Interferon-alpha, beta and tumor necrosis factor-alpha enhance the frequency of miniature end-plate potentials at rat neuromuscular junction.

The effects of the two cytokines, rat interferon-alpha, beta and human tumor necrosis factor-alpha, were studied at the rat neuromuscular junction by using classical electrophysiological techniques. Both cytokines in a similar way at concentrations of 2,000 and 35,000 U/ml, respectively, increased transiently and with a relatively long delay (15 to 25 min) the frequency of miniature endplate potentials. The observed effects may be related to complex second messenger mechanisms and contribute to modulation and plasticity of neurotransmission.

Blockage of nicotinic acetylcholine receptors by 5-hydroxytryptamine.

The action of 5-hydroxytryptamine (5HT) on nicotinic acetylcholine receptor (nAChR) channels was investigated in mouse myotubes, human cloned TE671/RD cells, and Xenopus laevis oocytes. The decay of the ACh-activated whole-cell currents was reversibly accelerated in the presence of 5HT (10(-5) to 10(-3) M), in a dose-dependent manner. 5HT also reduced the size and accelerated the decay of currents elicited by ACh in Xenopus oocytes injected with mRNA extracted from C2 myotubes or Torpedo electroplaques, or oocytes injected with cloned mouse muscle AChR subunit mRNAs. The effect of 5HT was promptly reversed after washout, or by depolarizing the oocyte beyond -10 mV. In patch-clamp recordings from myotubes, bath-application of 5HT did not exert an indirect influence on the ACh-activated channels within the patch membrane. In contrast, when the patch membrane was exposed to 5HT (10(-6) M), ACh unit responses appeared as bursts of short pulses. It is concluded that the regulation of ACh responses by 5HT results from a fast noncompetitive blocking action of nAChR-channels. These results show that ligand-gated channels, activated by their specific neurotransmitter, may be regulated by a different neurotransmitter through a direct action on the receptor molecule.

Interleukin-2 lengthens extrajunctional acetylcholine receptor channel open time in mammalian muscle cells.

The effect of interleukin-2 (rIL-2) on nicotinic acetylcholine receptors (nAChR) was examined on cultured muscle fibres isolated from the flexor digitorum brevis muscle (FDB) of the rat and on aneural mouse cultured C2 myotubes. Intracellular measurement of the sensitivity to iontophoretically applied ACh demonstrated that the sensitivity of the extrajunctional nAChRs in cultured fibres showed a transient increase after application of rIL-2 (2,000-3,000 units/ml). Cell-attached patch-clamp experiments on the same fibres proved that rIL-2 (2,000 units/ml) induces a significant increase in the mean open time of the extrajunctional nAChR channel. The other channel parameters were not significantly modified. The same applied also to aneural mouse patch-clamped C2 myotubes exposed to rIL-2 (2,000 units/ml). In freshly dissociated fibres no effects on nAChR channels were observed following rIL-2 application. 125I-rIL-2 binding experiments on either 7-day cultured or freshly dissociated adult muscle fibres showed that a specific binding with a Kd of 2.07 +/- 0.4 nM develops in cultured fibres but fails to occur immediately after dissociation. It is concluded that rIL-2 modulates the duration of extrajunctional nAChR channels in both myotubes and adult muscle cells, and that this effect is probably due to the activation of a second messenger system.

Effect of calcitonin gene-related peptide on synaptic transmission at the neuromuscular junction of the frog.

The effects of calcitonin gene-related peptide (CGRP) on synaptic mechanisms were studied at the frog neuromuscular junction by using classical electrophysiological techniques. CGRP reduced the quantal content of evoked neurotransmitter release, as well as the sensitivity of postsynaptic nicotinic acetylcholine receptors (AChRs). No effect on the frequency of the miniature end-plate potentials or on the desensitization of the AChRs could be observed. Both the measured effects may depend on the stimulation of the cyclic AMP second messenger system.

Regulation of acetylcholine receptor function by the phorbol ester TPA in rat skeletal muscle.

(1) The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of the protein kinase C (PrkC), on the function of junctional nicotinic acetylcholine receptors (nAChR) was examined on muscle fibres isolated from the M. flexor digitorum brevis of the rat. (2) In the presence of TPA the sensitivity of the whole endplates to iontophoretically applied ACh exhibited multiphasic oscillations: an early decrease followed by a delayed increase and, at the end again, a decrease to below pretreatment levels. This effect was more pronounced as the TPA concentration was increased in the range of 0.1-1 microM and was blocked by the PrkC-inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7). (3) TPA (0.1-0.5 microM) shortly applied to patch-clamped fibres caused a slight decrease in nAChR-channel slope conductance without affecting the mean lifetime. In a patch the opening frequency increased over time, after an initial decrease. (4) It is concluded that specific activation of the PrkC may be of regulatory significance on nAChR function.

Influence of protein kinase C-stimulation by a phorbol ester on neurotransmitter release at frog end-plates.

1. The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the stimulation-evoked neurotransmitter release has been investigated by measuring the quantal content (m) of end-plate potentials at frog neuromuscular junctions (Rana temporaria, M. sartorius). 2. After addition of TPA (0.1 up to 1 mumol/l) to the Ringer solution the m-values increased in a concentration-dependent manner up to more than 3 times the control values. 3. Inhibition of the activity of the protein kinase C through the inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) blocked this effect of TPA. 4. The TPA effect was much more conspicuous when the m-value was reduced by raising the extracellular Mg2+ concentration. Between the control m-values and the n-fold increase in the m-value enhanced by TPA a hyperbolic relation was observed. 5. It is concluded that protein kinase C stimulation affects predominantly the spontaneous release of neurotransmitter at the frog neuromuscular junction and only very poorly the stimulation-evoked one.

Presynaptic effects of the pardaxins, polypeptides isolated from the gland secretion of the flatfish Pardachirus marmoratus.

The effects of the two toxic proteins Pardaxin I and II isolated from the gland secretion of the flatfish Pardachirus marmoratus on frog neuromuscular transmission have been investigated and compared to those of the gland secretion. Pardaxin I and II showed pre- but not postsynaptic neurotoxic effects. They increased the frequency of the spontaneous release of transmitter quanta in a dose-dependent and temperature-influenced way up to more than 100 times control values. At the same time the quantal content of the evoked end-plate potentials was greatly elevated. Pardaxin I was about 5 times more effective than Pardaxin II, and both were roughly in the same range of efficacy as the original gland secretion (w/v). The glycosteroids isolated from the same gland secretion were relatively ineffective in promoting neurotransmitter release; however, at high doses they had postsynaptic effects, as shown by a diminution of the amplitude of the evoked end-plate potentials. They did not reinforce the effect of the Pardaxins. At higher doses both the Pardaxins and the gland secretion induced depolarization of postsynaptic membranes, muscle cell contractions which could not be blocked by (+)-tubocurarine or by tetrodotoxin, and eventually also physical disruption of muscle cells. No effects on nerve conductance were observed. Pore-forming activity of the Pardaxins has already been demonstrated. It is suggested that their presynaptic effects are a result of a possible affinity to the nerve terminals, of their hydrophobicity and mainly of this pore-forming activity. These toxins might be valuable tools in neuroscience research.

Postsynaptic effects of the phorbol ester TPA on frog end-plates.

The effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of protein kinase C (PKc), were examined on the frog neuromuscular junction. The depolarization elicited by iontophoretically applied acetylcholine (ACh) was reversibly decreased by 20-60% when muscle fibres were exposed to 1-5 X 10(-7) M TPA. Liposome-delivered phosphatidylcholine (100 micrograms/ml) prevented this effect. A similar decrease in ACh-sensitivity was produced by diacylglycerol (diolein), a physiological activator of PKc, but in this case the decrease was only partially reversible. In TPA-Ringer, the peak size of miniature end-plate potentials exhibited a small decrease; miniature end-plate currents were reduced in size and their decay time constant became longer and relatively independent of membrane potential. The possibility that these TPA-induced actions are mediated by activation of PKc is discussed.

Effects of phorbol ester on spontaneous transmitter release at frog neuromuscular junction.

Spontaneous transmitter release was studied at frog neuromuscular junctions exposed to the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of protein kinase C (PrkC). TPA at concentrations between 10(-7) and 10(-6) M induced a dose-dependent increase in miniature end-plate potential frequency. This frequency increase was enhanced by raising [Ca]o, diminished by lowering the temperature of the bath and virtually abolished in Ca2+ -free Ringer. The TPA effect was only poorly reversible after washing. TPA was ineffective in increasing spontaneous release of transmitter at phosphatidylcholine-pretreated neuromuscular junctions. It is suggested that PrkC might play a role in neuromuscular transmission processes.

Influence of divalent cations on the phospholipase-independent action of beta-bungarotoxin at frog neuromuscular junctions.

The influence of different divalent cations on the phospholipase-independent inhibition of transmitter release caused by beta-bungarotoxin (beta-BuTx), has been investigated by measuring the frequency of spontaneous miniature end-plate potentials (m.e.p.p.s) at frog neuromuscular junctions. After adding the toxin to normal calcium Ringer solution the m.e.p.p. frequency fell quickly to very low values. This was followed by an increase in frequency characterized by bursts of m.e.p.p.s. The temperature had a negligible effect on the speed of the first inhibition. In Ringer solutions where calcium had been substituted by other divalent cations (5 mM) in the presence of ethyleneglycol-bis-(beta-aminoethylether)N, N'-tetraacetic acid (EGTA, 1 mM), this beta-BuTx-induced decrease in m.e.p.p. frequency was markedly slower. The potency of cations in promoting the initial phase of toxin action was in the sequence: calcium greater than magnesium greater than strontium congruent to cobalt greater than manganese. This phospholipase-independent inhibition of transmitter release followed approximately first-order kinetics, suggesting that it depends mainly on toxin concentration. In the absence of any divalent cations in the Ringer solution beta-BuTx had practically no effect on m.e.p.p. frequency. It appears that beta-BuTx requires divalent cations in order to bind to motor nerve terminals and exert its initial inhibitory action on spontaneous release of transmitter quanta.

Effects of obidoxime chloride on native and sarin-poisoned frog neuromuscular junctions.

The effect of the oxime reactivator obidoxime chloride (obidoxime) on single frog neuromuscular junctions has been studied in order to clarify its action on the acetylcholine receptor (AChR) and on the acetylcholine esterase (AChE), both before and after blocking its enzymatic activity with the organophosphorus compound sarin. Experiments iontophoretic application of obidoxime to end-plates demonstrated that it has a weak direct depolarizing effect. Furtheron, the drug is shown to possess a potentiating effect on the ACh-induced depolarization. After the AChE activity had been inhibited with sarin, obidoxime on the contrary decreases the depolarization induced by ACh. Both effects are fully reversible. It is concluded that obidoxime acts as an inhibitor of the AChE and as a partial antagonist of the AChR. The antagonistic effect on the receptor is usually masked by the predominating anticholinesterase effect. The effect of obidoxime on miniature end-plate potentials in long-time experiments on sarin-poisoned muscles, showed only weak signs of recovery from the action of the AChE inhibitor. Only focally higher concentration of the drug produced a more marked but short term recovery of the mepps, which is, however, supposed to be dependent on the AChR antagonism. It is still unclear how much of the varying therapeutic usefulness of obidoxime in clinical cases is due to its AChE reactivation and how much to the antagonistic effect on the AChR.

Antibodies to beta-bungarotoxin and its phospholipase inactive derivative.

Antisera were raised against the presynaptic neurotoxin beta-bungarotoxin and against its phospholipase-inactive derivative, modified by reaction with p-bromophenacyl bromide. The cross-reactivity of the antisera to other phospholipase A2 enzymes and polypeptide neurotoxins was examined. The antisera inhibited both the neurotoxic effects of beta-bungarotoxin at the frog motor endplate and the enzymatic activity of the toxin on model phospholipid membranes, although it is unlikely that the catalytic active centre is the locus of any major determinant.

A further study of the phospholipase-independent action of beta-bungarotoxin at frog end-plates.

1. The effect of beta-bungarotoxin (beta-BuTx) at the frog neuromuscular junction has been investigated further in order to distinguish more clearly between phospholipase- independent and phospholipase-dependent actions on transmitter release. 2. Inhibition of the enzymatic activity, by substitution of strontium for calcium, allowed determination of the dose-response curve of the early rapid decrease in transmitter release caused by the toxin. In the presence of strontium ions there was, however, still about 7% residual enzymatic activity, and electrophysiological evidence of it could be seen in room-temperature experiments at high concentrations of beta-BuTx. This residual enzymatic activity could be suppressed by lowering the temperature to 5 degrees C. 3. In normal calcium-Ringer solution beta-BuTx produced the typical triphasic effect on the amplitude of end-plate potentials (e.p.p.s). Lowering the temperature markedly delayed an then diminished the secondary transient increase. There was, however, comparatively little temperature influence on the first rapid decrease in e.p.p. amplitude. Enzymatic assays confirmed the temperature dependence of the toxin's phospholipase activity on model phospholipid substrates. 4. The kinetics of the phospholipase-independent action of beta-BuTx were examined in strontium-Ringer compared to calcium-Ringer solution, as well as in calcium-Ringer at different temperatures. Both the time to onset of inhibition and the time to 50% inhibition of the e.p.p., during the first phase of toxin action, are temperature-dependent and briefer in calcium than in strontium-Ringer solution. It is suggested that calcium is more effective than strontium in promoting this phospholipase- independent interaction of beta-BuTx with the nerve terminal membrane.

Effect of p-nitrophenyl diazonium fluoroborate on cholinergic mechanisms.

Electrophysiological experiments were done to investigate the effect o p-nitrophenyl diazonium fluoroborate (p-NPD) on motor endplates of the frog's m. cutaneus pectoris. The compound has no direct depolarizing effect on the postsynaptic membrane and stabilizes it irreversibly when added to the bath. Longtime iontophoretical applications of p-NPD produce a biphasic effect: initially a potentiation of the depolarizations due to acetylcholine (ACh) (both iontophoretically applied and presynaptically liberated), and subsequently an inhibition of the response to ACh. When the acetylcholinesterase (AChE) is inactivated previously, only the inhibiting effect of the compound is demonstrable. The association constant of p-NPD to purified AChE and to membrane fragments of electroplax was determined by biochemical methods. The compound's affinity to the AChE was found to be about 20 times greater than to the acetylcholine receptor (AChR). Iontophoretical application of p-NPD to cholinergic neurons in the hippocampal cortex of the cat also produced the characteristic biphasic effect on ACh-induced activity of these investigated neurons. The results suggest that the biphasic effect depends on the capacity of p-NPD to combine with both the AChE and the AChR. The AChE is first inhibited with low concentrations thereby potentiating the ACh response. At higher concentrations the AChR's are progressively inhibited too, thereby diminishing the excitability of the postsynaptic membrane up to a complete block.