude of ACh potentials and accelerated the slow decay of depolarization, induced by the continuous application of ACh. beta-Eudesmol (40 microM) and phencyclidine (10 microM) decreased both the open time and opening frequency, without affecting the single channel conductance. Bupivacaine (10 microM) decreased only the open time. Chlorpromazine (10 microM) decreased only the opening frequency. These results indicate that the blocking effect of beta-eudesmol on nerve-evoked contraction, was due to blockade of nicotinic ACh receptor channels at the neuromuscular junction. Like phencyclidine, beta-eudesmol blocked the nicotinic ACh receptor channel in both the open and closed conformations, and accelerated the desensitization of the nicotinic ACh receptor.     

An electrophysiological study on the effects of Pa-1G (a phospholipase A(2)) from the venom of king brown snake, Pseudechis australis, on neuromuscular function.

The effects of Pa-1G, a phospholipase A(2) (PLA(2)) from the venom of the Australian king brown snake (Pseudechis australis) were determined on the release of acetylcholine, muscle resting membrane potential and motor nerve terminal action potential at mouse neuromuscular junction. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations revealed that Pa-1G (800 nM) significantly reduced the amplitude of endplate potentials within 10 min exposure. The quantal content of endplate potentials was decreased to 58+/-6% of control after 30 min exposure to 800 nM Pa-1G. The toxin also caused a partial depolarisation of mouse muscle fibres within 60 min exposure. Extracellular recording of action potentials at motor nerve terminals showed that Pa-1G reduced the waveforms associated with both sodium and potassium conductances. To investigate whether this was a direct or indirect effect of the toxin on these ionic currents, whole cell patch clamp experiments were performed using human neuroblastoma (SK-N-SH) cells and B82 mouse fibroblasts stably transfected with rKv1.2. Patch clamp recording experiments confirmed that potassium currents sensitive to alpha-dendrotoxin recorded from B82 cells and sodium currents in SK-N-SH cells were not affected by the toxin. Since neither facilitation of acetylcholine release at mouse neuromuscular junction nor depression of potassium currents in B82 cells has been observed, the apparent blockade of potassium currents at mouse motor nerve endings induced by the toxin is unlikely to be due to a selective block of potassium channels.     

Decrease in calcium currents induced by aminoglycoside antibiotics in frog motor nerve endings.

1. The effects of the aminoglycoside antibiotics, streptomycin, neomycin and gentamicin were examined on perineural currents and evoked acetylcholine (ACh) release at frog motor nerve endings. 2. In the standard solutions used previously to measure Ca2+ currents, streptomycin reduced the peak amplitude of the Ca2+ component of the perineural current. 3. In a solution in which changes in both Ca2+ currents and evoked ACh release can be recorded simultaneously, both Ca2+ currents and evoked ACh release were reduced by aminoglycosides in the potency order neomycin > streptomycin > gentamicin. This potency sequence is similar to that reported previously for these agents as inhibitors of neurally-evoked contractions of mammalian skeletal muscle. 4. These data suggest that the presynaptic inhibitory effects of aminoglycoside antibiotics at the neuromuscular junction occur as a consequence of a reduction in Ca2+ currents in the motor nerve terminal.     

Effects of low ozone-oxygen concentrations on the acetylcholine release at the mouse neuromuscular junction

The use of a mixture of low concentrations of ozone (O3) with oxygen (O2) have been proved to be useful in different human pathological conditions. Owing to a lack of both pharmacological and epidemiological basic studies, the scientific consideration of this therapeutic potential is still inappropriate. Here, we started, from an electrophysiological point of view, a study on the possible effects of low O3 doses on the acetylcholine (ACh) release at the neuromuscular junction. Indeed, some experimental conditions indicate a positive effect either in maintaining cytosolic calcium (Ca2+) homeostasis or in increasing the efficacy of the intracellular antioxidant systems. Furthermore, a positive action on the kinetics of some antioxidant enzymes must be taken into account as a possible molecular mechanism in the regulation of the function of cellular homeostasis. Our data demonstrate a reduction of evoked ACh release in the mouse neuromuscular junction. O3 affects neither the spontaneous ACh release nor the kinetics of the ACh-receptor-channel complex. The results are compatible with a reduction of intracellular Ca2+ and proved a molecular action of O3.     

Inhibition of skeletal muscle nicotinic receptors by the atypical antipsychotic clozapine

We have previously observed that certain atypical antipsychotic drugs reduce the amplitude and duration of miniature end-plate currents (EPCs) at the frog neuromuscular junction (Effects of atypical antipsychotics on vertebrate neuromuscular transmission, Nguyen, Q.-T., Yang, J., Miledi, R. Neuropharmacology 42, 2002, 670-676), therefore suggesting that these drugs act on nicotinic acetylcholine receptors. In this study we examined the effects of the atypical antipsychotic clozapine on nicotinic receptors of frog neuromuscular end-plates or in Xenopus oocytes expressing the alpha(1)beta(1)gamma delta mouse skeletal muscle nicotinic receptor. At neuromuscular junctions, postsynaptic currents were reduced by micromolar concentrations of clozapine. This compound also acted presynaptically by increasing the quantal content of EPCs of muscles without noticeably affecting paired-pulse facilitation. In oocytes, clozapine inhibited alpha(1)beta(1)gamma delta receptors with an IC(50) of 10 microM and a Hill coefficient of 1. Blockage of alpha(1)beta(1)gamma delta receptors by clozapine bears several hallmarks of open-channel blockers, including faster response decays, strong voltage dependence of the block, large rebound currents upon wash, and reduction of peak responses even at saturating concentrations of acetylcholine. However, clozapine increased the EC(50) for acetylcholine and its blocking effect was enhanced by preincubation. These results suggest that clozapine antagonizes muscle nicotinic receptors by blocking open channels, and possibly also by another mechanism which still remains to be investigated.     

吡喹酮对神经肌肉接头传递的作用

吡喹酮可使电刺激腓总神经所引起的胫前肌收缩反应明显加强。如给腓总神经施加超强刺激,静脉注射筒箭毒碱可减弱肌肉收缩反应,吡喹酮却能加强筒箭毒碱的抑制作用,使神经肌肉传递迅即完全阻断。采用微电极细胞内记录技术研究吡喹酮对离体大鼠膈神经隔肌的作用,发现吡喹酮不明显影响膈肌细胞的静息膜电位。低浓度吡喹酮(2×10^-4M)可使微终板电位(mepp)频率明显增加。高浓度吡喹酮8×10^-4M则使mepp振幅逐渐变小和消失。吡喹酮在使神经肌肉传递阻滞不断加深的同时,尚能使终板电位的振幅变小,终致完全消失。     

Mechanism for acetylcholine receptor localization at nerve-muscle synapse

This paper summarizes the hypothesis proposed to explain the mechanism for AChR localization at the neuromuscular synapse. Two theories have been proposed to explain the neuronal control of extrajunctional AChR. One theory claimed that motoneurons decreased the ACh sensitivity of the extrajunctional membrane through neurotrophic influence. However, direct electrical stimulation of denervated muscles resulted in a decrease of extrajunctional ACh sensitivity, supporting the other hypothesis that loss of extrajunctional AChR of the innervated muscle is directly related to muscle activity per se. AChR clusters (high density of AChR) at the neuro-muscular junction were supposed to result from the association of nerves with preexisting AChR clusters. However, Xenopus nerve-muscle cocultures clearly demonstrated that AChR clusters at the neuromuscular junction were formed after the nerve came in contact with the muscle membrane. Two hypothesis are proposed for nerve-induced formation of AChR clusters. Preferential insertion of AChR into the end-plate was suggested by the finding that AChR messenger RNA was more abundant near to than far from the end-plate in adult muscle fibers. On the other hand, in cultured and embryonic muscles, AChR clusters were formed at nerve-muscle junctions through receptor redistribution which was mediated by the passive diffusion-trap mechanism.     

Effects of edrophonium on end-plate currents in frog skeletal muscle

The effects of edrophonium on end-plate currents measured at frog neuromuscular junctions were assessed using the two microelectrode voltage clamp technique. Low concentrations of edrophonium (10^?5 M and 2.5 × 10^?5 M) increased the peak amplitude and the kinetic parameters of end-plate currents measured in curare-Ringer and in 5 mM Ca²⁺–10 mM Mg²⁺-Ringer solutions. At the highest concentration tested (6 × 10^?5 M), edrophonium decreased the peak amplitude and maximum rate of rise but had little effect on the time to peak. Time to half decay was the only parameter studied which continued to increase as edrophonium concentration was increased from 10^?5 M to 6 × 10^?5 M. Normalized values of the parameters for both 10^?5 M and 2.5 × 10^?5 M edrophonium revealed no significant difference between end plates bathed in curare-Ringer and those bathed in 5 mM Ca²⁺–10 mM Mg²⁺-Ringer.It is suggested that the low concentration of edrophonium inhibit the postjunctional acetylcholinesterase thereby causing an increase in transmitter concentration in the end-plate region, but that the highest concentration (6 × 10^?5 M) has a direct desensitizing action as well as the anticholinesterase action on the end-plate membrane. This desensitizing action is present although no depolarization of the end-plate membrane is observable.     

Activation of protein kinase C potentiates postsynaptic acetylcholine response at developing neuromuscular synapses.

1. Phorbol 12-myristate 13-acetate (TPA, 1 microM) and phorbol 12,13-dibutyrate (PDBu, 2 microM), activators of protein kinase C (PKC), increased the mean amplitude and decay time of the spontaneous synaptic currents of Xenopus nerve-muscle coculture, whereas, 4 alpha-phorbol (2 microM) which is an inactive phorbol analogue had no effect. 2. Staurosporine (0.5 microM) and H-7 (10 microM), inhibitors of PKC, inhibited the potentiation effects of TPA on the spontaneous synaptic currents. 3. Effects of TPA on the postsynaptic acetylcholine (ACh) sensitivity were examined by iontophoresis of ACh to the surface of embryonic muscle cells of 1-day-old Xenopus cultures. TPA increased both the amplitude and decay time of ACh-induced whole-cell currents in isolated myocytes. 4. TPA concentration-dependently increased the mean open time of low-conductance ACh channels but did not affect those of high-conductance ACh channels. PDBu but not 4 alpha-phorbol exhibited similar effects to TPA. Staurosporine and H-7 inhibited the increasing effects of TPA. 5. These results suggest that activation of PKC might be involved in synaptogenesis at developing neuromuscular synapses by the postsynaptic potentiation of ACh sensitivity.     

Presynaptic action of uranyl nitrate on the phrenic nerve-diaphragm preparation of the mouse

The contraction of the diaphragm of the mouse, induced by nerve stimulation, can be potentiated by uranyl nitrate [UO2(NO3)2, 0.2-0.8 mM]. At concentrations greater than 0.8 mM, uranyl nitrate also directly enhanced the contractions induced by electrical stimulation of muscle. The effects of uranyl nitrate in potentiating the twitch were augmented by low calcium (0.25-0.5 mM) and antagonized by high levels of calcium (5-10 mM). Electrophysiological studies on the effects of uranyl nitrate on the diaphragm in the mouse have revealed that the frequency of miniature end-plate potentials (MEPP) but not the amplitude was increased; the amplitude and quantal content of end-plate potential (EPPs) were also markedly increased by uranyl nitrate. The most peculiar phenomenon induced by uranyl nitrate was that repetitive end-plate potentials, as well as repetitive action potentials, of the diaphragm of the mouse were triggered by single nerve stimulation in the presence of uranyl nitrate. The duration of muscle action potentials was significantly prolonged. Recordings of the evoked compound action potentials from nerve axons showed that uranyl nitrate not only prolonged the duration of the axonal compound action potentials but also induced antidromic activity with a single stimulation. This latter effect of uranyl nitrate was blocked by d-tubocurarine. All of these findings suggest that uranyl nitrate potentiated the contractions of the diaphragm of the mouse, possibly through the activation of stimulus-activated repetitive discharges which resulted in the repetitive end-plate potentials and muscle action potentials.     

Ephedrine: a postsynaptic depressant drug at the mammalian neuromuscular junction

The actions of ephedrine were studied using intracellular recording techniques in guinea pig intercostal muscle fiber. After application of ephedrine (10^?5 ?10^?4 M), miniature end-plate potential (MEPP) amplitude was decreased in a concentration-dependent manner. A greater depression in amplitude of end-plate potentials (EPP) was observed in preparations immobilized with d-tuboeurarine. In addition, ephedrine depressed the transient depolarization produced by iontophoretically applied acetylcholine (ACh).In normal Krebs solution and high Mg²⁺ solution, 10^?4 M ephedrine increased MEPP frequency but did not alter the quantal content of EPPs in high Mg²⁺ blocked muscle. In these preparations, EPP amplitude did not change significantly. These results suggested that in the absence of a postsynaptic receptor blocking agent, a slight presynaptic action of ephedrine may offset the decreased responsiveness of the postsynaptic membrane. In addition, no significant alterations of resting membrane potential or the electrical characteristics of the membrane were recorded.Pretreatment of a mouse phrenic nerve-diaphragm preparation with ephedrine did not protect against the irreversible blockade by α-bungarotoxin. These results suggested that ephedrine may depress ACh potentials, MEPPs, and EPPs in dTc-blocked muscle by acting postsynaptically at a site on or associated with the ACh receptor other than the ACh recognition site.