Antagonism by beta-eudesmol of neostigmine-induced neuromuscular failure in mouse diaphragms.

beta-Eudesmol, a sesquiterpenol extracted from a Chinese herb, Atractylodes lancea, at 10-80 microM, did not affect muscle action potentials, miniature and evoked endplate potentials and acetylcholine-induced depolarization in the presence or absence of neostigmine in mouse phrenic nerve-diaphragms. However, the tetanic fade, muscle fasciculation and twitch potentiation induced by neostigmine were effectively antagonized by 20 microM beta-eudesmol. When trains of pulses were applied to the nerve in the presence of neostigmine, beta-eudesmol reduced the incidence of explosive depolarization of the endplate from 95% to 35-67% of junctions, and shortened the duration when it occurred. Moreover, both the maximal and steady-state depolarizations during repetitive stimulation were reduced while the amplitudes of steady-state endplate potentials were increased. The results suggest that beta-eudesmol antagonized neostigmine-induced neuromuscular failure mainly by a presynaptic action to depress the regenerative release of acetylcholine during repetitive stimulation. The mechanism of antagonism is obviously not tubocurarine-like and it is unrelated to desensitization of acetylcholine channels.     

Reversals of the neostigmine-induced tetanic fade and endplate potential run-down with respect to the autoregulation of transmitter release.

1. In order to shed more light on the role of presynaptic cholinoceptors in the modulation of transmitter release, the effects of tubocurarine, choline and hexamethonium on neostigmine-induced tetanic fade and run-down of endplate potentials (e.p.ps) in response to indirect stimulation with trains of pulses were studied in the intact and cut isolated phrenic nerve-diaphragm preparation of the mouse, respectively. 2. Tubocurarine, choline and hexamethonium reduced both the tetanic fade and e.p.p. run-down caused by neostigmine, despite the fact that they themselves also induced these two effects. 3. At a given degree of postsynaptic inhibition, choline and hexamethonium caused less e.p.p. run-down and reversed the neostigmine-induced tetanic fade and e.p.p. run-down better than tubocurarine. Moreover, the e.p.p. run-down caused by choline or hexamethonium, but not that induced by tubocurarine, was reciprocally reversed by neostigmine. 4. Tubocurarine, choline and hexamethonium significantly decreased the endplate depolarization induced by repetitive nerve stimulation in the presence of neostigmine. The remaining depolarization continued to grow during repetitive stimulation in the presence of choline or hexamethonium, but not, however, in the presence of tubocurarine; a finding which suggests that choline and hexamethonium but not tubocurarine may be displaced from the receptor by the accumulated acetylcholine. 5. The mutual reversal by neostigmine and cholinoceptor antagonists of e.p.p. run-down may implicate the presence of a positive (physiological) and a negative (pharmacological) feedback regulation for evoked transmitter release via nicotinic cholinoceptors in the mammalian motor nerve, depending on the concentration of acetylcholine within the synaptic cleft.     

Pharmacological relevance of peripheral type benzodiazepine receptors on motor nerve and skeletal muscle.

1. Effects of agonists and antagonists of peripheral and central benzodiazepine receptors (pBZR and cBZR) on neuromuscular transmission were studied in mouse isolated phrenic nerve-diaphragm preparations. 2. Ro5-4864, a pBZR agonist, had no effect on the neuromuscular transmission but increased muscle contractility and antagonized the tetanic fade induced by neostigmine. 3. Ro5-4864 inhibited the regenerative tonic endplate depolarization caused by repetitive stimulation in the presence of neostigmine without affecting the amplitude and decay time of miniature and evoked single endplate potentials. 4. All the effects of Ro5-4864 were shared by PK11195, a pBZR antagonist, but not by clonazepam and flumazenil, a cBZR agonist and antagonist, respectively. 5. It is suggested that peripheral type benzodiazepine receptors modulate presynaptic function and muscle contraction.     

Transmitter-mediated local contracture of the endplate region of the focally innervated mouse diaphragm treated with anticholinesterase.

1. Local contraction of the endplate region in response to nerve stimulation was studied in isolated mouse diaphragms. In normal preparations, muscle contractions involved the whole length of the muscle fibre with rise and decay times in the order of tens of ms whether evoked with a single or train of pulses. 2. When acetylcholinesterase was inhibited with neostigmine, tetanic stimulation produced a twitch-like phasic contraction and a delayed tonic contracture. A brief train of pulse (10 ms, 300 Hz) was enough to trigger a full size tonic contracture which reached an amplitude about one tenth that of control tetanus and had a duration of about 4 s. 3. Tetanic stimulation evoked a non-propagating prolonged depolarization at the endplate region lasting for about 1 s following a few muscle action potentials. 4. mu-Conotoxin, a specific inhibitor of muscle Na+ channel, selectively abolished the phasic contraction and the muscle action potentials leaving the tonic contracture and the prolonged depolarization unaffected. 5. Both the tonic contracture and the prolonged depolarization were highly sensitive to blockade by tubocurarine (IC50 0.05-0.1 microM) and vesamicol (1 microM, an inhibitor of packaging acetylcholine into synaptic vesicles), were attenuated by increasing Ca2+ concentration and were prolonged by decreasing Ca2+. 6. The results suggest that prolonged activation of endplate nicotinic receptors by endogenously released transmitter can produce substantial contractions of the endplate region when acetylcholinesterase are inhibited. The source of Ca2+ for the contraction seems to come mainly from intracellular stores.     

Mechanisms underlying the vecuronium-induced tetanic fade in the isolated rat muscle

The cellular mechanisms underlying the effects of vecuronium on the tetanic contraction were studied in vitro with a combination of myographic and electrophysiologic techniques. We used the isolated sciatic nerve extensor digitorum longus muscle preparation of the rat. Indirect twitches were evoked at 0.1 Hz pulses and tetani at 50 Hz pulses. Trains of end-plate potentials were generated at 50 Hz. The electrophysiological variables used in the analysis of the end-plate potentials were: amplitude, tetanic run-down, quantal size and quantal content. The myographic study demonstrated that vecuronium at 0.4 microM caused tetanic fade, but left the twitch unaffected. Regarding electrophysiology, vecuronium (0.4 microM) decreased the amplitude of end-plate potentials and increased their tetanic run-down. These changes were due to significant reductions in both the quantal content of the end-plate potentials and the quantal size. It is concluded that vecuronium has both pre- and postsynaptic effects at the neuromuscular junction, and that it induces fade of the tetanic contraction via a summation of these effects.     

Cellular mechanisms of atracurium-induced tetanic fade in the isolated rat muscle

Although atracurium is a widely used neuromuscular blocker, we still lack knowledge regarding some of its cellular mechanisms of action. Thus, similar to other clinically used blockers atracurium induces, both in vivo and in vitro, fade of the tetanic contraction. However, the cellular mechanisms underlying this tetanic fade have never been systematically studied. In the present work these mechanisms were investigated in vitro. A sciatic nerve extensor digitorum longus muscle preparation of the rat was used. A combination of myographical and electrophysiological techniques was employed. Indirect twitches were evoked at 0.1 Hz and tetanic contractions at 50 Hz. Trains of end-plate potentials were evoked at a frequency of 50 Hz. The electrophysiological variables used in the analysis of the trains of end-plate potentials were: peak amplitude of the first end-plate potential in the train, peak amplitude of plateau end-plate potentials in the train, tetanic run-down of the end-plate potentials' train, quantal content of first and plateau end-plate potentials in the train, quantal size. In the myographical study atracurium, at a concentration of 2.4 microM, induced a complete fade of the tetanic contraction while only slightly affected the twitch. In the electrophysiological study atracurium, at the same 2.4 microM concentration, significantly decreased the amplitude of both first end-plate potentials in the train (control: 14.4 mV; atracurium: 3.2 mV) and plateau end-plate potentials (control: 10.8 mV; atracurium: 2.4 mV) and reinforced the tetanic run-down of the train of end-plate potentials, evaluated as the percent loss in amplitude of plateau end-plate potentials compared to first end-plate potentials in the trains (control: 25.2%; atracurium: 33.2%). Atracurium also significantly decreased the quantal content of first end-plate potentials in the train (control: 231; atracurium: 68), the quantal content of plateau end-plate potentials (control: 159; atracurium: 42) and the quantal size (control: 0.119 mV; atracurium: 0.075 mV). In relative terms the decrease in quantal content was about twice as large as the decrease in quantal size. This indicates that the fade of the tetanic contraction induced by atracurium (2.4 microM) is due to both pre- and postsynaptic blocking effects, the presynaptic one being stronger.     

Mechanisms Underlying the Vecuronium-Induced Tetanic Fade in the Isolated Rat Muscle

Abstract The cellular mechanisms underlying the effects of vecuronium on the tetanic contraction were studied in vitro with a combination of myographic and electrophysiologic techniques. We used the isolated sciatic nerve extensor digitorum longus muscle preparation of the rat. Indirect twitches were evoked at 0.1 Hz pulses and tetani at 50 Hz pulses. Trains of end-plate potentials were generated at 50 Hz. The electrophysiological variables used in the analysis of the end-plate potentials were: amplitude, tetanic run-down, quantal size and quantal content. The myographic study demonstrated that vecuronium at 0.4 μM caused tetanic fade, but left the twitch unaffected. Regarding electrophysiology, vecuronium (0.4 μM) decreased the amplitude of end-plate potentials and increased their tetanic run-down. These changes were due to significant reductions in both the quantal content of the end-plate potentials and the quantal size. It is concluded that vecuronium has both pre- and postsynaptic effects at the neuromuscular junction, and that it induces fade of the tetanic contraction via a summation of these effects.     

在不均匀牵拉的离体大鼠膈肌标本上梭曼的突触前和突触后作用

在离体大鼠膈肌上制备了一种不均匀牵拉肌肉以制动,便于用微电极记录终板电位(EPP)的标本(INSMP),避免了常规制动方法带来的制动药物、台氏液Ca~(2+),Mg~(2+)浓度改变或钳压肌肉导致膜电位(MP)下降的种种干扰。INSMP的MP和小终板电位(MEPP)正常,EPP幅度高达30mV以上,是研究药物对接头作用的较好标本。在INSMP上,梭曼(5.5μM)使MEPP频率加快,串刺激(50Hz)诱发的平均串EPPs幅度及其平均ACh 量子含量减少80％和77％。小剂量三碘季铵酚和六烃季铵可部分对抗梭曼的作用。梭曼引起强直收缩抑制主要原因为终板区蓄积的ACh作用于突触前N受体、负反馈地抑制ACh 量子释放;次要原因为作用突触后N受体、使其对ACh敏感性降低。     

Neuromuscular blocking profile of the vecuronium analogue, Org-9487, in the rat isolated hemidiaphragm preparation.

1. The neuromuscular effects of the short-acting aminosteroid muscle relaxant Org-9487 have been studied in the in vitro rat phrenic nerve/hemidiaphragm preparation by use of twitch tension and electrophysiological recording techniques. 2. Org-9487 (5-100 microM) produced a concentration-dependent decrease in the amplitude of twitches (0.1 Hz) and tetanic contractions (50 Hz) evoked by motor nerve stimulation. The compound produced fade of force during both 50 Hz stimulation and train-of-four stimulation at 2 Hz, indicating a prejunctional component of action. 3. Anticholinesterases only partially reversed the effect of Org-9487 on twitch responses. This was possibly because, at the concentrations required to block twitches in the rat, Org-9487 itself was found to possess significant anticholinesterase activity. 4. Org-9487 (3 microM) increased the rundown of endplate current amplitudes during a 2 s train of 50 Hz nerve stimulation. This was because Org-9487 increased the quantal content of the first endplate current in the train without affecting acetylcholine release towards the latter part of the train. 5. Org-9487 (10 microM) produced a voltage-dependent decrease in the time constant of decay of endplate currents at 32 degrees C and 0.5 Hz, indicative of a block of endplate ion channels. The blocking rate constant increased with membrane hyperpolarization.     

The effects of tetraethylammonium during twitch and tetanic stimulation of the phrenic nerve diaphragm preparation in the rat

Tetraethylammonium (TEA) (2.6 x 10(-3) M) potentiated the twitches of the indirectly- or directly-stimulated phrenic nerve diaphragm of the rat at 37 degrees C by prolonging the action potential of the sarcolemma, due to an inhibition of the repolarizing K+ current. With indirect stimulation, TEA caused a use-dependent inhibition of tetanic contractions, induced every 10 min by 10 sec of 50 Hz stimulation, and a post-tetanic depression of the twitches was observed after about 40 min. Recording of the electromyogram (EMG) and compound action potentials of the phrenic nerve, localized the two inhibitory effects to the neuromuscular junction. They were caused by different mechanisms of action. Choline (3.6 x 10(-4) M) antagonized the depression of the twitch but not the use-dependent inhibition. Lowering the temperature to 20 degrees C reduced the depression of the twitch, whereas the use-dependent inhibition was enhanced. The release of transmitter was probably normal during tetanic stimulation; a post-synaptic desensitization of acetylcholine (ACh) receptors caused the inhibition. Microelectrode recordings of endplate potentials supported this conclusion. The depression of the twitch was due to a presynaptic depletion of transmitter. This was confirmed by inducing an additional depletion and depression of the twitch with N-ethyl-maleimide (2.5 x 10(-5) M). Since the depression of the twitch was antagonized by choline, the depletion was probably due to an inhibited uptake of choline into the nerve terminals.     

Reduction of quantal size and inhibition of neuromuscular transmission by bafilomycin A

The energy for uphill transport of neurotransmitters into synaptic vesicles is created by bafilomycin A- and concanamycin A-sensitive vacuolar H(+)-ATPase (V-ATPase). Both blockers (at 0.1-5 microM) depressed twitch tension and induced tetanic fade of mouse diaphragm on stimulation of the phrenic nerve. Axonal impulse conduction and depolarization of motor endplate by exogenous acetylcholine were not inhibited. The IC(50)s for bafilomycin A and concanamycin A were 1.1+/-0.2 and 0.7+/-0.1 microM, respectively. Contractile response evoked by stimulation of diaphragm, muscle resting membrane potential and membrane resistance were not altered. V-ATPase blockers decreased quantal size and shifted the distribution of miniature endplate potentials (mepps) to low amplitude direction. The increase of mepp events in high KCl medium was suppressed slightly. The blockers depressed endplate potentials (epps) with IC(50)s of 0.7+/-0.2 microM (bafilomycin A) and 0.4+/-0.1 microM (concanamycin A). On high frequency stimulation, the coefficient of variance and run-down of epps were increased. The inhibitory effects on mepps and epps were irreversible and augmented by nerve stimulation. The results suggest that inhibition of V-ATPase reduces the acetylcholine content of synaptic vesicles, leading to suppression of neuromuscular transmission.     

The neuromuscular transmission fade (Wedensky inhibition) induced by L-arginine in neuromuscular preparations from rats.

L-Arginine (4.7-18.8 mM) and 3-(4-morpholinyl)-sydonone imine hydrochloride (SIN-1; 1.15 mM) induced an increase in tetanic fade caused by indirect stimulation (180-200 Hz) of muscle. However, Wedensky inhibition, different from control, was not observed when the preparations treated with d-tubocurarine were directly stimulated by the same frequency. D-Arginine (9.4 mM) was ineffective in changing R values caused by indirect stimulation (180-200 Hz) of muscle. N(omega)-Nitro-L-arginine (73 mM) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM) did not produce any effect on Wedensky inhibition but did antagonize the tetanic fade induced by L-arginine (9.4 mM). The SIN-1 effect was antagonized by previous administration of ODQ (108 microM), which alone did not produce any effects on R values. These results indicate that NO acting at the presynaptic level increases the Wedensky inhibition induced by high frequency of stimulation applied on motor nerves, and its effect may be produced through the cGMP-GC pathway.     

The effects of hyoscine and anticholinesterases on cholinergic transmission to the smooth muscle cells of the avian gizzard

1. Intracellular recordings were made from smooth muscle cells of the pigeon and chick gizzards. Hyoscine (10(-6) g/ml.) blocked, within 2 min, the excitatory junction potentials (E.J.P.s) evoked by transmural stimulation. As the amplitudes of E.J.P.s decreased, their durations also decreased. Hyoscine had no effect on the inhibitory junction potentials (I.J.P.s) evoked by transmural stimulation.2. Initially, physostigmine or neostigmine (10(-7) - 5 x 10(-6) g/ml.) caused a marked increase in the amplitudes of E.J.P.s, without affecting their time courses. Sometimes, anticholinesterases caused a single stimulus to evoke multiple E.J.P.s.3. After several minutes in the presence of high concentrations (10(-6) - 5 x 10(-6) g/ml.) of anticholinesterases the muscle cells were depolarized by about 15-20 mV, and thus the amplitudes of I.J.P.s evoked by transmural stimulation were larger than under control conditions. E.J.P.s summed at much lower stimulation frequencies than normal, an observation which indicated that high concentrations of anticholinesterases increased the durations of the E.J.P.s. Repetitive stimulation evoked a membrane depolarization which persisted for as long as 10 sec after stimulation was stopped.4. These results are discussed in terms of the action of the drugs at the nervesmooth muscle junction and on ganglion cells in the myenteric plexus.     

Effects of toxin II from the sea anemone Anemonia sulcata on contractile and electrical responses of frog skeletal muscle fibres

The effects of Anemonia sulcata toxin II (ATX-II) were studied on mechanical and electrical activities of frog muscle fibres isolated from semitendinosus or tibialis anterior muscles of Rana temporaria (2.8-7.7 degrees C). In concentrations ranging between 7.7 and 100 microM, ATX-II greatly potentiated the isometric twitch of single muscle fibres in a time-dependent manner. Increase in twitch amplitude by ATX-II was associated with an increase in time to peak tension and time from peak tension to half relaxation. ATX-II caused no change in maximum force production during fused tetanus, but the tension was maintained for several seconds after the cessation of stimulation. Such long tetanic contractions were also obtained in low-Na Ringer solution, but their duration was somewhat shorter. No specific action of ATX-II was detected on relaxation kinetics during a tetanus. The twitch potentiating effect of ATX-II was markedly increased by 3,4-diaminopyridine. Action potentials recorded from single muscle fibres in the presence of ATX-II showed a delayed onset of repolarization with a reduced rate of fall. In addition, ATX-II caused repetitive spontaneous firing of action potentials after the cessation of tetanic stimulation. ATX-II (3.3 - 10 microM) also increased action potential duration by reducing the rate of repolarization in surface fibres of formamide-treated sartorius or cutaneous pectoris muscles (20 degrees C) stimulated indirectly or directly. The potentiation of twitch force and the prolongation of contractions caused by tetanic stimulation can be attributed to the membrane action of ATX-II, which leads to prolongation of action potentials, to repetitive muscle firing and to the appearance of plateau potentials.     

Effects of Chlorhexidine on the Isolated Rat Phrenic Nerve-Diaphragm Preparation

Dose-dependent inhibitory effect of chlorhexidine (2.5 × 10^?5–5.0 × 10^?4) g/ml on neuromuscular transmission were localized by tension and electromyogram recording during indirect stimulation. Subsequent direct stimulation showed an additional inhibition which might be located to the sarcolemma. In addition, contracture was observed at the highest concentrations. Microelectrode experiments showed a miniature endplate potential frequency increase (at 1.0 × 10^?6 g/ ml), suggesting a presynaptic action. Increasing the dose to 5.0 × 10^?6 g/ml disclosed a decrease of the miniature endplate potential amplitude, indicating a reduction of the postsynaptic receptor sensitivity, which might cause neuromuscular inhibition. This was confirmed by endplate potential recording in cut preparations. The endplate potential showed a moderate degree of use-dependent inhibition which did not usually cause a tetanic fade. Experiments with curare, decamethonium and neostigmine indicated that chlorhexidine probably caused a decamethonium-like inhibition. A small depolarization of the sarcolemma was probably not the cause of the directly elicited inhibition or contracture. The contracture was potentiated in preparations made myotonic by pretreatment with para-hydroxy-mercuribenzoate. It could not be inhibited by the excitation-contraction uncoupler dantrolene, but it was dependent on extracellular Ca²⁺. Chlorhexidine-induced influx of Car²⁺ might thus cause the contracture.     

Effects of anticholinesterases and of sodium fluoride on neuromyal desensitization

Desensitization of amphibian neuromyal junction was obtained by either repetitive, brief (2 msec) iontophoretic pulses of acetylcholine (ACh) or by a prolonged (40 sec) iontophoretic application of ACh. When ACh was applied repetitively, ACh potentials diminished gradually at a rate dependent on ACh pulse frequency; the recovery time constant amounted to approximately 7 sec following 30 sec of brief ACh pulses applied at 20 Hz. In the case of prolonged, 40 sec ACh pulses, repolarization occurred within the duration of the pulse. Subsequent test (2 msec) pulses of ACh showed a diminished endplate response.Anticholinesterase drugs, the organophosphorus tetraethylpyrophosphate (TEPP) and the carbamate, neostigmine, markedly accelerated desensitization. This occurred even when the iontophoretic current was decreased following anticholinesterase treatment in order to generate ACh potentials similar in amplitude to those recorded prior to the treatment. These and additional results indicated that anticholinesterases exert a direct, desensitizing effect independently of the possibility of their causing accumulation of iontophoretically applied ACh.Sodium fluoride (NaF) employed in concentrations of 0.1 to 5 mM, significantly delayed the onset of desensitization whether the latter was induced by repetitive or prolonged pulses of ACh; it also accelerated the recovery of the endplate from desensitization. At concentrations ranging from 0.05 to 2 mM, NaF also antagonized the acceleration of desensitization induced by either TEPP or neostigmine; in the combined presence of the anticholinesterase and NaF, the rate constant of desensitization approximated to that recorded under control conditions. This action by NaF was exerted after prolonged (30 min) TEPP treatment; thus, this effect was not due to the reactivating potential of NaF. Repetitive (at 50 Hz) indirect stimulation of the endplate produced progressive diminution of the EPP's, with a plateau occurring at 60 sec. This was concomitant with marked diminution of the response to test pulses of ACh and a slight decrease in the quantal content of the EPP's; thus, it was not due to diminution of the release of ACh. The rate of this phenomenon was markedly increased by anticholinesterases and markedly decreased by NaF.It is emphasized in the Discussion that the antidesensitizing action of NaF does not depend on its chelation of Ca²⁺ and that it may be due to the direct action of NaF on the recentor.     

Muscarinic inhibition of [3H]-noradrenaline release on rabbit iris in vitro: effects of stimulation conditions on intrinsic activity of methacholine and pilocarpine.

1. Rabbit isolated irides were loaded with [3H]-noradrenaline and superfused with Tyrode solution. The inhibition by the muscarinic agonists (+/-)-methacholine and pilocarpine of the [3H]-noradrenaline overflow into the superfusate evoked by field stimulation (pulses of 1 ms duration, 75 mA) was measured as an index of activation of presynaptic muscarinic receptors. 2. The fractional rate of release per pulse during the first stimulation period (S1) was low with 360 pulses at 3 Hz, intermediate with 360 pulses at 10 Hz and high with 1200 pulses at 10 Hz. Upon repetitive stimulation (7 periods at 20 min intervals), the fractional rates of release per pulse during S7 no longer differed, suggesting a 'long-term' regulation of [3H]-noradrenaline release depending on the stimulation conditions. 3. The evoked [3H]-noradrenaline overflow was depressed by (+/-)-methacholine in a concentration-dependent manner. The EC50 ranged from 0.29 to 0.42 microM. Methacholine nearly abolished the transmitter release evoked at 3 Hz but reduced that induced at 10 Hz by only 50%. Under the latter condition the methacholine concentration-inhibition curve was bell-shaped and no muscarinic inhibition was observed in the presence of methacholine 30 microM. After washout of methacholine the evoked [3H]-noradrenaline release was temporarily enhanced. 4. Atropine 0.1 microM enhanced the [3H]-noradrenaline overflow (evoked by stimulation with 360 or 1200 pulses at 10 Hz), probably antagonizing a presynaptic inhibition by endogenous acetylcholine. The inhibition by methacholine was competitively antagonized by atropine 0.1 microM (apparent -log KB = 8.5-9.0). 5. Depending on the concentration, pilocarpine reduced the [3H]-noradrenaline overflow evoked by 360 pulses at 3 Hz up to 63%. However, at 10 Hz stimulation frequency the compound was inactive as an agonist but competitively antagonized the presynaptic inhibition induced by methacholine. The KB under the latter condition (0.95 microM) was very close to the EC50 value determined at 3 Hz (0.85 microM). 6. The results demonstrate a muscarinic inhibition of noradrenaline release from the rabbit isolated iris. The activation by pilocarpine of the presynaptic receptors provides an alternative explanation for the miosis induced in the rabbit in vivo, which might be the result of a decreased sympathetic tone in the iris dilator muscle.     

Acetylcholine hydrolysis during neuromuscular transmission in the synaptic cleft of skeletal muscle of mouse and chick

Inhibition of acetylcholinesterase (AChE) by more than 80% by neostigmine or physostigmine resulted in a failure of tetanic contraction (100 Hz) in the isolated mouse nerve-diaphragm preparation. In the chick biventer cervicis muscle, however, the tetanic contraction was well maintained and even outlasted the period of nerve stimulation after inactivation of AChE. The concentration of (+)tubocurarine for 70% block of the indirect twitch response of the mouse diaphragm at 0.1 Hz was increased from 0.67 to 0.99 to 1.21-2.03 microM in the presence of neostigmine (0.15-1.5 microM) which inhibited AChE by 70% or more, while that to depress the tetanic contraction (50 Hz) was increased from 0.38 to 0.42 to 0.53-0.69 microM. In the chick muscle, physostigmine at 2.4 microM increased the concentration of (+)tubocurarine for 70% block of the twitch response from 1.68 to 4.14 microM, whereas that for block of the response to exogenous acetylcholine (ACh) was increased from 1.47 to 74.6 microM. On single stimulation, the relative peak concentrations of acetylcholine (ACh) at the postsynaptic receptor site of the mouse diaphragm and chick biventer cervicis were estimated to be increased by about 110 and 120% respectively, after complete inhibition of AChE. In the chick muscle, physostigmine increased the relative concentration of ACh by about 40-fold at the receptor site for exogenously applied ACh. It is concluded that the intrinsic ACh released from the nerve terminal is hydrolyzed by about 50% during the time of diffusion across the synaptic cleft whereas most of exogenous ACh is hydrolyzed before reaching the target.     

Effects of the sea anemone Anemonia sulcata toxin II on skeletal muscle and on neuromuscular transmission

Effects of anemone toxin II (ATX II) have been analysed on the neuromuscular junction of the frog and different twitch muscles. Amplitudes of evoked endplate potentials and endplate currents are increased by ATX II, without effects on the amplitudes of miniature endplate potentials and endplate currents resulting from ionophoretically applied transmitter. The increase in evoked transmitter release is due to an increase in quantal content caused by an effect of the toxin on the presynaptic action potentials. ATX II is also effective on muscle fibers. The action potentials of frog twitch muscles are reversibly prolonged by ATX II. Their rate of rise and amplitudes are increased, while there is no effect on resting membrane potential. Similarly, action potentials of fast twitch muscle (extensor digitorum longus, EDL) of the mouse are reversibly prolonged by ATX II. In slow twitch muscle (soleus, SOL) of the mouse the toxin induces repetitive action potentials following the generation of a single action potential. Tetrodotoxin resistant action potentials of both denervated EDL and SOL are greatly and irreversibly prolonged by ATX II. The effects on muscle are due to a Na+ channel specific action of ATX II. Na+ current inactivation is slowed with the time constant tau h increasing towards positive membrane potentials. The steady state inactivation curve hoo was shifted to more positive potentials and its slope reduced.     

Selective antagonism to succinylcholine-induced depolarization by alpha-bungarotoxin with respect to the mode of action of depolarizing agents.

1. The interactions of alpha-bungarotoxin or tubocurarine with the neuromuscular block and endplate depolarization induced by succinylcholine (SCh) in the phrenic nerve-diaphragm preparation of mice were studied in order to elucidate the role of depolarization by SCh in the neuromuscular blockade. 2. The SCh concentrations required to depress the indirect twitch response by 20% and the evoked endplate potential in cut muscle preparations by 80% were 10 microm and 6 microM, respectively, while only 2 microM SCh was needed to induce maximal endplate depolarization from -80 mV to about -60 mV. 3. SCh blocked the neuromuscular transmission synergistically with either alpha-bungarotoxin or tubocurarine. There was an initial partial reversal of the neuromuscular inhibition caused by tubocurarine, but not that by alpha-bungarotoxin. 4. alpha-Bungarotoxin (0.025 microM) antagonized SCh (10 microM)-induced depolarization more effectively than it depressed miniature endplate potentials and the antagonism was insurmountable by increasing SCh concentration. By contrast, tubocurarine preferentially depressed miniature endplate potentials and antagonized SCh-depolarization competitively. 5. The above difference was attributed to the irreversible nature of alpha-bungarotoxin binding to acetylcholine receptors, to the slow diffusion of the toxin molecule into the synaptic cleft and thus to the more rapid binding with perijunctional receptors compared with junctional ones. 6. It is concluded that the sustained depolarization of the endplate by SCh results largely from an action on the perijunctional receptor in mice and, unlike cats, the neuromuscular block by SCh is not due to the depolarization per se but rather to a direct attenuation of endplate potential.