Interactions of gephyrotoxin with the acetylcholine receptor-ionic channel complex. I. Blockade of the ionic channel

The novel tricyclic alkaloid, gephyrotoxin ( GyTX ), found in the skin secretions of the frog Dendrobates histrionicus , potentiates and blocks the indirectly elicited muscle twitch in a concentration-dependent manner. GyTX prolongs the falling phase of the muscle action potential and decreases delayed rectification, supporting the idea that the alkaloid blocks the voltage-sensitive potassium conductance of the electrically excitable membrane. The peak amplitude of the end-plate currents (EPC) and miniature end-plate currents ( MEPC ) were depressed, but no significant deviation from linearity relative to control was seen in the current-voltage relationship. The decay time constant of the EPC (tau EPC) was markedly shortened by GyTX , the effect being greater at 10 degrees than at 22 degrees. The relationship between the log of tau EPC and membrane potential disclosed a linear relationship at all concentrations tested, but a progressive loss of voltage sensitivity of tau EPC was seen when GyTX concentrations were increased. Also, the plot of 1/tau EPC against GyTX concentration revealed a linear relationship. The lack of voltage and time dependence suggests that GyTX has little effect on the ACh receptor-ionic channel complex in the closed conformation. Single-channel conductance studied by means of fluctuation analysis did not change after GyTX application, but the channel lifetime decreased by about 40% at clamp potentials of -105 mV and at a toxin concentration of 7.5 microM. Repetitive nerve stimulation led to a pronounced " rundown " in the EPCs which was frequency-dependent. These findings were taken as evidence that GyTX interacts with the acetylcholine receptor complex, causing a blockade of its channel mainly in the open conformation.     

Interactions of bupivacaine with ionic channels of the nicotinic receptor. Electrophysiological and biochemical studies

The actions of the tertiary local anesthetic bupivacaine were studied on the nicotinic receptor-ionic channel complex (AChR) using electrophysiological and biochemical methods. Voltage clamp studies of the frog sartorius and cutaneous pectoris neuromuscular junction revealed a concentration-dependent depression of the decay time constant of the end-plate (tau EPC) and spontaneous miniature end-plate (tau MEPC) currents. The relationship of the reciprocal of either tau EPC or tau MEPC and bupivacaine concentration up to 100 microM was linear. Voltage dependence of EPC over the range +60 to -150 mV was reduced, whereas both EPC and MEPC decays were adequately described by a single exponential function at all concentrations tested. Peak MEPC and EPC amplitudes were also depressed in a concentration-dependent manner such that 100 microM bupivacaine reduced peak amplitude by about 50%. The current-voltage relationship remained linear under all conditions tested. Nerve-evoked responses were difficult to study at concentrations greater than 100 microM because of apparent blockade of nerve conduction. Extracellular recording of the MEPC afforded results similar to those obtained with EPCs. The tau MEPC could be reduced to less than 300 mu sec at a bupivacaine concentration of 400 microM. Fluctuation analysis showed that bupivacaine at concentrations of 10 and 25 microM did not change channel conductance but decreased single-channel lifetime to 76% and 39% of control values, respectively. Biochemical studies were performed on Torpedo californica membrane fragments using [3H]phencyclidine ([3H]PCP) and [3H]perhydrohistrionicotoxin ([3H]H12-HTX) as channel probes. Bupivacaine inhibited the binding of [3H]PCP and [3H]H12-HTX with inhibition constants (Ki) of 32 and 25 microM, respectively. The corresponding inhibition constants for bupivacaine methiodide were 1.8 and 3.2 microM. The preincubation of the membranes with carbamylcholine increased the affinity of bupivacaine for the ionic channel sites 5- to 8-fold and the affinity of bupivacaine methiodide 3- to 4-fold. Bupivacaine, however, had no affinity for the agonist recognition site as determined by [3H]ACh and [125I]alpha-bungarotoxin bindings. The electrophysiological and biochemical studies indicate that bupivacaine reacts primarily with the ionic channel of the nicotinic AChR. The results are consistent with a sequential model in which the drug interacts with the sites at the ionic channel of AChR in its open conformation, producing species with little or no conductance. From the present studies there is no evidence for an interaction of bupivacaine with the agonist binding site or closed states of AChR.     

Electrophysiological estimation of the actions of acetylcholinesterase inhibitors on acetylcholine receptor and cholinesterase in physically isolated Aplysia neurones.

1. The actions of representative cholinesterase inhibitors on the acetylcholine responses of physically isolated single neurones from the pedal ganglion of Aplysia californica were studied, using electrophysiological techniques and rapid agonist application to analyse both the inhibitory actions on the acetylcholine receptor-channel complex and the degree of inhibition of acetylcholinesterase activity on the same neurone. The inhibitors used were physostigmine, edrophonium and diisopropylfluorophosphate (DFP). 2. When selected neurones were suddenly exposed to 50 microM acetylcholine by a 'concentration clamp' technique a large Na-dependent inward current was initiated, and decayed in the continued presence of acetylcholine without external perfusion. However, if perfusion of the acetylcholine solution was reinitiated the current increased somewhat, indicating that the decay of current was due to some combination of receptor desensitization and local depletion of acetylcholine at the membrane by acetylcholinesterase. 3. With simultaneous application of acetylcholine (50 microM) and physostigmine (0.1 to 100 microM) there was a dose-dependent reduction of peak amplitude of the acetylcholine response. However, physostigmine at low concentrations (0.1 to 10 microM) caused a time-dependent increase in the current amplitude alone with a time- and dose-dependent inhibition of acetylcholinesterase activity. At the highest concentration of physostigmine (100 microM) acetylcholinesterase activity was abolished but the current peak was very depressed. After removal of physostigmine from the bathing solution, the current amplitude decreased toward the control at the two lower concentrations as the inhibitory actions on acetylcholinesterase activity were almost reversible, while at the two higher concentrations (10 and 100 microM) the current increased and the inhibition of acethylcholinesterase remained. 4. When acetylcholine (50 microM) and edrophonium (0.1 to 10 microM) were applied simultaneously, edrophonium caused a dose-dependent increase in the peak amplitude that was correlated with a dose-dependent inhibition of acetylcholinesterase activity. Prolonged exposure to edrophonium did not change the peak amplitude and there was no time-dependent change in the inhibition of acetylcholinesterase activity. At the highest concentration of edrophonium used (100 microM), simultaneous application with acetylcholine augmented the peak amplitude relative to control, but to a lesser extent than 10 microM. Prolonged exposure to the highest concentration of edrophonium caused a time-dependent reduction in the peak amplitude.(ABSTRACT TRUNCATED AT 400 WORDS)     

The acetylcholine receptor of the neuromuscular junction recognizes mecamylamine as a noncompetitive antagonist

The secondary amine, mecamylamine, interacts with the nicotinic receptor ionic channel complex as a noncompetitive antagonist. Mecamylamine (1-10 microM) blocked indirect muscle twitches with no discernible effect on the membrane potential, overshoot, or amplitude of the action potential. It also produced a voltage- and concentration-dependent depression of the peak amplitude of the endplate currents (EPC) and induced nonlinearity in the current-voltage relationship. The decay time constant of the EPC (TEPC) was significantly shortened. The linear relationship between the reciprocal of TEPC and the drug concentration suggested an open channel blockade. Patch-clamp studies, in agreement with the noise analysis results, revealed that mecamylamine (1-8 microM) shortened the lifetime of the open channels. Further, the single channel studies showed that at high concentrations mecamylamine reduced the double exponential nature of the distribution of open times characteristic of channels recorded from myoballs. Closed times had a complex distribution that could not be fitted to a single exponential function because of the presence of short closures or "flickers" during the open state. Although the frequency of channel openings progressively decreased with increasing drug concentration, the single channel conductance remained unchanged at all the concentrations tested. Biochemical studies showed that mecamylamine (up to 100 microM) did not block [3H]acetylcholine binding to the nicotinic receptor of the Torpedo electroplax, but inhibited the binding of [3H]perhydrohistionicotoxin to its channel site, both in the resting and the activated state. These results suggested that, at the nicotinic receptors of the neuromuscular junction, mecamylamine acted as a noncompetitive blocker, binding primarily to the receptor's open channel conformation. Most of the alterations of EPCs were consistent with the predictions of a sequential model for open channel blockade. Biochemical and patch-clamp results, however, could not be fully explained by this model and provided some evidence of the existence of additional blocked states most likely through pathways into desensitized species. In contrast to a competitive antagonism of acetylcholine receptors reported at autonomic ganglia, there was no such action of the drug at the neuromuscular junction; thus, mecamylamine is a useful tool to characterize the nicotinic receptors from different synapses.     

Effects of emetine and dehydroemetine at the frog neuromuscular junction

Emetine and dehydroemetine caused concentration-dependent reduction of the quantal content of the endplate potential (EPP) at the frog neuromuscular junction. At lower concentrations, the drugs had presynaptic action only, as they decreased the amplitude of the EPP without significantly affecting that of the spontaneous miniature EPP (MEPP). At higher concentrations, the drugs had postsynaptic effects as well. Studies of low frequency facilitation indicated that although at low concentrations the effect of emetine on quantal content was independent of frequency of stimulation, at higher concentrations the effect became markedly frequency-dependent and many of the stimuli (at 4 and 8 Hz) failed to evoke EPPs. In the voltage-clamped transected cutaneous pectoris muscle emetine (10(-4) M) depressed the peak amplitude of the endplate current (EPC). Emetine markedly shortened the time constant of decay of the EPC (tau) at more negative holding potentials but did not change the single exponential character of the decay. The results indicate that the acute effects of emetine involve blocking neuromuscular transmission by acting prejunctionally at low concentrations and pre- as well as postjunctionally at higher concentrations. The prejunctional effect is due to interference with evoked release and probably, mobilization of acetylcholine. The postjunctional effect is due to blockade of the acetylcholine-activated ionic channel.     

Activation and block of the adult muscle-type nicotinic receptor by physostigmine: single-channel studies

The plant-derived acetylcholinesterase inhibitor physostigmine has previously been shown to act on the nicotinic acetylcholine receptor (nAChR) causing either direct activation or potentiation of currents elicited by low concentrations of nicotinic agonists, or, at higher concentrations, channel block. We examined mouse adult-type muscle nAChR activation by physostigmine and found that channel activation by physostigmine exhibits many characteristics common with channel activity elicited by nicotinic agonists. Single-channel conductance was indistinguishable, and mutants known to slow channel closing in the presence of nicotinic agonists had a similar effect in the presence of physostigmine. However, physostigmine is a very inefficacious agonist. The presence of physostigmine did not alter the effective opening rate for a subsaturating dosage of carbachol, suggesting that physostigmine does not interact with the nicotinic agonist binding site. Mutations to a residue (alphaLys125) previously identified as part of the putative binding site for physostigmine reduced the duration of openings elicited by physostigmine, but the effects were generally small and, in most cases, nonsignificant. At higher concentrations, physostigmine blocked channel activity. Block manifested as a reduction in the mean open time and the emergence of a closed state, with a mean duration of 3 to 7 ms. The properties of block were consistent with two equivalent blocking sites per receptor with microscopic binding and unbinding rate constants for physostigmine of 20 microM(-1) s(-1) and 450 s(-1) (K(D) = 23 microM). These observations indicate that physostigmine is able to activate muscle nAChR by interacting with a site other than the nicotinic ligand binding site.     

The relationship between cholinesterase inhibition in the chick biventer cervicis muscle and its sensitivity to exogenous acetylcholine.

The effect of physostigmine has been studied on cholinesterase in homogenates of chick biventer cervicis muscles and on the contractile responses of the intact muscles to acetylcholine and carbachol. The concentration of physostigmine required to produce the maximum increase in sensitivity to acetylcholine almost completely inhibited the cholinesterase in muscle homogenates. This concentration of physostigmine had no effect on muscle contractures elicited by carbachol. By taking account of the combined effects of acetylcholine diffusion and enzymic hydrolysis, a quantitative theoretical relationship has been derived between the level of cholinesterase activity in cylindrical muscles and the fractional occupancy of the acetylcholine receptors in these muscles in the presence of different concentrations of exogenous acetylcholine. This theory attributes the thousand-fold increase in sensitivity to exogenous acetylcholine produced by anticholinesterases in chick biventer cervicis muscles largely to an alteration in acetylcholine concentration gradient within the muscle and accounts satisfactorily for the shift in the dose-response curve for acetylcholine which occurs after treatment of the muscles with various concentrations of physostigmine.     

The relationship between cholinesterase inhibition in the chick biventer cervicis muscle and its sensitivity to exogenous acetylcholine

The effect of physostigmine has been studied on cholinesterase in homogenates of chick biventer cervicis muscles and on the contractile responses of the intact muscles to acetylcholine and carbachol. The concentration of physostigmine required to produce the maximum increase in sensitivity to acetylcholine almost completely inhibited the cholinesterase in muscle homogenates. This concentration of physostigmine had no effect on muscle contractures elicited by carbachol. By taking account of the combined effects of acetylcholine diffusion and enzymic hydrolysis, a quantitative theoretical relationship has been derived between the level of cholinesterase activity in cylindrical muscles and the fractional occupancy of the acetylcholine receptors in these muscles in the presence of different concentrations of exogenous acetylcholine. This theory attributes the thousand-fold increase in sensitivity to exogenous acetylcholine produced by anticholinesterases in chick biventer cervicis muscles largely to an alteration in acetylcholine concentration gradient within the muscle and accounts satisfactorily for the shift in the dose-response curve for acetylcholine which occurs after treatment of the muscles with various concentrations of physostigmine.     

Meproadifen reaction with the ionic channel of the acetylcholine receptor: potentiation of agonist-induced desensitization at the frog neuromuscular junction

The actions of the nicotinic noncompetitive antagonist meproadifen on both the acetylcholine (ACh) receptor-ion channel complex and electrically excitable membrane were examined in frog sciatic-nerve sartorius muscle preparations. Meproadifen (10-25 microM) blocked the nerve-evoked twitch without affecting the directly evoked twitch, the threshold, overshoot, amplitude, rate of rise, or falling phase of the directly elicited action potential in muscle. This suggests that this agent, at the concentrations that affect the nicotinic receptor, had negligible effect on the excitable membrane. In addition, the drug did not affect either the quantal content or quantal size of the end-plate potential. Meproadifen caused a voltage- and time-dependent decrease in the peak amplitude of the end-plate current (EPC) without significantly shortening the time constant of EPC decay. The voltage- and time-dependent effects of meproadifen were more pronounced at more negative potentials, as evidenced by hysteresis loops and nonlinearity in the current-voltage relationship of the EPC. Both hysteresis and nonlinearity in the current-voltage relationship of the EPC were eliminated when brief conditioning pulses were used for stepwise changes of membrane potentials. The decay time constant of the EPC in the presence of meproadifen remained an exponential function of time. Meproadifen blocked iontophoretically elicited EPCs but did not affect single-channel lifetime, conductance, or the decay time constant of the miniature EPC. Thus, the blockade was more marked on iontophoretically elicited EPCs than on miniature EPCs. Meproadifen also caused desensitization of both the junctional and extrajunctional ACh receptors, but, more important, meproadifen accelerated steady-state desensitization by several-fold (compared with the agonist). The marked depression of peak EPC amplitude and miniature EPC, its high affinity for the binding sites in the presence of the agonist, and acceleration of agonist-induced desensitization suggest that meproadifen interacts with the ACh-bound but nonconducting state of the ACh receptor-ion channel complex. Therefore, it appears that meproadifen interacts with the closed ionic channel of the ACh receptor in its resting and activated but nonconducting states, and only slightly affects the open conformation of the ionic channel.     

Release of non-neuronal acetylcholine from the human placenta: difference to neuronal acetylcholine

The synthesis and release of non-neuronal acetylcholine, a widely expressed signaling molecule, were investigated in the human placenta. This tissue is free of cholinergic neurons, i.e. a contamination of neuronal acetylcholine can be excluded. The villus showed a choline acetyltransferase (ChAT) activity of 0.65 nmol/mg protein per h and contained 500 nmol acetylcholine/g dry weight. In the absence of cholinesterase inhibitors the release of acetylcholine from isolated villus pieces amounted to 1.3 nmol/g wet weight per 10 min corresponding to a fractional release rate of 0.13% per min. The following substances did not significantly modify the release of acetylcholine: oxotremorine (1 microM), scopolamine (1 microM), (+)-tubocurarine (30 microM), forskolin (30 microM), ouabain (10 microM), 4alpha-phorbol 12,13-didecanoate (1 microM) and tetrodotoxin (1 microM). Removal of extracellular calcium, phorbol 12,13-dibutyrate (1 microM) and colchicine (100 microM) reduced the acetylcholine release between 30% and 50%. High potassium chloride (54 mM and 108 mM) increased the acetylcholine release slightly (by about 30%). A concentration of 10 microM nicotine was ineffective, but 100 microM nicotine enhanced acetylcholine release gradually over a 50-min period without desensitization of the response. The facilitatory effect of nicotine was prevented by 30 microM (+)-tubocurarine. Inhibitors of cholinesterase (physostigmine, neostigmine; 3 microM) facilitated the efflux of acetylcholine about sixfold, and a combination of both (+)-tubocurarine (30 microM) and scopolamine (1 microM) halved the enhancing effect. In conclusion, release mechanisms differ between non-neuronal and neuronal acetylcholine. Facilitatory nicotine receptors are present which are activated by applied nicotine or by blocking cholinesterase. Thus, cholinesterase inhibitors increase assayed acetylcholine by two mechanisms, protection of hydrolysis and stimulation of facilitatory nicotine receptors.     

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.     

Mechanisms of the inhibition of endplate acetylcholine receptors by antiseptic chlorhexidine (experiments and models)

Mechanisms of the inhibition of evoked multiquantal endplate currents (EPC) by chlorhexidine (CHX) were studied in electrophysiological experiments and by mathematical modeling to discriminate between possible channel, receptor, and non-receptor effects of this common antiseptic drug. Experiments were carried out on the isolated neuromuscular preparation of the cut m. sartorius of the frog Rana ridibunda. The nerve-stimulation-evoked endplate currents were measured by standard double microelectrode technique. For the mathematical simulation, a method based on the solution of a system of ordinary differential equations was used. CHX in milimolar concentrations suppressed the amplitude and shortened the evoked EPC. Recovery of the EPC amplitude was very slow, and EPC shortening persisted during 30–40 min washout of the drug. There is no indication that CHX competes for acetylcholine or carbachol binding site(s). A comparison of the experimental data with mathematical simulation made it possible to construct a reliable kinetic scheme, which describes the action of CHX. CHX induces a combined slow blockade of the open ionic channel and long-lasting allosteric inhibition of the nicotinic acetylcholine receptor. The very slow washout of the drug in terms of EPC amplitude and virtually no recovery of the shortened EPC time course might substantiate certain caution to avoid unintentional high-dose application during its antibacterial application.     

The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptor-ionic channel complex. II. Patch clamp studies

Patch clamping of myoballs to record single channels was performed to examine the interaction of the anticholinesterase agent pyridostigmine (Pyr) with the acetylcholine (ACh) receptor-ion channel complex. Single ACh channel currents were recorded from tissue-cultured muscle cells of neonatal rats (myoballs). Pyr (50-100 microM) decreased the frequency of channel-opening events activated by ACh, and induced a modified form of the ACh channel currents. Channel conductance was lower in the presence of Pyr, and channel lifetime remained unaltered or only slightly prolonged. In addition, channel openings were frequently interrupted by fast flickers in the presence of Pyr. Higher concentrations (200 microM-1 mM) of the drug induced irregular waves of bursting activity during the initial phase of the application, and, subsequently, significantly reduced the frequency of channel openings. Infrequent channel openings with low conductance were observed in the patch when the micropipette was filled with Pyr alone. These results suggest that, in addition to its anticholinesterase activity, Pyr reacts with the ACh receptor, and both alone or in combination with ACh induces an altered, desensitized species of the nicotinic receptor-ion channel complex.     

Interactions of gephyrotoxin with the acetylcholine receptor-ionic channel complex. II. Enhancement of desensitization

The actions of the tricyclic alkaloid gephyrotoxin ( GyTX ) on the extrajunctional and junctional acetylcholine (ACh) sensitivity and desensitization were studied in the chronically denervated rat soleus muscle and cutaneous pectoris muscle of the frog. At low concentrations, GyTX greatly depressed the extrajunctional ACh sensitivity of the chronically denervated soleus muscles. In addition, GyTX produced a strong inhibition of junctional end-plate potentials evoked by ACh. Junctional and extrajunctional desensitizations induced by microiontophoretically applied ACh were greatly enhanced by the alkaloid in a frequency-dependent manner. These effects were readily reversible. The interaction of GyTX with binding sites on the acetylcholine receptor-channel (AChR) complex was studied on electroplax membranes from Torpedo californica. GyTX binds to the AChR complex at a site distinct from the ACh binding site, as revealed by its lack of inhibition of [125I]alpha-bungarotoxin ( [125I]BGT) binding. On the other hand, GyTX at a concentration range between 1 microM and 100 microM significantly increased the potency of the agonist carbamylcholine as an antagonist of binding of [125I]BGT. At low micromolar concentrations, GyTX inhibited the binding of [3H]perhydrohistrionicotoxin and [3H] phencyclidine to sites associated with the ionic channel of the AChR complex. The affinity of GyTX for these sites was increased 3- to 5-fold by carbamylcholine. Results of electrophysiological and binding studies indicate that GyTX not only blocks the open channel of the AChR but also enhances desensitization of the AChR complex by increasing receptor affinity for agonists.     

Comparison of the actions of carbamate anticholinesterases on the nicotinic acetylcholine receptor

Neostigmine (Neo), pyridostigmine (Pyr), and physostigmine (Phy) at low concentrations inhibited acetylcholine (ACh) esterase, thereby indirectly potentiating ACh enhancement of [3H]perhydrohistrionicotoxin (H12-HTX) binding to the channel sites of the nicotinic ACh receptor of Torpedo membranes. However, at higher concentrations, they inhibited ACh action due to their direct binding to the ACh receptor. They displaced binding of [3H]ACh and 125I-alpha-bungarotoxin (alpha-BGT) to the receptor sites with the following order of decreasing potency: Neo greater than Phy greater than Pyr. Furthermore, Neo and Pyr potentiated [3H] H12-HTX binding to the receptor's channel sites. Preincubation of ACh receptors with any of the three carbamates reduced the rate of binding of 125I-alpha-BGT and increased the potency of carbamylcholine in inhibiting 125I-alpha-BGT binding, suggesting that the three carbamates act as partial agonists and potentiate receptor desensitization. Although none of the three carbamates inhibited [3H]H12-HTX binding to the receptor's closed channel conformation, only Phy was a potent inhibitor of [3H]H12-HTX binding to the carbamylcholine-activated conformation. The potency of Phy was not due to the absence of positive charge since Phy methiodide acted similarly. The data suggest that the major action of the three carbamates at nicotinic cholinergic synapses is inhibition of ACh-esterase. Their interactions with the nicotinic ACh receptor are with its "receptor" as well as allosteric "channel" sites, but they differ in their effects. Neo and Pyr act mainly as partial agonists, while Phy is mostly an inhibitor of the channel in the activated receptor conformation.     

Requirement of a colchicine-sensitive component of the cytoskeleton for acetylcholine receptor recovery.

1. The effect of colchicine treatment on acetylcholine receptor function was examined in potassium depolarized, voltage-clamped snake twitch fibre endplates. Receptor function was assessed by analysis of miniature endplate currents (m.e.p.c.) as well as acetylcholine (ACh)-induced single channel currents. 2. Pretreatment of snake muscle fibres with colchicine (10 microM to 100 microM) for 16-18 h had no effect on m.e.p.c. amplitude or decay rates. At higher concentrations (1 mM), there was a slight decrease in the average m.e.p.c. amplitude. 3. Colchicine produced a concentration-dependent decrease in the extent of m.e.p.c. amplitude recovery following a 10 min exposure to 540 microM carbachol. Exposure of 100 microM colchicine-treated preparations to 0.5 microM staurosporine further reduced the extent of m.e.p.c. amplitude recovery following carbachol exposure. 4. The decrease in m.e.p.c. amplitude following carbachol exposure was not due to a shift in the m.e.p.c. reversal potential. In addition, the distribution of m.e.p.c. amplitudes remained unimodal in both control and colchicine (100 microM)-treated preparations following carbachol exposure. 5. In addition to the normal, large conductance (approximately 48 pS) ACh-activated channels, a population of small conductance (approximately 29 pS) channels was observed in colchicine-treated preparations following exposure to carbachol. In preparations treated with both colchicine and staurosporine and then exposed to carbachol, the conductance of these small channels was identical to that of colchicine or staurosporine alone. 6. We suggest that prolonged exposure of snake twitch fibre endplates to agonist results in the activation and desensitization of ACh receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further studies on the site of action of nicotinic blocking drugs

The effects of combinations of tubocarine and physostigmine, and of physostigmine and edrophonium alone, have been studied on the responses of the chick biventer cervicis muscle to nerve stimulation and to exogenous acetylcholine. The effects of hexamethonium, tetraethylammonium and triethylcholine have also been studied on the dose-response curves for acetylcholine, determined in the presence of low concentrations of physostigmine.Suitable combinations of tubocurarine and physostigmine reduced responses of the muscle to nerve stimulation, whilst leaving the responses to acetylcholine unaffected, whereas physostigmine and edrophonium augmented responses to acetylcholine, whilst leaving responses to nerve stimulation unaffected. These results indicate that the multiply innervated fibres of the muscle are particularly sensitive to inhibition of cholinesterase.Hexamethonium, tetraethylammonium and triethylcholine, drugs that are known to exhibit actions similar to that of the combination of tubocurarine and physostigmine, and also to possess anticholinesterase activity, depressed responses to acetylcholine determined in the presence of concentrations of physostigmine sufficiently low to possess a specific inhibitory action on cholinesterase. The results indicate that the neuromuscular block produced by large concentrations of these drugs is probably post-junctional in origin.     

A further study on the kinetics of the subcellular current events at the mouse end-plate in the presence of cimetidine and ranitidine

The cholinomimetic activity of Cimetidine and Ranitidine has been demonstrated by several authors. In the aim to better understand the phenomenon, we analyse the miniature end-plate current decay time. The prolongation of the decay phase of the synaptic current induced by the "selective" H2-antagonist Ranitidine, and to a lesser extent and at higher concentrations by Cimetidine, resembles that of the cholinesterase inhibitors. These agents usually prolong the quantal conductance change having little or no effect on the channel lifetime. The results of our previous experiments, which data were obtained by analyzing the "voltage" events, either spontaneous or evoked, of a classic frog preparation, showed a marked alteration of the temporal parameters. These effects, obtained at higher drug concentrations than those used in the present work, are now better defined by deriving extracellularly the "current" events. The results are also compared with those obtained by assaying the cholinesterase inhibitor Eserine, under the same experimental conditions.     

Physostigmine, tacrine and metrifonate: the effect of multiple doses on acetylcholine metabolism in rat brain

The effects of two consecutive intramuscular doses of three cholinesterase inhibitors (physostigmine, tetrahydroaminoacridine and metrifonate) were compared in rats. The results revealed major differences in biochemical effects on the brain of the rat including the extent and duration of inhibition of cholinesterase, inhibition of release of acetylcholine and increase in levels of acetylcholine. Side effects were also markedly different in the time of appearance, duration and severity. These results suggest that there are significant differences in the mechanisms of action of various cholinesterase inhibitors. Since all three cholinesterase inhibitors are currently used in the experimental treatment of Alzheimer's disease, these findings have potential implications for the symptomatic therapy of these patients.     

Halothane and isoflurane alter acetylcholine activated ion channel kinetics

The effects of halothane and isoflurane on the acetylcholine activated ion channel were studied in the frog sartorius muscle using the two electrode voltage clamp technique. The miniature end-plate currents (MEPCs) were recorded and evaluated for amplitude, duration of growth phase and time constant of decay (tau). Both halothane and isoflurane decreased tau in a dose dependent manner. Depression of current amplitude was also dose dependent. The ED50 value indicates tau is more affected than the amplitude of MEPC. Biexponential decay phases were seen in a small fraction of cells exposed to a low concentration of halothane but not isoflurane. Biexponential decay, when seen, was more prominent at less negative clamped membrane potential. The normal linear relationship between membrane potential and tau was not altered by anesthetics.