On the effect of the ionophore X-537A on neuromuscular transmission in the rat

In the rat phrenic nerve-diaphragm muscle preparation, X-537A at 6×10^?6 to 3×10^?5 M (1) depolarized muscle fibre membranes, (2) caused an occasional transient increase in and ultimate block of spontaneous transmitter release, (3) did not increase the amplitude of the end-plate potential (epp) but abruptly blocked stimulus-evoked transmitter release, and (4) produced an increase in the occurrence of “giant” miniature epp's (mepp's). The possibility is discussed that the sporadically raised mepp frequency was due to an ionophore-induced depolarization of nerve terminals. The increased occurrence of “giant” mepp's apparently reflected a X-537A-induced spontaneous multiquantal release of acetylcholine. This was not dependent on extracellular calcium but appeared to be of presynaptic origin.     

Effect of the "calcium ionophore" A-23187 on transmitter release at the frog neuromuscular junction.

The ionophore A-23187 when added to the usual Ca²⁺-Ringer at the frog neuromuscular junction has almost no effect on the frequency of miniature end-plate potentials (min.e.p.p.s). The ionophore does increase the rate of Ca²⁺ efflux from frog muscle, so it is in effective concentrations in the Ringer. When added to Ringer containing Ni²⁺ instead of Ca²⁺, the ionophore increases the min.e.p.p. frequency. We suggest that the ionophore can carry divalent cations into the terminal, but there are mechanisms to keep the Ca²⁺ low.Apparently these mechanisms are unable to rapidly eject or sequester Ni²⁺.     

An analysis of miniature synaptic events recorded from locust muscle--variations in amplitude and time courses.

A study of miniature post-synaptic potentials (min. e.p.s.p.'s) in metathoracic extensor tibiae muscle fibres at neuromuscular junctions of adult locusts (Schistocerca gregaria) has been undertaken. Extracellular min. e.p.s.p.'s recorded from single junctional sites and their "marked" intracellular min. e.p.s.p. counterparts varied both in time course and amplitude and at many sites a small proportion of abnormal miniatures were observed, i.e., of large amplitude and/or of long duration. Positive correlations between the rise times and 1/2-decay times of the extracellular min. e.p.s.p.'s and between rise times and amplitudes and 1/2-decay times and amplitudes of "marked" intracellular events were found. Comparison of results obtained from differently innervated muscle fibres demonstrates that the occurrence of abnormal miniatures is independent of the type of innervation i.e., "fast" or "slow" excitatory motoneurons. Mechanisms that lead to the occurrence of abnormal miniatures are discussed in relation to the presence of large vesicles in the terminals of the excitatory motoneurons but the occurrence of these events could equally well be explained by the "post-synaptic saturation hypothesis".     

Broad-range effects of ionophore X-537A on pollen tubes of Lilium longiflorum

The effects of the broad-range cationophore X-537A on pollen tubes of Lilium longiflorum were investigated, using both light and electron microscopy. Pollen tube growth is completely inhibited within 30 min after the application of 5·10^-5 M ionophore X-537A; cytoplasmic streaming is stopped only after 60 min of ionophore treatment. Ultrastructurally, X-537A effects are a vacuolation of Golgi cisternae and a general vacuolation. The wall is thickened at the very tip. Coated vesicles and coated regions are enriched close to and at the plasma membrane. The results indicate that pollen tube tip growth needs a specific ion distribution.Abbreviations CTC chlorotetracycline - DMSO dimethylsulfoxide     

Miniature End-plate Potentials at Mammalian Neuromuscular Junctions Poisoned by Botulinum Toxin

IT is generally accepted that botulinum toxin entirely blocks transmitter release from motor nerve terminals without affecting nerve conduction or the sensitivity of the muscle membrane to acetylcholine. In particular, it has been reported that with both acute and chronic intoxication with type A botulinum, miniature end-plate potentials (m.e.p.p.s.) disappear completely from a muscle at about the time that transmission is blocked^1,2. The action of botulinum toxin has been reinvestigated following acute application of toxin to the rat diaphragm in vitro and chronic paralysis of rat soleus muscle following a single intramuscular injection of toxin; miniature potentials have been observed to persist following blockade of neuromuscular transmission.     

Some effects of the ionophore X-537A on the isolated rat tail artery

The effects of micromolar concentrations of the ionophore X-537A (RO 2-2985) were studied using isolated preparations of the rat tail artery. The ionophore causes complete release of catecholamines from adrenergic nerves, which is the sole cause of the transient contractile response. The amines are released by a nonexocytotic process which seems to be related to the ability of X-537A to act as an efficient transmembrane carrier of Na+, k+, and H+. The ionophore also causes an almost complete and irreversible loss of the cocaine-sensitive component of metaraminol uptake by the tissue. X-537A dissipates the transmembrane concentration gradients of Na and K in the smooth muscle component of the preparation. This effect is unrelated to the release of endogenous catecholamines, and it can also be observed after the Na pump has been inhibited with ouabain. It is fully reversible, though not readily, and it can be induced repeatedly. In catecholamine-depleted strips, X-537A dissipates the transmembrane Na+ and K+ gradients without causing any change in tension. Stimulation of the rate of O2 consumption by X-537A in catecholamine-depleted tissue is reversible, and it is unaffected by ouabain and (or) removal of external Ca2+.     

Habitual exercise enhances neuromuscular transmission efficacy of rat soleus muscle in situ.

Rat motor nerve terminals and the endplates they interact with exhibit changes to varying patterns of use, as when exposed to increased activation in the form of endurance exercise training. The extent to which these changes affect neuromuscular transmission efficacy is uncertain. In this study, the effects of habitual exercise on the electrophysiological properties of neuromuscular transmission in rat soleus muscle were investigated using a novel in situ approach. Consistent with previous reports, miniature endplate potential frequency was enhanced by habitual exercise. Other passive properties, such as resting membrane potential, miniature endplate potential amplitude, and "giant" miniature endplate potential characteristics were unaltered by the training program. Full-size endplate potentials were obtained by blocking soleus muscle action potentials with mu-conotoxin GIIIb. Quantal content values were 91.5 and 119.9 for control and active groups, respectively (P < 0.01). We also measured the rate and extent of endplate potential amplitude rundown during 3-s trains of continuous stimulation at 25, 50, and 75 Hz; at 50 and 75 Hz, we found both the rate and extent of rundown to be significantly attenuated (10--20%) in a specific population of cells from active rats (P < 0.05). The results establish the degree of activity-dependent plasticity as it pertains to neuromuscular transmission in a mammalian slow-twitch muscle.     

Effect of tetanus toxin on mechanisms by which calcium ions regulate transmitter secretion at the neuromuscular junction

Phrenicodiaphragmal rat preparations were used to study the transmitter secretion by intracellular recording of end plate potentials (EPP) and miniature EPP (MEPP). In tetanus toxin-poisoned terminal, the regulatory effect of the external gradient of Ca2+ was abolished as evidenced by the fact that spontaneous secretion did not differ from that in calcium-free solution in health, as the external concentration of Ca2+ rose from 0 to 20 mM. Calcium ionophore A 23187 in intact terminals activated spontaneous release of the transmitter, but did not affect the poisoned terminal. Ouabain enhanced spontaneous secretion both in health and in poisoning. 4-Aminopyridine (4-AP) did not change the frequency of MEPP, while "giant" MEPPs that reflect spontaneous synchronization of the release of quants occurred both in health and in poisoning. 4-AP potentiated the reactivation effects of rhythmic stimulation of poisoned synapses, particularly with reference to the evoked release and led to the recovery of transmission. It is likely that tetanus toxin fixed by gangliosides of the presynaptic membrane prevents, in this particular case, the functioning of both endo- and exogenous ionophoroses that transport Ca2+ to the "active zones", without affecting their asynchronous supply from the intracellular depots.     

金褐霉素（Aureofuscin）对神经末梢...

By means of the intracellular recording technique, the effect of aureofuscin (20 micrograms/ml, oversaturation solution) on the ACh release from motor nerve terminals and on muscle cell membrane potential were investigated in phrenic nerve diaphragm preparations of the mice. The results showed that (a) aureofuscin reduced the resting membrane potential of the muscle cell slightly; (b) the frequency of miniature end-plate potentials and the mean quantal content of end-plate potentials increased at first and then recovered approximately to the control level; (c) the depolarization produced by aureofuscin in the muscle cell membrane was reversible and the aureofuscin-invoked facilitation in miniature end-plate potential discharges was Ca(2+)-dependent; and (d) aureofuscin did not block neuromuscular transmission.     

Electrical and mechanical responses of chromatophore muscle fibers of the squid,Loligo opalescens,to nerve stimulation and drugs

Summary The muscle fibers of brown and red chromatophores in the skin of the squid, Loligo opalescens, respond to motor nerve stimulation with non-propagating excitatory postsynaptic potentials (e.p.s.p.'s) of fluctuating amplitude. Depending on the strength of stimulation several size classes of e.p.s.p.'s are found, indicating polyneuronal innervation. Facilitation and summation are minimal even though the reversal potential of the e.p.s.p.'s is close to zero.Acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) have no effect on membrane characteristics of the muscle fiber, but ACh greatly augments the spontaneous quantal release of transmitter [increase in the frequency of miniature postsynaptic potentials (m.p.s.p.'s)] and thereby causes tonic contraction (miniature tetanus). 5-HT reduces the frequency of miniature potentials and abolishes tonic contraction. Inhibition of cholinesterase by eserine does not affect the amplitude or time course of e.p.s.p.'s and of m.p.s.p.'s. High concentrations of cholinergic blocking agents (atropine, banthine) reduce the postsynaptic effects of nerve stimulation in some cases. The natural transmitter substance of the motoneurones may not be ACh. The action of 5-HT appears to be intracellular.Neighboring muscle fibers are electrically coupled through low resistance pathways. These are most likely provided by the close junctions that form part of the myo-muscular junctions. The specific membrane resistance of the regular muscle fiber membrane was found to range from 1,056 to 1,320 Ohm×cm², that of the close junctions ranges from 12.8 to 22.6 Ohm×cm². The area occupied by close junctions is small, however, and only 10% of the current injected into one cell passes into the next. Some of the e.p.s.p.'s observed in a given muscle fiber most likely represent the electrotonic spread of the e.p.s.p.'s of the neighbor fibers. Of the six classes of e.p.s.p.'s observed in some muscle fibers, only two may originate in these fibers themselves.Chromatophores in aged preparations often exhibit pulsations. These are caused by spike potentials arising within muscle fibers whose membranes have become electrically excitable. Each spike is preceded by a generator depolarization. The electrical coupling of neighboring muscle cells permits conduction of the spike potentials throughout the set of muscle fibers of a pulsating chromatophore. Altered conditions within such preparations also lead to tonic contractions and contractures that are not necessarily accompanied by electrical activity. Several arguments are presented in support of the hypothesis that the tonic condition of nerve terminals (characterized by enhanced spontaneous transmitter release) and of muscle fibers (characterized by inability to relax) is due to an abnormal condition of intracellular calcium (lack of Ca-binding by sarcoplasmic reticulum or other storage sites).No evidence could be found for an inhibitory innervation of the chromatophore muscles. The nerve-induced relaxation of tonically contracted muscle fibers is caused by the action of motoneurones.Preliminary experiments on muscle fibers of the anterior byssus retractor muscle of Mytilus support the hypothesis that the tonic behavior (catch) of other molluscan muscles is due to mechanisms similar to those found in the chromatophore muscles.This investigation was supported by Public Health Service Grant No. NB 04145 from the National Institute of Neurological Diseases and Blindness. We are grateful to the director of the Friday Harbor Laboratories, Prof. R. L. Fernald for providing space and facilities for this investigation.Supported by a Training Grant GM 1194 from the National Institute of General Medical Sciences.     

The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A

The divalent ionophores A23187 and X-537A induce parthenogenesis in sea urchin eggs. This results from their ability to mobilize intracellular Ca²⁺, which is implicated in both artificial parthenogenesis as well as the natural fertilization process. A23187 causes expulsion of cortical granules and elevation of the fertilization membrane within 0.5–9 min followed by an initiation of cell cleavage. The broader spectrum ionophore X-537A is less potent, but the production of cytoplasmic aberrations are more apparent. In contrast to the sperm-activated egg, the initial phase of ionophore induced activation is accompanied either by relatively insignificant changes in membrane resistance, or an increase.     

CHANGES IN THE FINE STRUCTURE OF THE NEUROMUSCULAR JUNCTION OF THE FROG CAUSED BY BLACK WIDOW SPIDER VENOM

Application of black widow spider venom to the neuromuscular junction of the frog causes an increase in the frequency of miniature end-plate potentials (min.e.p.p.) and a reduction in the number of synaptic vesicles in the nerve terminal. Shortly after the increase in min.e.p.p. frequency, the presynaptic membrane of the nerve terminal has either infolded or "lifted." Examination of these infoldings or lifts reveals synaptic vesicles in various stages of fusion with the presynaptic membrane. After the supply of synaptic vesicles has been exhausted, the presynaptic membrane returns to its original position directly opposite the end-plate membrane. The terminal contains all of its usual components with the exception of the synaptic vesicles. The only other alteration of the structures making up the neuromuscular junction occurs in the axon leading to the terminal. Instead of completely filling out its Schwann sheath, the axon has pulled away and its axoplasm appears to be denser than the control. The relation of these events to the vesicle hypothesis is discussed.     

Action of botulinum A toxin and tetanus toxin on synaptic transmission

Intracellular recordings of the spontaneous activity from mammalian spinal cord neurons in culture demonstrated different sensitivities of excitatory and inhibitory synaptic transmission for the action of tetanus toxin (Tetx) and botulinum toxin type A (Botx). The effects of Tetx and Botx on spontaneous and nerve-evoked transmitter release were compared under identical experimental conditions in experiments on in vitro poisoned mouse diaphragms. At 37 degrees C completely paralyzed endplates are characterized by a very low frequency of spontaneous miniature endplate potentials (m.e.p.p.s) and by a 100% failure to evoke endplate potentials (e.p.p.s) in response to single nerve stimuli. Striking differences in the action of both toxins have been observed when the very low transmitter release probabilities of paralyzed nerve-muscle preparations were increased by tetanic nerve stimulation and/or application of potent K+-channel blockers and/or by reduction of temperature to 25 degrees C. While Botx did not change the short latency between nerve impulse and postsynaptic response, Tetx produced a temporal dispersion of the quantal release suggesting that the toxins act at different sites in the chain of events that result in transmitter release. To find further evidence to support the different actions of the toxins the spontaneous transmitter release was studied in more detail. Tetx blocked preferentially the release of so-called large mode m.e.p.p.s without affecting the frequency of the small mode ones. In contrast, Botx strongly inhibited both the small and large mode m.e.p.p.s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological study of mediator secretion in the frog neuromuscular synapse under a colchicine block of axoplasmic transport

Two weeks after colchicine nerve treatment the evoked transmitter release was blocked in part of the frog sartorius synapses, with spontaneous activity being absent from some of them. In the synapses with evoked and spontaneous transmitter release preserved within this period of time, the magnitudes of the absolute refractory phase of nerve terminals were significantly higher than the control ones, while in part of synapses, the frequency of miniature end plate potentials (MEPP) was considerably increased. Nerve stimulation (5 imp.s-1) led to a rise of the amplitude of evoked potentials and of MEPP frequency followed by irreversible blockade of synaptic activity. It is concluded that substances transported by rapid axonal flow control the level of membrane potential of nerve terminals and are fairly important for presynaptic membrane integrity.     

Regulation of tetanus toxin-inhibited transmitter secretion in the neuromuscular junction in a calcium-free medium

The effect of Ca2+ removal from the external medium on regulation of the release of the synaptic transmitter in the tetanus toxin (TT)-inhibited neuromuscular junctions was studied on a rat phrenicodiaphragmal preparation with the aid of the conventional microelectrode technique of recording synaptic activity. As the external concentration of calcium was decreased from 2 to 0 mM, the frequency of miniature end plate potentials remained unchanged in the preparations isolated 3 to 3.5 h after intramuscular injection of TT (10(5) MLD for mouse). TT considerably reduced activation of the transmitter release, caused in intact synapses by ouabain (0.1 mM) and repetitive stimulation of the diaphragmatic nerve (50 imp/s). The data obtained indicate that in the TT-inhibited motor nerve terminals, the level of the transmitter release does not depend on the external concentration of calcium and that TT damages some of the intracellular sources of calcium.     

Effect of prostaglandins E1 and F2 alpha on neuromuscular transmission in the frog sartorius muscle.

End plate potentials (e.p.p.s.) and miniature end plate potentials (m.e.p.p.s.) were recorded intracellularly at the neuromuscular junction of the frog sartorius muscle. Addition of as little as 8.5 x10(-8)M PGE1 reduced the mean m.e.p.p. frequency. The mean amplitude of m.e.p.p.s was not changed, the mean amplitude of the e.p.p.s and the quantum content of the transmitter released by a nerve impulse was slightly reduced. A decrease in mean m.e.p.p. frequency was also seen in response to the administration of 8.5 x 10(-8)M PG2 alpha. The mean amplitude of e.p.p.s and m.e.p.p.s and the quantum content remained unchanged. The possible presynaptic mode of action of PGs in the preparation of discussed.     

Effects of the ionophores,X-537A and A-23187 on catecholamine release from the in vitro frog adrenal

1. The ionophore X-537A increases the rate of catecholamine release from the $\text{in vitro}$ frog adrenal.2. The ratio of epinephrine/norepinephrine measured during X-537A stimulation was the same as that during spontaneous release.3. Even when Ca⁺⁺ was removed from the Ringer, X-537A stimulated catecholamine release, but depolarization by elevated extra-cellular K⁺ was no longer effective.4. X-537A also increases the release of dopamine β-hydroxylase, suggesting that the ionophore acts, at least in part, by stimulating the exocytosis of the chrommaffin granule contents.5. Therefore, it is questionable whether the release of catecholamines by X-537A is owing to its action as a Ca⁺⁺- ionophore.6. The divalent cation ionophore, A-23187 (50μM), did not affect the rate of catecholamine release.     

Transmitter release in botulinum-poisoned muscles

Examination of miniature end-plate potentials (m.e.p.ps) in rat skeletal muscle poisoned in vivo by botulinum toxin type A reveals the presence of two populations of potentials. One population which corresponds to m.e.p.ps in unpoisoned muscles and to quantal end-plate potentials. The frequency of these m.e.p.ps is greatly reduced by botulinum toxin. The second population of m.e.p.ps has quite different characteristics. These m.e.p.ps have a more variable, but generally much larger amplitude, and their time to peak is longer than normal m.e.p.ps. The frequency of these m.e.p.ps increases during poisoning and reaches 0.3-1 Hz after 10-14 days. In addition to the variability in amplitude and time-to-peak these m.e.p.ps differ from those at unpoisoned junctions by being unaffected by procedures which alter extra- or intracellular Ca2+ concentrations. The appearance of this Ca2+-insensitive spontaneous quantal secretion of acetylcholine is apparently not a direct effect of the toxin but secondary to blockade of impulse transmission since it also appears at unpoisoned end-plates when transmission is impaired for other reasons. Procedures which increase the intracellular Ca2+ concentration in nerve terminals restore transmitter release from botulinum toxin poisoned nerves. Furthermore, the block caused by the toxin is very temperature-dependent, a reduction in temperature relieving the block. Since presynaptic Ca2+ currents are unaltered by the toxin it is proposed that the block of transmission is due to a reduction in the calcium content of the nerve terminal to a level where the amount of Ca2+, which normally enters, is insufficient to activate transmitter release.     

Depolarization reverses age-related decrease of spontaneous transmitter release.

The effect of increasing extracellular Ca concentration on spontaneous transmitter release was studied at soleus nerve terminals of young (10 mo) and old (24 mo) C57BL/6J mice depolarized by high extracellular K concentration ([K]o). By using intracellular recording, miniature end-plate potentials (MEPPs) were first recorded in a normal [K]o Krebs solution. Subsequently, MEPPs were recorded in high [K]o Krebs solutions with four different Ca concentrations: Ca-free/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 0.5, 1.5, and 2.5 mM Ca. In both the normal [K]o Krebs and the Ca-free-high [K]o Krebs solutions, MEPP frequency was lower at old than at young nerve terminals. In the three high [K]o Krebs solutions with Ca, MEPP frequency was progressively higher at old than at young nerve terminals with higher Ca concentrations. Periodic oscillations were observed in MEPP frequency of depolarized nerve terminals. The period of oscillation was inversely proportional to spontaneous transmitter release. These results demonstrate that when the nerve terminal is depolarized, permeability of the terminal membrane to Ca increases because of opening of voltage-dependent Ca channels. In the present study resting MEPP frequency was lower at old than at young terminals. On depolarization, MEPP frequency became higher at old than at young terminals. The study demonstrates that voltage-dependent Ca entry increases during aging at the soleus nerve terminal.     

Inhibitory Miniature Potentials in the Stretch Receptor Neurons of Crayfish

Intracellular microelectrodes inserted into the soma of crayfish stretch receptor neurons record frequent fluctuations of the membrane potential. Time course, amplitude, and interval distribution indicate that they are miniature potentials. At the average resting potential the polarity of the miniature potentials depends on the anion used in the microelectrode: KCl electrodes record depolarizing, K citrate or K₂SO₄ electrodes, hyperpolarizing miniature potentials. The inhibitory postsynaptic potentials (i.p.s.p.'s) show a similar polarity change. The reversal potentials of i.p.s.p.'s and miniature potentials are equal and within 10 mv of the resting potential, more negative with K citrate (or K₂SO₄), less negative with KCl electrodes. Reversal can be accomplished by changing the membrane potential by stretching or by current passing. Injection of Cl^- into the soma or replacement of external Cl by propionate results in an abrupt increase of the amplitude of the miniature potentials lasting for several minutes. The miniature potentials like the i.p.s.p.'s are reversibly abolished by the application of picrotoxin and γ-aminobutyric acid. They are not affected by tetrodotoxin, nor by acetylocholine, eserine, or atropine. It is concluded that the miniature potentials represent a spontaneous quantal release of transmitter substance from inhibitory nerve terminals, and that the transmitter substance predominantly increases the Cl^- permeability of the postsynaptic membrane. The effect of the spontaneously released transmitter on the behavior of the receptor neuron is considerable. The membrane conductance is increased by up to 36% and the excitability is correspondingly depressed.