Calcium channel effectors are potent non-competitive blockers of acetylcholine receptors.

Nicardipine and other calcium channel effectors (CCEs) were studied for their effects on nicotinic acetylcholine receptor (nAChR) activity. While CCEs had no effect on frog (Rana pipiens) skeletal muscle contractions resulting from nerve stimulation or direct stimulation of the muscle, nicotinic agonist-induced contractures were blocked. Nicardipine did not affect nAChR single-channel open time or amplitude, corroborating data from endplate currents (EPCs); EPC amplitudes and decays were unaffected. All the CCEs tested, however, non-competitively blocked nAChRs. The block of nAChRs resulted in a shortened agonist-induced depolarization and thus a diminished contracture response. An increase in cultured mouse skeletal muscle (C-2) cell single-channel closed times was observed with the intracellular addition of nicardipine, verifying a direct block of nAChRs. Using single-channel analysis, nicardipine's site of action, or at least access to its site of action, was determined to be at the intracellular side of the receptor. A direct action of the CCEs on the nAChR was also shown by their ability to block phencyclidine (PCP) binding to Torpedo nobiliana membranes. All the CCEs blocked specific binding of [3H]-PCP to its binding site on the nAChR-channel complex, with bepridil and nicardipine being the most potent. These data are compatible with a model in which nicardipine and other CCEs, at concentrations which do not alter nAChR channel open time or conductance, block the effects of superfused nicotinic agonist on nAChRs either by stabilizing the formation of the nAChR desensitized state or by effecting a slow channel block.

Echothiophate and cogeners decrease the voltage dependence of end-plate current decay in frog skeletal muscle.

The effects of three irreversible anticholinesterase agents, echothiophate (217MI), tertiary methylamine analog of 217MI (217AO) and Tetram, on end plate currents (e.p.c.s) of Rana pipiens cutaneous pectoris muscle were studied using electrophysiological techniques. All three compounds (217MI, 1-10 microM; 217AO, 1-25 microM; and Tetram, 1-50 microM) decreased the rate of e.p.c. decay (alpha) to the same extent as neostigmine (10 microM), a reversible anticholinesterase agent. Decay remained a single exponential at all membrane potentials. 217MI and its derivatives greatly reduced the normal voltage dependence of alpha represented by the slope (H = mV-1) of log alpha vs. membrane potential, in contrast to neostigmine which had no effect on H. Suppression of Ach release by the addition of 4 mM Mg++ to end-plates did not alter the reduction of H by 217AO indicating that the anticholinesterase-induced decrease in H is not simply due to an increased interaction between Ach and its receptors. Additionally, the pretreatment of end-plates with methanesulfonyl fluoride, also an irreversible cholinesterase agent, did not modify the effects of 217AO and Tetram on H. 217MI and its derivatives, at low concentrations which altered H, did not affect [3H]PCP or [125I]alpha-bungarotoxin binding to Torpedo californica Ach receptor-rich membranes. It is concluded that these agents alter H by an effect on the Ach receptor ion channel complex unrelated to either esterase inhibition or channel block.

Augmentation of succinylcholine-induced neuromuscular blockade by calcium channel antagonists.

The block of endplate currents (EPCs) by succinylcholine, in frog cutaneous pectoris muscles, is significantly potentiated by certain organic calcium channel antagonists, at concentrations at which the calcium channel antagonists have no effect on EPCs themselves. Succinylcholine alone blocked the current in a dose-dependent manner with an IC50 = 2.7 microM in voltage-clamped muscle fibers. The ability of succinylcholine to block endplate currents was potentiated by nicardipine (3.5- and 31-fold by 1 and 10 microM, respectively), by bepridil (4.3-fold by 1 microM), and by verapamil (7.7-fold by 10 microM). The dihydropyridine nifedipine (10 microM), did not potentiate the succinylcholine effect. Since the calcium channel antagonists do not affect endplate currents directly at these concentrations, a channel blocking or receptor antagonistic effect of these drugs is not indicated. We therefore suggest that the enhancement of block by succinylcholine may be due to the increased desensitization of the receptor. Furthermore, this enhancement of desensitization may explain the effects of these calcium channel antagonists seen in whole animal studies, where the contractures caused by acetylcholine and succinylcholine, applied systemically, are blocked.

Voltage clamp analysis of the effect of cationic substitution on the conductance of end-plate channels.

The effect of two commonly used sodium substitutes, tris and glucosamine, on the amplitude and kinetics of miniature end-plate currents (MEPCs), acetylcholine (ACh) induced end-plate currents (EPC) and EPC fluctuations was studied in voltage clamped single muscle fibres from a monolayer preparation of the cutaneous pectoris muscle. Total replacement of sodium with each substitute shifted the reversal potential from -4.7 mV (normal sodium solution) to -3.6 mV (tris) and -49.0 mV (glucosamine). In tris and glucosamine substituted solutions the current (MEPC or EPC) - voltage relation became markedly nonlinear, with peak current decreasing with membrane hyperpolarization. Peak current at +40 mV, was unaltered in tris solutions and reduced in glucosamine substituted solutions. MEPCs decayed with a single exponential time course and the EPC fluctuation spectra were characterized by single Lorentzian functions in both normal sodium solution and each substituted solution. Analysis of EPC fluctuations demonstrated that both tris and glucosamine decrease single channel conductance and increase channel lifetime. Both effects were enhanced by either membrane hyperpolarization or by increasing the concentration of each substitute. In the presence of each cationic substitute, single channel conductance increased and mean channel lifetime decreased with membrane depolarization. Analysis of the data according to the constant field assumptions (Goldman, Hodgkin, Katz equation) provided an inadequate description of experimental currents obtained at hyperpolarized membrane potentials with total ion substitution. Shifts in reversal potential with partial substitution were, however, adequately predicted by the GHK equation. These results suggest that tris and glucosamine ions interact with end-plate channels to reduce cation permeability and decrease channel closing rates. The dependence of this block on the level of membrane potential suggests that these cations bind to site(s) within open end-plate channels.

Ketamine and ditran block end-plate ion conductance and [3H]phencyclidine binding to electric organ membrane.

Alterations by ketamine (10-100 microM) and ditran (50-100 microM) of end-plate currents were studied using transected cutaneous pectoris muscles. Both drugs reduced peak current and shortened the time constant for end-plate current decay (tau). Ketamine was more effective at pH 5.3 than at 7.4 or 9.1. Recovery from blockade was asymmetrical in that tau recovered more quickly than did peak current when the drugs were removed from the bath. By contrast, 4-aminopyridine antagonized the depression of peak current by ketamine, but not the reduction of tau. Both ketamine and ditran disrupted the voltage dependence of tau. The binding to microsacs prepared from electric organs of [3H]phencyclidine ([3H]PCP) was blocked by ketamine and ditran. In microsacs treated with carbachol, the IC50 for ketamine block of [3H]PCP binding was 6.6 X 10(-6) M. For ditran, the IC50 for block of [3H]PCP binding in the presence of carbachol was 1.7 X 10(-6) M. The binding of [alpha-125I]bungarotoxin to the microsacs or to the cultured chick myotubes was reduced only slightly by ketamine. Because ketamine has no effect on transmitter release and little effect on [alpha-125I]bungarotoxin binding, it is concluded that, like PCP, ketamine and ditran block open channels in the end-plate. In addition, the asymmetrical recovery of end-plate current parameters suggests that ketamine may block closed channels. The recovery from block of closed channels (caused by either a direct action on closed channels or a very slow channel unblocking rate) proceeds more slowly than does the block of open channels.

Cyclic guanosine 3':5'-monophosphate accumulation and 45Ca-uptake by rat superior cervical ganglia during preganglionic stimulation.

Repetitive preganglionic nerve stimulation increases cyclic guanosine 3':5'-monophosphate (cGMP) content in rat superior cervical ganglia by a mechanism requiring Ca++ but resistant to blockade by cholinergic receptor antagonists. Similarly, 45Ca-uptake during prolonged preganglionic nerve stimulation is unaffected by hexamethonium or atropine. These findings indicate that nerve stimulation increases cGMP accumulation and 45Ca-uptake by a noncholinergic mechanism Substance P, met-enkephalin and luteinizing hormone-releasing factor have little or no effect on cGMP content. By contrast, bethanechol causes a 3-fold increase in cGMP content and postganglionic cell firing. Thus, as reported by others, muscarinic receptor activation increases ganglionic cGMP[. 4-Aminopyridine causes an increase in cGMP of resting ganglia that requires Ca++ and the nerve terminal is blocked by tetrodotoxin but unaffected by atropine or hexamethonium. Ouabain also increases ganglionic cGMP content by a process that requires Ca++ and the nerve terminals. Like preganglionic nerve stimulation, 4-aminopyridine and ouabain cause cGMP accumulation in the nerve terminals or in the ganglion cells as a consequence of releasing a noncholinergic transmitter. The uptake of Ca++ by ganglion cells is not an adequate stimulus for cGMP accumulation because the nicotinic receptor agonist dimethylphenylpiperazinium increases 45Ca-uptake but has no effect on cGMP formation in ganglia.

Postsynaptic inhibition of neuromuscular transmission by trifluoperazine.

The effect of trifluoperazine (TFP) on neuromuscular transmission was investigated on chick biventer cervicis and frog cutaneous pectoris and sartorius nerve-muscles. In the chick, TFP inhibited indirectly elicited twitches in a frequency-dependent manner. Inhibition was much more rapid at higher frequencies of stimulation. Directly elicited twitches, KCl contracture and action potentials of desheathed frog sciatic nerve and sartorius muscles were unaffected by TFP, suggesting an action of TFP on neuromuscular transmission. TFP depressed end plate potential amplitude and miniature end plate potential (MEPP) amplitude without affecting MEPP frequency. When MEPP frequency was increased by high Na+ Ringer, depression of MEPP amplitude was much more rapid. Similarly, at high frequencies of stimulation (100 Hz), TFP rapidly depressed end plate currents. TFP inhibited contractures induced by bath-applied acetylcholine (ACh); depressed ACh potentials produced by iontophoretically applied ACh; decreased ionic current and time constant of decay of end-plate currents of transected muscle; and inhibited [alpha-125I]bungarotoxin binding to ACh receptor. These data suggest that TFP acts postsynaptically in a frequency-dependent manner to inhibit neuromuscular transmission. Based on recent evidence that TFP is a potent antagonist of calmodulin and that calmodulin is localized mainly to postsynaptic regions, we postulate that the postsynaptic inhibitory actions of TFP may be mediated through antagonism of calmodulin, which in turn may regulate ACh receptor function.

Characterization of end-plate conductance in transected frog muscle: modification by drugs.

Cutaneous pectoris muscles of Rana pipiens were transected distal to the innervated region. Within 10 min, membrane potentials (Em's) of -33 +/- 2.5 mV and end-plate potentials (3-15 mV) were recorded unaccompanied by muscle action potentials or twitch. The fall in Em was associated with a net loss of [K+]i and a net gain of [Na+]i. Although input resistance fell by 50% and the space constant was slightly reduced in the transected muscle fibers, end-plates could be adequately voltage-clamped with two microelectrodes. End-plate currents (e.p.c.s) with rise times of 350 to 700 musec were recorded as a function of holding potential (Vm). The current-voltage relationship of peak e.p.c.s over the range of -70 to +20 mV was linear and the reversal potential (-6.6 +/- 2.2 mV) was the same as that found for intact muscle fibers. The decay phase of e.p.c.s could be described as a single exponential at all Vm's and had a voltage and temperature dependence similar to that described for e.p.c.s of glycerol-treated muscles. Tubocurarine (0.3 microM) caused a significant decrease in the time constant (tau) of e.p.c. decay and e.p.c. amplitude. The depression of e.p.c. amplitude by tubocurarine was reversed by 4-aminopyridine while the decrease of tau was not. Atropine (10(-4) M) caused a monotonic shortening of e.p.c.s at a Vm of -90 mV but e.p.c.s recorded at +50 mV were biphasic. Lidocaine, a quaternary nitrogen analog of lidocaine (QX314), lobeline and hexafluorenium were studied also in transected muscle and their effects on the parameters of e.p.c. are described. Both lobeline (50 microM) and hexafluorenium caused a decrease of tau and eliminated the voltage dependence of tau at negative Vm's. The transected muscle can be used for the study of conductance kinetics of end-plate and for the study of drug action uncomplicated by the presence of other drugs of Mg++ to eliminate contraction.

An attempt to distinguish between the actions of neuromuscular blocking drugs on the acetylcholine receptor and on its associated ionic channel.

The effects of lobeline and tubocurarine on the voltage-clamped endplates of frog sartorius and cutaneous pectoris muscles were examined at room temperature (20-23 degrees C). Like tubocurarine, lobeline causes nondepolarizing neuromuscular blockade. The half-time of decay (t((1/2))) of endplate currents (e.p.c.s) recorded at a holding potential (V(m)) of -90 mV was significantly shorter in endplates treated with lobeline (50 muM; mean t((1/2)) +/- SEM = 0.41 +/- 0.02 ms) or tubocurarine (11.4 muM; t((1/2)) = 0.64 +/- 0.04 ms) than in those treated with Mg(2+) (13 mM; t((1/2)) = 1.39 +/- 0.11 ms) or a low concentration of tubocurarine (3 muM; t((1/2)) = 0.87 +/- 0.05 ms). Similarly, lobeline (10 muM) shortened the t((1/2)) of untreated miniature e.p.c.s by 35%; tubocurarine, however, abolished miniature e.p.c.s at the concentration required to observe its actions on e.p.c. decay kinetics. The t((1/2)) of e.p.c.s recorded from preparations treated with Mg(2+) (13 mM), tubocurarine at low concentrations (3 muM), or untreated miniature e.p.c.s was logarithmically related to V(m), being slower at more hyperpolarized values. By contrast, the t((1/2))s of e.p.c.s recorded in either lobeline (50 muM) or tubocurarine (11.4 muM) were independent of voltage in the range -150 to -80 mV. The ability of lobeline to shorten t((1/2)) and to remove the voltage dependence of t((1/2)) was partially antagonized by Mg(2+) (13 mM). As expected, when lobeline or tubocurarine was removed from the bath or when acetylcholine release from the motor nerve terminals was increased by 4-aminopyridine (20 muM) and Ca(2+) (10 mM) (in the presence of lobeline or tubocurarine), the amplitude of e.p.c.s increased as a function of time. However, the t((1/2)) of the decay phase of the e.p.c.s remained shortened (i.e., unaltered from the earlier treatment). These results suggest that both tubocurarine and lobeline have at least two distinct postjunctional actions including: (i) a block of the acetylcholine receptor and (ii) a block of the ionic channel associated with the acetylcholine receptor.

Effects of McN-A-343, a cholinomimetic drug, on endplate currents in the frog.

The muscarinic ganglion stimulating agent, McN-A-343 has unusual blocking actions on endplate currents (EPCs) at frog neuromuscular junctions. McN-A-343 caused depolarization by a curare-sensitive process, blocked neuromuscular transmission, depressed EPCs and reduced the time for EPC decay. These results are explained best by a nicotinic agonist action of McN-A-343 on the acetylcholine receptor to cause ion flow and the blockade by McN-A-343 of the open ion channels. The actions of McN-A-343 are similar to those of decamethonium (C-10) described by others. Unlike C-10, however, McN-A-343 did not alter the exponential character of the EPC or alter the voltage dependency of the EPC. The prototypical nicotinic agonist, dimethylphenylpiperazinium had no effect on EPC parameters of endplate clamped at -90mV.

Pre- and postjunctional neuromuscular blockade by carbachol.

Carbachol, when applied to the bathing Ringer solution of frog sartorius muscles, caused depolarization of the endplate and a blockade of endplate potentials (EPP's), miniature EPP's (mepp's) and the iontophoretic acetylcholine potential. In muscles treated with an analog of hemicholinium-3, alpha, alpha'bis(dimethylammonium acetaldehyde diethylacetal)-p-p'-diacetylbiphenyl dibromide (DMAE), depolarization of the endplate by carbachol was blocked and the blockade by carbachol of the iontophoretic acetylcholine potential was prevented. These responses to carbachol were attributed to a postjunctional action that was antagonized by DMAE. In contrast, the blockade by carbachol of EPP's and mepp's was enhanced in DMAE-treated muscles at a time when carbachol-induced depolarization was blocked. This response to carbachol was attributed to a pre-junctional action. Carbachol either blocked transmitter release by a mechanism that was insensitive to DMAE or enhanced the prejunctional blocking actions of DMAE. Succinylcholine had actions similar to carbachol. DMAE prevented depolarization by succinylcholine but enhanced neuromuscular blockade by succinylcholine. SKF 525-A (beta-diethylaminoethyl diphenylpropylacetate hydrochloride), like DMAE, prevented depolarization but not transmission blockade caused by carbachol.