The effects of tetraphenylboron on spontaneous transmitter release at the frog neuromuscular junction.

1 The effects of tetraphenylboron (TPB) on spontaneous transmitter release were studied in the frog sartorius muscle preparation. 2 TPB (0.001-1 mM) produced a time-dependent increase in miniature endplate potential (m.e.p.p.) activity that was not sustained. TPB (0.1 mM) produced similar effects on m.e.p.p. frequency in normal Ringer solutions, in the absence of Ca2+ or Cl- and in the presence of excess Ca2+ and of tetrodotoxin. The effect of TPB (0.01 mM) was reduced but not abolished in the absence of Ca2+. 3 As m.e.p.p. frequency fell from its peak level in TPB (0.04 mM) m.e.p.p. amplitude was reduced. The reduction of m.e.p.p. amplitude was not prevented by choline (30-300 muM). 4 When m.e.p.p. activity fell below the noise level in the presence of TPB (0.1 mM), lanthanum (0.5 mM) was ineffective in promoting measurable m.e.p.p. activity. 5 The effects of TPB were slowly reversible by washing. 6 The results indicate that TPB acts to reduce the nerve terminal stores of acetylcholine, probably by a combination of rapid release and concomitant inhibition of transmitter storage.     

The effects of adenosine triphosphate and adenosine diphosphate on transmission at the rat and frog neuromuscular junctions

1 The effects of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were investigated on evoked end-plate potentials (e.p.ps) and on miniature end-plate potentials (min. e.p.ps) recorded from muscle fibres of the rat diaphragm and the frog sartorius.

2 ATP and ADP decreased the quantum content of the e.p.ps and the frequency of the min. e.p.ps. The maximum effects produced by the two substances were similar.

3 The potency of ATP was found to be similar to that of adenosine. In the presence of adenosine, in a concentration producing its maximum effect, the addition of ATP had no further effect. This is compatible with the idea that ATP acts in the same way as adenosine.

     

Effects of sea-anemone toxin (ATX-II) on the frequency of miniature endplate potentials at rat neuromuscular junctions.

Soleus and extensor digitorum longus muscles were isolated from rats. The muscles were exposed to ATX-II, a toxin isolated from extracts of the sea-anemone Anemonia sulcata . The toxin caused a dose-dependent increase in the frequency of miniature endplate potentials in both types of muscle. The increase in frequency could be reversed by the application of tetrodotoxin (TTX), and could be prevented by prior exposure of the muscles to TTX. It is concluded that ATX-II causes a sodium-dependent depolarization of the nerve-terminal membrane.     

The effects of vesamicol on trains of endplate currents and on focally recorded nerve terminal currents at mammalian neuromuscular junctions.

1. The effects of vesamicol, an inhibitor of vesicular acetylcholine (ACh) storage, were studied on trains of endplate currents (e.p.cs) in the cut rat hemidiaphragm nerve-muscle preparation and on trains of focally recorded nerve terminal current waveforms in the mouse triangularis sterni nerve-muscle preparation. 2. In the rat, 0.1 and 1 microM (-)-vesamicol produced an enhancement of the rundown of e.p.c. amplitudes during trains of high frequency (50 Hz) nerve stimulation. However, 1 microM (+)-vesamicol had no effect on the rundown of e.p.c. amplitudes. 3. In the mouse, high concentrations of (-)-vesamicol (10-100 microM) produced a concentration- and stimulation-dependent decrease in the amplitude of the second negative-going deflection of focally recorded nerve terminal current waveforms. 4. At 1 mM, (-)-vesamicol produced a stimulation-independent decrease in the amplitude of the first negative-going deflection of the nerve terminal current waveforms, an increase in signal delay and evidence of nerve conduction failure. These all indicate a local anaesthetic-like block of nodal Na(+)-channels. 5. In contrast to its effects on trains of e.p.cs, the effects of vesamicol on the nerve terminal current waveforms were not stereoselective, the (+)-isomer being equipotent with the (-)-isomer. 6. Low concentrations of the Na(+)-channel blocking toxin, tetrodotoxin (15-60 nM), produced similar changes in the focally recorded nerve terminal current waveforms to those seen with vesamicol. 7. It is concluded that the stereoselective rundown of e.p.c. amplitudes produced by (-)-vesamicol is due to an effect, either direct or indirect, on ACh mobilization within motor nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)     

The importance of phospholipase A2 in the early induction by crotoxin of biphasic changes in endplate potentials at the frog neuromuscular junction

In Ca2+-Ringer, crotoxin induced a rapid fall in amplitude of endplate potentials followed by a marked secondary rise but, in Ca2+-free, Sr2+-EGTA Ringer, crotoxin failed to elicit either response. In Ca2+-washout experiments, the onset of giant miniature endplate potentials indicated that binding of crotoxin occurred in Sr2+-EGTA Ringer. Facilitation of endplate potential amplitude due to a closely spaced twin impulse was increased to 162% of control near the peak of the crotoxin-induced secondary phase. These findings are consistent with the phospholipase A2 subunit acting specifically at the active zone.     

Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions

L. , B. J. and I. C. H. . Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions. Toxicon 28, 1479–1489, 1990.—Addition of crotoxin to the frog neuromuscular junction in either 0.9 mM Ca²⁺ plus tubocurarine or 0.5 mM Ca²⁺ Ringer solution produced a triphasic change in the amplitude of nerve-evoked endplate potentials (e.p.p.s) and, with 0.5 mM Ca²⁺, a biphasic change in miniature endplate potential (m.e.p.p.) frequency. The secondary phase of rising e.p.p. amplitude was associated with an increase in facilitation of e.p.p. amplitude with closely spaced twin impulses; the increase in spontaneous release lagged behind that of evoked release. When a calcium chelator, BAPTA, was loaded into presynaptic nerve terminals to buffer cytosolic free Ca²⁺, both e.p.p. amplitude and twin-impulse facilitation were increased by crotoxin to a similar extent relative to that in the control without BAPTA. The duration of the increase in twin-impulse facilitation was reduced but the duration of the increase in e.p.p. amplitude was unaffected by BAPTA loading. The presence of BAPTA did not alter the characteristic changes in spontaneous release in response to crotoxin. These results suggest that the augmentation of evoked and spontaneous transmitter release by crotoxin is not primarily due to changes in cytosolic Ca²⁺. The response time between stimulus and e.p.p. peak was lengthened in all phases due to prolongation of the interval between stimulus and e.p.p. onset and, in the secondary and tertiary phases, slowing of e.p.p. rise-time. The protein kinase C inhibitor, H-7, produced complex changes in e.p.p.s. under control conditions but did not alter the triphasic response characteristic of intoxication. These results suggest that crotoxin initiates a primary disturbance in the phasic release process leading to a series of time-gated changes which include transient facilitation then uncoupling of phasic release and generalized acceleration of spontaneous release.     

The actions of adenosine and some analogues on evoked and potassium stimulated release at skeletal and autonomic neuromuscular junctions

Summary The actions of the nucleosides adenosine, 1-methyladenosine, 1-methylisoguanosine and 2-chloradenosine on transmitter release at the mammalian neuromuscular junction and the vas deferens have been examined. All the nucleosides depressed the evoked release of acetylcholine at the neuromuscular junction, the order of potency being 2-chloroadenosine > 1-methylisoguanosine > 1-methyladenosine adenosine. This correlated reasonably with the potency of these compounds in depressing spinal reflexes in anaesthetized mice (Buckle and Spence 1981). Neither adenosine (0.5 and 1.0 mmol l^–1) or 1-methylisoguanosine (10 and 20 mol l^–1) had any effect on the elevation of minature endplate potential frequency caused by 12 mmol l^–1 K⁺ at the neuromuscular junction. In the guinea-pig vas deferens, however, 1-methylisoguanosine and adenosine were approximately equipotent in depressing overflow of radioactively labelled noradrenaline. The actions of the nucleosides have been compared with their effects on adenylate cyclase and their ability to resist uptake and deamination. It is concluded that the relative potencies of the nucleosides are not determined solely by their ability to survive in the extracellular fluid.     

Cadmium: effects on transmitter release at the frog neuromuscular junction

Cd2+ competitively antagonizes Ca2+ in the evoked release of acetylcholine from nerve terminals in the frog sciatic-sartorius neuromuscular junction. The dissociation constant between Cd2+ and its receptor sites was calculated to be 1.7 microM. In agreement with previous work we found that brief exposures to 100-500 microM Cd2+ had little or no effect on spontaneous transmitter release. However, in contrast to other reports, prolonged exposures to 100-100 microM Cd2+ or tetanic nerve stimulation during brief exposures to high concentrations of Cd2+ produced substantial increases in spontaneous transmitter release. We conclude that Cd2+, when present at low concentrations, is a specific Ca2+ channel blocker. At higher concentrations, Cd2+ is either relatively impermeable to the presynaptic nerve membrane or, if it does enter the nerve terminal, it is less effective than such metals as Pb2+, La3+ or Hg2+ in increasing the rate of spontaneous transmitter release.     

The action of lindane in accelerating the spontaneous release of transmitter at the frog neuromuscular junction

Summary Lindane (5×10^–5M) causes a progressive and marked rise in MEPP frequency at the frog neuromuscular junction. Concentrations over a range of 5×10^–6M to 5×10^–4M were tested. The results suggest that it has two actions in promoting this effect. Its major effect is probably to cause an increase in Ca²⁺-permeability and a rise in Ca²⁺ entry. Its second, smaller effect, which persists in the absence of extracellular Ca²⁺, is probably also because of a rise in [Ca²⁺]_i. The ways in which these effects might be produced and the significance of these findings for explaining the known pharmacological actions of lindane are discussed.     

The action of verapamil on the rate of spontaneous release of transmitter at the frog neuromuscular junction.

Verapamil is known to reduce Ca2+ entry in a variety of cells. At 10(-5) M it produces a small reduction in MEPP frequency at the frog neuromuscular junction, whereas the rate of spontaneous release rises following treatment at a concentration of 10(-4) M. This latter effect is augmented by raising [Ca2+]0 to 9 mM or, more especially, by raising the temperature from 17 to 23 degrees C. It is argued that both these opposing effects are related to the action of verapamil in modifying [Ca2+]i at the presynaptic terminals and it is suggested that the drug can affect both inward Ca2+ flux (so reducing the steady-state position of [Ca2+]i) and also, at higher concentration, either inhibit the membrane Ca2+ pump or cause the release of Ca2+ from intracellular Ca2+ stores (so raising [Ca2+]i).     

Blocking effects of blended paeoniflorin or its related compounds with glycyrrhizin on neuromuscular junctions in frog and mouse

The combined effects of paeoniflorin (PF), a main component of paeony roots, and glycyrrhizin (GLR), a main component of licorice roots, were investigated on isolated sciatic nerve-sartorius muscle preparations in frogs, or on isolated or in situ phrenic nerve-diaphragm muscle preparations in mice, intending to explain the effects of "Shakuyaku-Kanz?-T?, composed of both these Chinese drugs, on clinical neuropathy. PF and GLR used together blocked indirectly stimulated twitchings at concentrations which when used alone induced no blocking effects. PF and GLR at a combining ratio of 1:2 (weight concentrations) corresponding to the amounts contained in "Shakuyaku-Kanz?-T?, was more potent than when they were used at the ratio of 1:1 or 2:1. The synergistic effects induced by GLR were also confirmed for the other components, paeoniflorigenone or oxypaeoniflorin, which are contained in paeony roots, and for succinylcholine. The blocking effect of d-tubocurarine were not increased by GLR. Concludedly, PF and GLR were found to cause the pharmacological blend effect. The two combined compounds were mainly therapeutic components in "Shakuyaku-Kanz?-T?".     

The effects of ethanol and meperidine on auditory evoked potentials

The effects of ethanol and meperidine on the auditory evoked potential (AEP) to stimuli of different intensities were investigated. Sixteen normal male volunteers received ethanol, 0.8 ml/kg, 100 mg meperidine, and a placebo on different days in a double-blind study. AEPs were recorded from Fz, Cz and Pz electrode placements. The stimuli were 500 msec 1000 Hz tones at 50, 60, 70 and 80 dB sound pressure level presented in a pseudo-random sequence. Meperidine had no significant effect on AEP variables. Ethanol reduced AEP activity between 24 and 250 msec but had no effect on the sustained potential measured between 300 and 450 msec.     

The effects of tetraphenylboron on neuromuscular transmission in the frog.

1 The effect of tetraphenylboron (TPB) were studied on the frog sciatic nerve-sartorius muscle preparation. 2 TPB (0.01-1 mM) blocked indirectly elicited twitches of the preparation. 3 TPB (0.01-0.1 mM) produced no depolarization but lowered membrane resistance. TPB increased miniature endplate potential (m.e.p.p.) frequency, the rate of rise of the endplate potential (e.p.p.), and slowed the rate of rise and rate of fall of the muscle action potential. 4 In Mg2+ solutions the quantal content of e.p.ps was initially increased by TPB (0.01 mM). This was followed by a decrease of e.p.p. and m.e.p.p. amplitudes, accompanied by a lack of e.p.p. failures. 5 Larger concentrations of TPB (0.1 mM) produced an increase in e.p.p. amplitude followed by the sudden abolition of e.p.ps. This effect was associated with abolition of the nerve terminal spike. 6 TPB (0.1 mM) exhibited no postjunctional blocking action. 7 The results indicate that TPB acts prejunctionally, initially causing an increased release of acetylcholine. Subsequently, transmitter output is reduced by a reduction of quantal size.     

Dual effect of 2,4-dinitrophenol on the spontaneous release of transmitter at the frog neuromuscular junction.

The uncoupling agent 2,4-Dinitrophenol (DNP) has a dual effect on the spontaneous release of transmitter at the frog neuromuscular junction, causing an initial fall in miniature endplate potential (MEPP) frequency followed by a dramatic rise. The latter effect is probably associated with the release of Ca²⁺ from the mitochondria. The initial fall in MEPP rate is independent of [Ca²⁺]₀ but is largely suppressed by pretreatment with theophylline; it is suggested that DNP also combines with another intracellular Ca²⁺-store, so reducing Ca²⁺ leakage and causing a fall in the steady-state level of [Ca²⁺]_i. The results confirm that MEPP frequency is largely determined by [Ca²⁺]_i at the presynaptic terminals.     

Oxaliplatin induces hyperexcitability at motor and autonomic neuromuscular junctions through effects on voltage-gated sodium channels

Oxaliplatin, an effective cytotoxic treatment in combination with 5-fluorouracil for colorectal cancer, is associated with sensory, motor and autonomic neurotoxicity. Motor symptoms include hyperexcitability while autonomic effects include urinary retention, but the cause of these side-effects is unknown. We examined the effects on motor nerve function in the mouse hemidiaphragm and on the autonomic system in the vas deferens. In the mouse diaphragm, oxaliplatin (0.5 mM) induced multiple endplate potentials (EPPs) following a single stimulus, and was associated with an increase in spontaneous miniature EPP frequency. In the vas deferens, spontaneous excitatory junction potential frequency was increased after 30 min exposure to oxaliplatin; no changes in resting Ca(2+) concentration in nerve terminal varicosities were observed, and recovery after stimuli trains was unaffected.In both tissues, an oxaliplatin-induced increase in spontaneous activity was prevented by the voltage-gated Na(+) channel blocker tetrodotoxin (TTX). Carbamazepine (0.3 mM) also prevented multiple EPPs and the increase in spontaneous activity in both tissues. In diaphragm, beta-pompilidotoxin (100 microM), which slows Na(+) channel inactivation, induced multiple EPPs similar to oxaliplatin's effect. By contrast, blockers of K(+) channels (4-aminopyridine and apamin) did not replicate oxaliplatin-induced hyperexcitability in the diaphragm. The prevention of hyperexcitability by TTX blockade implies that oxaliplatin acts on nerve conduction rather than by effecting repolarisation. The similarity between beta-pompilidotoxin and oxaliplatin suggests that alteration of voltage-gated Na(+) channel kinetics is likely to underlie the acute neurotoxic actions of oxaliplatin.     

Butyrylcholinesterase and acetylcholinesterase activity and quantal transmitter release at normal and acetylcholinesterase knockout mouse neuromuscular junctions

1 The present study was performed to evaluate the presence and the physiological consequences of butyrylcholinesterase (BChE) inhibition on isolated phrenic-hemidiaphragm preparations from normal mice expressing acetylcholinesterase (AChE) and BChE, and from AChE-knockout mice (AChE(-/-)) expressing only BChE. 2 Histochemical and enzymatic assays revealed abundance of AChE and BChE in normal mature neuromuscular junctions (NMJs). 3 In normal NMJs, in which release was reduced by low Ca(2+)/high Mg(2+) medium BChE inhibition with tetraisopropylpyrophosphoramide (iso-OMPA) or bambuterol decreased ( approximately 50%) evoked quantal release, while inhibition of AChE with fasciculin-1, galanthamine (10, 20 micro M) or neostigmine (0.1-1 micro M) increased (50-80%) evoked quantal release. Inhibition of both AChE and BChE with galanthamine (80 micro M), neostigmine (3-10 micro M), O-ethylS-2-(diisopropylamino)ethyl-methylphosphono-thioate (MTP) or phospholine decreased evoked transmitter release (20-50%). 4 In AChE(-/-) NMJs, iso-OMPA pre-treatment decreased evoked release. 5 Muscarinic toxin-3 decreased evoked release in both AChE(-/-) and normal NMJs treated with low concentrations of neostigmine, galanthamine or fasciculin-1, but had no effect in normal NMJs pretreated with iso-OMPA, bambuterol, MTP and phospholine. 6 In normal and AChE(-/-) NMJs pretreatment with iso-OMPA failed to affect the time course of miniature endplate potentials and full-sized endplate potentials. 7 Overall, our results suggest that inhibition or absence of AChE increases evoked quantal release by involving muscarinic receptors (mAChRs), while BChE inhibition decreases release through direct or indirect mechanisms not involving mAChRs. BChE apparently is not implicated in limiting the duration of acetylcholine action on postsynaptic receptors, but is involved in a presynaptic modulatory step of the release process.     

Action of adenosine triphosphate on endplate potentials recorded from muscle fibres of the rat-diaphragm and frog sartorius

Evoked and spontaneous endplate potentials (e.p.ps) were recorded with intracellular electrodes from fibres of the rat diaphragm and the frog sartorius muscle. The amplitudes of the evoked e.p.ps were reduced to about one-half their control values in the presence of 0.1 mm and 0.2 mm of adenosine triphosphate (ATP). The amplitude of the spontaneous endplate potentials (miniature e.p.ps) was unaffected by ATP but their frequency was reduced.     

The effects on frog neuromuscular transmission of agents which act upon microtubules and microfilaments

The effects of colchicine, colchicine-like agents and cytochalasin B were analyzed on end-plate potentials (epps) recorded intracellularly from frog sartorius muscles in which neuromuscular transmission was partially blocked with low Ca²⁺— high Mg²⁺ Ringer solutions. Colchicine and podophyllotoxin decreased the mean quantal content of the epp (m) while griseofulvin increased m. Vinblastine sulfate and cytochalasin B had no effect on transmitter release in the concentrations employed in the study.