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.     

Effects of sodium selenite on neuromuscular junction of the mouse phrenic nerve-diaphragm preparation

The effects of sodium selenite on the neuromuscular junction of the phrenic nerve-diaphragm of the mouse were studied. Nerve-evoked twitches of the diaphragm of the mouse, the frequency of miniature endplate potentials, the quantal content of endplate potentials and the compound action potentials of the axon were measured. Sodium selenite induced a slight increase of the amplitude of the twitch, followed by twitch depression. The amplitude of the twitch, increased by selenite, became more prominent after the suppression of the twitch induced by cadmium ions, d-tubocurarine or magnesium ions. It appeared that the increased amplitude of twitch was due to the facilitation of transmitter release, since selenite significantly increased the frequency of miniature endplate potentials, and the amplitude and quantal content of endplate potentials; the amplitude and half decay time of miniature endplate potentials were unaffected. Twitch depression induced by selenite was enhanced by ammonium ions, high potassium and low magnesium and attenuated by high calcium. During the period of gradual depression of the twitch, selenite decreased the amplitude of compound action potentials of the phrenic nerve axon and caused the disappearance of endplate potentials. Ammonium ions enhanced the blockade of axonal conduction induced by selenite. Moreover, the depolarizing agents, ammonium and high potassium also induced an initial increase of twitch amplitude followed by depression of the twitch. These findings indicate that selenite probably alters the release of the transmitter by depolarizing the nerve membrane. The effects of selenite were antagonized by glutathione and cyanide, suggesting that the binding of selenite to sulfhydryl groups of the membrane was essential for inducing its pharmacological actions.     

The in vivo effect of lipopolysaccharide on the spontaneous release of transmitter from motor nerve terminals.

1. The in vivo effect of E. coli lipopolysaccharide (LPS) on the spontaneous release of transmitter was studied in the isolated phrenic nerve-diaphragm preparation of the mouse. 2. The resting membrane potential was decreased and frequency of miniature endplate potentials (m.e.p.ps) was increased by treatment with LPS. 3. Pretreatment of diaphragms with ouabain markedly increased the frequency of m.e.p.ps in control group but not in the LPS group. 4. When mice were treated with polymyxin B (a LPS neutralizer), pentoxifylline (an inhibitor of tumor necrosis factor-alpha formation) and NG-nitro-L-arginine (an inhibitor of nitric oxide (NO) synthase) the effects of LPS were reversed. 5. These results suggest that LPS increases the spontaneous transmitter release through, at least in part, the pathways of tumour necrosis factor-alpha and NO followed by an inhibition of the Na(+)-pump activity in the endplate area.     

The inhibitory effect of cadmium on neuromuscular transmission in the rat.

Cadmium (0.125-1 mM) was found to inhibit the isometric response of the isolated rat hemidiaphragm during indirect stimulation, but not during direct stimulation. This effect of cadmium (1 mM) was completely reversed by ethyleneglycol bis-(aminoethyl)-N,N,N',N'-tetra-acetic acid (2 mM) or by L-cysteine (2 mM) but only partially by increased calcium. Cadmium (10 micronM) significantly reduced the quantal release of transmitter in the isolated phrenic diaphragm and a concentration of 0.1 mM frequently caused a complete failure of the endplate response after 30 min. The effect of cadmium on neuromuscular transmission could not be readily reversed by washing with cadmium-free solution. Miniature endplate potential frequency and amplitude were not significantly affected by cadmium (0.1 or 0.5 mM). The results suggest that the effect of cadmium on the isolated phrenic nerve-diaphragm is due largely to inhibition of calcium function at presynaptic nerve terminals.     

Ca antagonistic and non-specific effects of diltiazem on the rat phrenic nerve diaphragm preparation

The calcium antagonist diltiazem (2.8 X 10(-4) M) blocked the twitches of a rat phrenic nerve diaphragm preparation after a period of twitch potentiation. Its ability to block twitches was greater during indirect than direct stimulation. Experiments on the isolated phrenic nerve indicated that the excitability of the nerve was blocked. Diltiazem (2.3-9.0 X 10(-5) M) caused a similar inhibition of indirectly and directly elicited tetanic contractions and EMG. Experiments with d-tubocurarine and lowered temperature disclosed a separate inhibition at the neuromuscular junction. High Ca2+ did not reverse the diltiazem-affected twitch or tetanic contractions, which suggests that they are non-specific effects. KCl (100 mM)-induced contractures were antagonized at low (2.3-4.5 X 10(-5) M) but not at high (1 mM) concentrations of diltiazem. Diltiazem depressed the initial phase of the two-phasic caffeine (10 mM) contracture and increased and accelerated the slow phase. Diltiazem greatly reduced the amplitude and duration of the caffeine-potentiated KCl contracture, and reduced and delayed the slow phase of the KCl-potentiated caffeine contracture. The effects on the combined contractures (caffeine-induced, KCl-potentiated) were partly antagonized by a high Ca2+ (2.2 X 10(-5) M) solution, which suggests that diltiazem has calcium antagonistic effects.     

An anti-curare effect of hexamethonium at the mammalian neuromuscular junction

1. Experiments were performed on the isolated phrenic nerve and diaphragm preparation of the rat.2. In preparations partly blocked with (+)-tubocurarine, the twitch amplitude increased after hexamethonium. This enhancement was not seen in preparations partly blocked with Mg(++) or with gallamine. High concentrations of hexamethonium produced failure of contraction.3. Extracellular endplate potentials were recorded from blocked preparations. The administration of hexamethonium resulted in an increased amplitude of these potentials only in curarized muscle.4. Hexamethonium had no anticholinesterase activity nor did it depolarize muscle cells or increase the quantal release of transmitter.5. It is concluded that hexamethonium exerts a specific anti-curare action. Experiments on the recovery of the twitch after washing out antagonists indicate that this process is limited by diffusion. The difference in rates of diffusion of hexamethonium and (+)-tubocurarine does not account for their interaction. The basis of the anti-curare action of hexamethonium is discussed.     

The effect of 3,3-dipyridylmethyl-1-phenyl-2-indolinone on the neuromuscular transmission in the rodent skeletal muscles.

The effects of a cognition enhancer, 3,3-dipyridylmethyl-1-phenyl-2-indolinone (DPMPI) (21.5-645 microM), on neuromuscular transmission were studied electrophysiologically on diaphragms of mouse and rat and the soleus muscle of rat. The drug DPMPI (21.5-645 microM) increased both direct and indirect twitch tension of mouse diaphragm. It also increased (a) the frequency of miniature endplate potentials and (b) the quantal content of endplate potential. However, DPMPI (64.5 microM) affected neither the amplitude of the directly elicited action potential of soleus muscle in the rat nor the magnitude of the resting membrane potential of mouse diaphragm, although DPMPI (215 microM) decreased the amplitude of the compound action potential of phrenic nerve. Based on these results, it is concluded that DPMPI had several effects on neuromuscular transmission, i.e. it (a) facilitated the transmitter releasing process of the motor nerve terminal, (b) decreased the conduction in the phrenic nerve and (c) increased the directly elicited twitch tension.     

Mechanisms of the inhibition by neostigmine of tetanic contraction in the mouse diaphragm.

Neostigmine (0.5-2 microM) caused fade of tetanic contractions (Wedensky inhibition) evoked by repetitive nerve stimulation. The mechanism underlying this action was studied in intact and cut isolated phrenic nerve-diaphragm preparations of mice. The fade was brought about by failure to elicit muscle action potentials. During fade, the muscle was unable to conduct directly evoked action potentials across the central endplate zone. Recovery of excitability occurred in 5 s with continued stimulation. In the presence of neostigmine, the resting membrane potential at endplate areas during repetitive stimulation decreased from -80 mV to less than -50 mV within the first 10 pulses at 75-200 Hz and thereafter recovered gradually to about -60 mV in the following 5 s during continuous stimulation. The quantal content of endplate potentials evoked by single stimulation was not reduced by neostigmine whereas that evoked by high frequency stimuli (75 Hz) was reduced to about 1/3 in 10 pulses. It is concluded that the fade of tetanic contraction caused by inhibition of acetylcholinesterase is induced by the inactivation of sodium channels in the area surrounding the endplates and that the sustained fade is due to a decrease of transmitter release. Both effects are the result of acetylcholine accumulation.     

Transmitter releasing action of selegiline ((-)-deprenyl) from peripheral sympathetic nerves under different experimental conditions

A high concentration of selegiline ((-)-deprenyl; 10(-4) M) potentiated low frequency (2 Hz) nerve stimulation-evoked release of [3H]noradrenaline from the isolated main pulmonary artery of the rabbit in the presence of neuronal (cocaine, 3 X 10(-5) M) and extraneuronal (corticosterone, 5 X 10(-5) M) uptake blockers, and inhibited the postsynaptic response. The transmitter-releasing action of 10(-4) M selegiline was inhibited by a moderate increase of external K+ (23.6 mM). Excess K+ by itself abolished the nerve-evoked release of [3H]noradrenaline but did not increase the resting outflow of radioactivity. Excess Ca2+ (7.5 mM) increased the stimulation-evoked transmitter release. In the presence of excess Ca2+, selegiline (10(-4) M) was effective in increasing the [3H]noradrenaline release in response to nerve-stimulation. Excess Ca2+ partly antagonized the postsynaptic inhibitory action of selegiline. In Ca2+-free, 1 mM EGTA-containing Krebs solution both the nerve-evoked 3H release and the transmitter releasing action of selegiline were abolished in agreement with the 'Ca-hypothesis'. The voltage-dependent K+-channel blocker, 4-aminopyridine (10(-5) M), increased the nerve-stimulation-evoked release of tritium from arteries. If selegiline was also present in the perfusion medium the nerve-evoked transmitter release further increased. 4-Aminopyridine completely antagonized the inhibitory action of selegiline on the postsynaptic contraction.     

Effects of butanedione monoxime on neuromuscular transmission.

1. The amplitude of endplate potentials was increased by concentrations of butanedione monoxime (BDM, 5-20 mM) that typically caused muscle paralysis. 2. Although BDM slowed the decay of spontaneous miniature endplate currents, the effect was insufficient to explain most of the large increase in amplitude of endplate potentials. 3. The quantal content of endplate potentials was increased by BDM in a reversible, concentration-dependent manner. 4. The frequency of miniature endplate potentials was not changed by 10 mM BDM in the presence of normal or raised potassium concentrations, indicating that BDM does not change quantal content by a direct effect on calcium channels or on steady-state intracellular calcium concentration. 5. A change in the time course of the extracellularly recorded nerve terminal action potential caused by BDM was similar to the change produced by 4-aminopyridine (4-AP). 6. The increase in quantal content produced by BDM was only slightly reduced in the presence of 1 mM tetraethylammonium (TEA) but was significantly reduced in the presence of 0.5 to 1 mM 4-AP. 7. It was concluded that BDM blocks a 4-AP-sensitive potassium conductance in motor nerve terminals and, by increasing the duration of the action potential in this way, increases evoked transmitter release.     

On the blockade of acetylcholine release at mouse motor nerve terminals by beta-bungarotoxin and crotoxin

1. beta-Bungarotoxin and crotoxin are phospholipose A2 neurotoxins, which block irreversibly the evoked release of acetylcholine from motor nerve terminals of mouse triangularis sterni preparations. 2. Extracellular recording of nerve terminal action potentials reveal that inhibition of transmitter release is not associated with failure of the action potential to invade nerve terminals. 3. When evoked transmitter release (measured as intracellularly recorded endplate potentials) was blocked by beta-bungarotoxin, spontaneous acetylcholine release was stimulated as in control experiments by K(+)-induced depolarization and by the Ca2(+)-ionophore A23187. 4. The site of action of the toxins remains to be elucidated but would appear to be associated with the coupling of action potential induced-depolarization to the release mechanism, rather than with the release mechanism itself.     

Accumulation of extracellular calcium at the endplate of mouse diaphragm after ecothiopate in vitro.

1. Experiments were carried out to investigate the accumulation from the extracellular medium of 45Ca2+ by the endplate region of skeletal muscle. 2. Mouse diaphragm muscle was incubated in physiological saline labelled with 45Ca at 37 degrees C for periods of up to 1.5 h. 3. The muscle was divided into junctional and non-junctional portions and the Ca from the extracellular fluid accumulated at the endplate determined from the 45Ca content of the portions. 4. The accumulation of extracellular Ca at the endplate region of muscles incubated in pysiological saline alone was nil, but there was accumulation in the presence of the anticholinesterase ecothiopate iodide 0.5 x 10(-6) M (ECO). Stimulation of the phrenic nerve at 0.02 Hz caused no further increase in accumulation but reduced the amount of spontaneous fasciculation. In tetrodotoxin (TTX) 10(-6) M, the accumulation was halved, and in 3.5 mM Mg2+ the accumulation was nil. Carbachol 10(-4) M resulted in an accumulation of Ca similar to that in ECO. 5. It is concluded that there was an accumulation of extracellular Ca following excitation of the nerve by stimulation at a low frequency and during the spontaneous fasciculations, and about half of the accumulation of extracellular Ca after ECO in the experiments was due to the postsynaptic action of ACh released non-quantally from the nerve terminals.     

The action of thallium acetate on phasic transmitter release in the mouse neuromuscular junction

Endplate potentials (EPP's) and miniature endplate potentials (MEPP's) were recorded from neuromuscular junctions of the mouse phrenic nerve-diaphragm preparation, blocked by high Mg⁺⁺ (12×10^–3 mol/l)-Ringer. Superfusion of the preparations with Mg⁺⁺-Ringer solutions containing thallium acetate (5×10^–4 mol/l Tlac) decreased phasic transmitter release as judged by EPP amplitudes as well as average quantal content, until total synaptic blockade (within about 300 min) occurred. Simultaneously MEPP amplitudes remained unchanged, whereas the frequency of MEPP's increased. When EPP amplitudes and/or quantal content were reduced by 50% (usually within about 180 min), superfusion with Mg⁺⁺-Ringer solution without Tlac did not restore phasic transmitter release. However, the increase in spontaneous transmitter release was reversible, as MEPP frequencies returned to normal values. 4-Aminopyridine (5×10^–4 mol/l 4-AP) as added to the bath solution in the state of 50%-reduced phasic release temporarily restored EPP amplitudes and average quantal content, whereas MEPP amplitudes remained unchanged.It is concluded that thallium irreversibly blocks phasic transmitter release, whereas spontaneous transmitter release is reversibly enhanced.     

A selective inhibitor of cAMP-specific phosphodiesterase, Ro 20-1724, has no effect on the quantal release of acetylcholine from the mouse phrenic nerve

The indirect twitch response of the mouse isolated phrenic nerve-diaphragm preparation, partially paralysed by tubocurarine, was restored only by about 10% by Ro 20-1724 at 2 to 280 microM. The solvent vehicle, dimethylsulphoxide, also showed the same effect to a similar extent. Intracellular recordings with glass microelectrodes revealed that Ro 20-1724 (40 microM) affected neither the resting membrane potential, the amplitude and frequency of miniature endplate potentials nor the amplitude of nerve-impulse evoked endplate potentials recorded in curarized preparations. The result indicates that Ro 20-1724 at a concentration four times the IC50 of phosphodiesterase inhibition has no effect on the quantal release of acetylcholine from a mammalian motor nerve and suggests that cAMP has no modulatory effect on the transmitter release.     

Microelectrode recording of the effects of agonists and antagonists on alpha-adrenoceptors on rat somatic nerve terminals.

The effects of apomorphine, catechol, clonidine, isoprenaline, (-)-and (+/-)-noradrenaline, phenylephrine, pyrogallol and xylazine were investigated on the frequency and amplitude of miniature endplate potentials (m.e.p.ps) and, with the exception of apomorphine, catechol and pyrogallol, on the amplitude of endplate potentials (e.p.ps) in the rat phrenic nerve diaphragm preparation. Clonidine, (-)-noradrenaline, phenylephrine and xylazine (each at 1.5 X 10(-5)M) increased m.e.p.p. frequency but not amplitude. The other drugs were ineffective, except isoprenaline (1.5 X 10(-5)M) which enhanced m.e.p.p. amplitude but not frequency. The increase in m.e.p.p. frequency was inhibited by phentolamine, prazosin and yohimbine (each 1.5 X 10(-9)M). Prazosin and yohimbine alone each reduced m.e.p.p. frequency but failed to abolish m.e.p.ps even at high concentrations (10(-3)M). Clonidine, (-)-noradrenaline, phenylephrine and xylazine (each 3 X 10(-6)M) enhanced e.p.p. amplitude; this enhancement was blocked by prazosin and by yohimbine (each 3 X 10(-6)M). In preparations fatigued by prolonged continuous nerve stimulation (5 Hz, 0.05 ms for 30 min), (-)-noradrenaline (3.3 X 10(-4)M) restored m.e.p.p. frequency. The results indicate that adrenoceptors on somatic nerve terminals interact with both alpha 1- and alpha 2-agonists and antagonists and show different characteristics from those at autonomic neuroeffector junctions. The alpha-adrenoceptors on somatic nerve terminals may have an ancilliary physiological role in influencing but not controlling transmitter release.     

Counter Effects of Higenamine and Coryneine,Components of Aconite Root,on Acetylcholine Release from Motor Nerve Terminal in Mice

Abstract

The counter effects of higenamine and coryneine, components of aconite root, on acetylcholine (ACh) release from motor nerve terminals in the mouse phrenic nerve–diaphragm muscle preparation were studied by a radioisotope method. Both nerve-evoked release and spontaneous release of [³H]-ACh from the preparation preloaded with [³H]-choline were measured. The change in the tetanic tension of muscle was simultaneously recorded in the same preparation. Higenamine (10 μM) augmented both the nerve-evoked and spontaneous ACh releases, and the muscle tension. The effects were inhibited by pretreatment with propranolol (10 μM), a β-adrenoceptor antagonist. Coryneine reduced the nerve-evoked release of ACh, accelerated the decay of tetanic tension (tetanic fade) at 30 μM, and it depressed the peak amplitude of tetanic tension at a higher concentration of 100 μM. These results suggest that of the two components contained in aconite root, higenamine increases ACh release via activation of β-adrenoceptor, and conversely coryneine depresses ACh release by preferentially acting at motor nerve terminal.     

An analysis of low level doses of cholinesterase inhibitors in cultured neurons and hippocampal slices of rats

Recent data indicate that the neurotoxic effects of organophosphate compounds, including those of the nerve agents VX and sarin, are not solely due to irreversible cholinesterase inhibition. In this study we applied the patch clamp technique to hippocampal neurons in culture and slices to investigate the effects of VX, sarin and huperzine A on transmitter release and the mechanisms related with such effects. The nerve agents VX and sarin at very low concentrations significantly reduced the evoked release of GABA and glutamate. This effect was dependent of the activation of muscarinic receptors. In the presence or absence of the Na(+)-channel blocker tetrodotoxin (TTX), VX increased the frequency of spontaneous glutamate and GABA-induced postsynaptic currents. The effect of VX on TTX-insensitive spontaneous currents appears to be unrelated to cholinesterase inhibition, because it could be detected even after cholinesterase was blocked by high concentrations of the nerve agent soman. The ability of the nerve gases to decrease evoked release of GABA and increase spontaneous transmitter release may underlie some of the neurotoxic effects of the compounds. Huperzine A did not affect spontaneous or evoked release of GABA and glutamate, suggesting that this compound may be a pure cholinesterase inhibitor and had no effect on postsynaptic GABAA or AMPA receptors.     

Counter effects of higenamine and coryneine, components of aconite root, on acetylcholine release from motor nerve terminal in mice

The counter effects of higenamine and coryneine, components of aconite root, on acetylcholine (ACh) release from motor nerve terminals in the mouse phrenic nerve-diaphragm muscle preparation were studied by a radioisotope method. Both nerve-evoked release and spontaneous release of [³H]-ACh from the preparation preloaded with [³H]-choline were measured. The change in the tetanic tension of muscle was simultaneously recorded in the same preparation. Higenamine (10 μM) augmented both the nerve-evoked and spontaneous ACh releases, and the muscle tension. The effects were inhibited by pretreatment with propranolol (10 μM), a β-adrenoceptor antagonist. Coryneine reduced the nerve-evoked release of ACh, accelerated the decay of tetanic tension (tetanic fade) at 30 μM, and it depressed the peak amplitude of tetanic tension at a higher concentration of 100 μM. These results suggest that of the two components contained in aconite root, higenamine increases ACh release via activation of β-adrenoceptor, and conversely coryneine depresses ACh release by preferentially acting at motor nerve terminal.     

A pharmacological study of the effect of carbamazepine on neuromuscular transmission in the rat diaphragm

The effects of carbamazepine (0.042-0.42 mM) on neuromuscular transmission were studied on the isolated rat phrenic nerve diaphragm preparation using standard pharmacological and electrophysiological methods. Carbamazepine decreased (1) the antidromic activity of the phrenic nerve, (2) the amplitude of the endplate potential (EPP) and miniature endplate potential (MEPP), (3) the quantal content of the endplate potential, (4) the indirectly-elicited twitch tension, (5) the muscle contracture in chronically denervated muscle induced by acetylcholine (ACh) and (6) the amplitude of the compound phrenic nerve action potential, in a concentration-dependent manner. The antidromic activity of the phrenic nerve was the most affected, while the phrenic nerve compound action potential was least affected. However, the IC50 for carbamazepine (the concentration of carbamazepine that inhibited 50% of the response) was in the same order of concentration, i.e. 0.11-0.3 mM. Compared with the effect of carbamazepine on the indirectly-elicited twitch tension with its actions described above, it is concluded that carbamazepine interfered with the neuromuscular activity by inhibiting pre- and postsynaptic process and conduction in the phrenic nerve.     

Block of potassium channels and facilitation of acetylcholine release at the neuromuscular junction by the venom of the scorpion, Pandinus imperator

Venom from the scorpion Pandinus imperator potently and selectively blocks voltage-gated K+ channels in bullfrog neurones (Pappone, P. A. and Cahalan, M. D. 1987, J. Neurosci. 7, 3300-3305). Its effects on neuromuscular transmission have now been assessed. Twitch tension studies on chick biventer cervicis preparations showed that the venom (1 microgram/ml and above) significantly augmented responses to nerve but not muscle stimulation; there was little change in postjunctional sensitivity to cholinoceptor agonists or K+-induced depolarization. Electrophysiological studies on mouse triangularis sterni preparations revealed that the venom had no effect on spontaneous transmitter release, but increased evoked transmitter release. Extracellular recordings of nerve terminal action potentials showed that the venom selectively reduced the component of the waveform associated with K+ currents. These results confirm that this venom can selectively block neuronal K+ currents, and they show that this can facilitate the release of acetylcholine at the neuromuscular junction.