Prion protein potentiates acetylcholine release at the neuromuscular junction.

Cellular prion protein (PrP(c)), the normal isoform of the pathogenic peptide (PrP(sc)) responsible of the transmissible spongiform encephalopaties (TSEs), is present in many neural tissues, including neuromuscular junctions (NMJ). To analyze if this protein could influence the synaptic transmission, we performed an electrophysiological approach to study the effect of cellular prion protein on a mammalian neuromuscular junction. The loose patch clamp (LPC) technique enables the study of the whole preparation including the pre- and the post-synaptic domains. In a mouse phrenic-diaphragm preparation, nanomolar concentrations of cellular prion protein were able to induce a very striking potentiation of the acetylcholine (ACh) release. The effect was mainly pre-synaptic with an increase of the amplitude of the miniature end-plate currents, probably calcium dependent. Moreover, an apparent facilitation of the synaptic transmission was noted. The results clearly indicate that cellular prion protein may play a key role in the function of the neuromuscular junction.     

Modulation of acetylcholine release at mouse neuromuscular junctions by interaction of three homologous scorpion toxins with K+ channels.

1. The effects of three scorpion toxins, charybdotoxin (CTX), iberiotoxin (IbTX), and noxiustoxin (NTX) have been studied on acetylcholine release and on K+ channels by means of twitch tension and electrophysiological recording techniques using isolated skeletal muscle preparations and by a radioligand binding assay using 125I-labelled dendrotoxin I (DpI) and rat brain synaptosomal membranes. 2. On chick biventer cervicis preparations, CTX and IbTX (125 nM) augmented the twitch responses to indirect muscle stimulation. Further, the increase (about 70-80% of control twitch height) was fast in onset, reaching a maximum within 25-30 min. NTX at 125 nM produced a slower augmentation of the twitch responses to indirect muscle stimulation, with the maximum response being seen after 40-50 min. 3. On mouse triangularis sterni preparations, CTX (300 nM after 35-40 min) and IbTX (100 nM after 15 min) increased quantal content of the evoked endplate potentials (e.p.p.) by about two fold. However, NTX (300 nM) caused only a small increase in e.p.p. amplitude, which was followed by repetitive e.p.ps in response to single shock nerve stimulation after 40-50 min. 4. Extracellular recording of nerve terminal current waveforms in triangularis sterni preparations revealed that CTX and IbTX (3-100 nM), but not NTX (100 nM), blocked the Ca(2+)-activated K+ current, IK-Ca. However, there was no major change in the portion of the nerve terminal waveform associated with voltage-dependent K+ currents, IKv. 5. In the radioligand binding assay, NTX potently displaced labelled [125I]-DpI, whereas CTX produced only partial displacement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic inhibition of spontaneous acetylcholine release induced by adenosine at the mouse neuromuscular junction

1. At the mouse neuromuscular junction, adenosine (AD) and the A(1) agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A(1) AD receptors through a mechanism that is still unknown. To evaluate whether the inhibition is mediated by modulation of the voltage-dependent calcium channels (VDCCs) associated with tonic secretion (L- and N-type VDCCs), we measured the miniature end-plate potential (mepp) frequency in mouse diaphragm muscles. 2. Blockade of VDCCs by Cd(2+) prevented the effect of the CCPA. Nitrendipine (an L-type VDCC antagonist) but not omega-conotoxin GVIA (an N-type VDCC antagonist) blocked the action of CCPA, suggesting that the decrease in spontaneous mepp frequency by CCPA is associated with an action on L-type VDCCs only. 3. As A(1) receptors are coupled to a G(i/o) protein, we investigated whether the inhibition of PKA or the activation of PKC is involved in the presynaptic inhibition mechanism. Neither N-(2[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89, a PKA inhibitor), nor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7, a PKC antagonist), nor phorbol 12-myristate 13-acetate (PHA, a PKC activator) modified CCPA-induced presynaptic inhibition, suggesting that these second messenger pathways are not involved. 4. The effect of CCPA was eliminated by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) and by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester epsilon6TDelta-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca(2+)-calmodulin. 5. To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K(+) concentrations. The effect of CCPA on ACh secretion evoked by 10 mm K(+) was prevented by the P/Q-type VDCC antagonist omega-agatoxin IVA. 6. CCPA failed to inhibit the increases in mepp frequency evoked by 15 and 20 mm K(+). We demonstrated that, at high K(+) concentrations, endogenous AD occupies A1 receptors, impairing the action of CCPA, since incubation with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A(1) receptor antagonist) and adenosine deaminase (ADA), which degrades AD into the inactive metabolite inosine, increased mepp frequency compared with that obtained in 15 and 20 mm K(+) in the absence of the drugs. Moreover, CCPA was able to induce presynaptic inhibition in the presence of ADA. It is concluded that, at high K(+) concentrations, the activation of A(1) receptors by endogenous AD prevents excessive neurotransmitter release.     

Interactions of lead and cadmium on acetylcholine release at the frog neuromuscular junction

The interactive effects of Cd2+ and Pb2+ on evoked and spontaneous transmitter release were studied in the sciatic nerve-sartorius muscle preparation of the frog (Rana pipiens). Either Pb2+ or Cd2+ competitively inhibited the actions of Ca2+ in bringing about evoked release, as measured by the endplate potential (EPP) amplitude. Combinations of Pb2+ and Cd2+ were additive in their effects on the EPP. The rate of spontaneous transmitter release was measured as the miniature endplate potential (MEPP) frequency. In contrast to their effects on the EPP, exposure of preparations to combinations of Pb2+ and Cd2+ actually increased the MEPP frequency less than exposures of the preparations to Pb2+ alone. The degree of reduction in MEPP frequency produced by Cd2+ depended upon the relative ratio of Pb2+ and Cd2+ ions. These results suggest that Pb2+ and Cd2+ ions competed for a common presynaptic receptor site during evoked release. They also suggest that Pb2+ ions may enter the nerve terminal, possibly through the Ca2+ channel, and that this entry is inhibited by Cd2+.     

Reversal of methylmercury-induced block of nerve-evoked release of acetylcholine at the neuromuscular junction

Acute bath application of micromolar concentrations of methylmercury (MeHg) blocks the nerve-evoked release of acetylcholine at the neuromuscular junction by presynaptic effects. The goal of the present study was to try to reverse this block of stimulus-evoked release. Experiments were conducted using the phrenic nerve hemidiaphragm of the rat and conventional intracellular microelectrode techniques. Myofibers were cut ("cut muscle") to prevent contractions elicited by stimulation of the phrenic nerve. End-plate potentials (EPPs) were recorded before and during MeHg application and during subsequent reversal attempts. MeHg (100 microM) blocked the EPP within 8-9 min of application. The time to block did not differ if Sr2+ (2 mM) was substituted for Ca2+ prior to exposure to MeHg. Washing the preparation with MeHg-free physiological saline at the time the EPP was blocked failed to reverse the block of synaptic transmission even during protracted washing. Increasing the extracellular [Ca2+] from 2 to 4 mM, or application of 4-aminopyridine (50 or 100 microM) failed to reverse block of the EPP. D-Penicillamine was also ineffective at reversing transmission block when applied at 0.4 mM; however, when applied at 1 mM D-penicillamine caused a return of EPPs in three of eight experiments within 5-20 min of wash. Longer periods of washing with D-penicillamine or use of higher concentrations of D-penicillamine were not effective in reversing transmission block in the refractory preparations. Increasing the intensity or duration of stimulation at the time of EPP block was uniformly successful in reversing MeHg-induced block; in 9 of the 10 preparations tested, EPPs could again be elicited from MeHg-blocked preparations merely by increasing the intensity and/or duration of stimulation, despite the continued presence of MeHg. Following reversal of transmission block by MeHg, continued exposure to MeHg resulted in a subsequent block of the EPP within approximately 3 min. During this subsequent block of the EPP by MeHg, increasing extracellular [Ca2+] or adding 4-aminopyridine did restore synaptic transmission. These results indicate that a temporary reversal of MeHg-induced block of synaptic transmission can be produced and that this effect does not require extracellular Ca2+ during the initial stages, but does seem to require extracellular Ca2+ during later stages.     

Factors influencing the twin-pulse facilitation of the release of transmitter at the mouse neuromuscular junction

1. The effects of several conditions and agents on the twin-pulse facilitation of the release of transmitter at the mouse neuromuscular junction in low-Ca²⁺ high-Mg²⁺ bathing solutions were examined.2. Twin-pulses gave two endplate potentials (epps) with first (m1) and second (m2) quantal contents. The ratio of m2/m1 was taken as a measure of the degree of facilitation.3. The mean value of this ratio was >1. Individual ratios fluctuated widely at junctions with smaller values of m1 but were focused around 1 at junctions with larger values of m1. Thus, some populations of junctions with smaller values of m1 contributed to an increment in the mean ratio.4. The mean ratio was virtually constant irrespective of changes in the spontaneous and evoked release of transmitter at temperatures between 20 and 36°C and at external concentrations of Ca²⁺ from 0.4 to 0.8 mM.5. 4-Aminopyridine(4-AP) slightly but significantly increased this ratio with increases in m1 and m2 at temperatures of 24 and 36°C. Ouabain slightly but significantly reduced the ratio, with increases in m1 and m2. The steadiness of the ratio was maintained in the presence of caffeine, high K⁺, neomycin or omega-conotoxin irrespective of changes in m1 and m2, except in the case of omega-contoxin.6. Spontaneous output at 36°C increased in the presence of 4-aminopyridine, ouabain, caffeine, high K⁺ or neomycin.7. These results indicate that maintenance of a stable value of the ratio of m2 to m1 is a dominant feature. The enhancement of the facilitation by 4-AP implies a contribution of another depolarization-dependent factor which may include a prolongation of the period of inflow of Ca²⁺ at the terminal.     

Effects of low ozone-oxygen concentrations on the acetylcholine release at the mouse neuromuscular junction

The use of a mixture of low concentrations of ozone (O3) with oxygen (O2) have been proved to be useful in different human pathological conditions. Owing to a lack of both pharmacological and epidemiological basic studies, the scientific consideration of this therapeutic potential is still inappropriate. Here, we started, from an electrophysiological point of view, a study on the possible effects of low O3 doses on the acetylcholine (ACh) release at the neuromuscular junction. Indeed, some experimental conditions indicate a positive effect either in maintaining cytosolic calcium (Ca2+) homeostasis or in increasing the efficacy of the intracellular antioxidant systems. Furthermore, a positive action on the kinetics of some antioxidant enzymes must be taken into account as a possible molecular mechanism in the regulation of the function of cellular homeostasis. Our data demonstrate a reduction of evoked ACh release in the mouse neuromuscular junction. O3 affects neither the spontaneous ACh release nor the kinetics of the ACh-receptor-channel complex. The results are compatible with a reduction of intracellular Ca2+ and proved a molecular action of O3.     

Pharmacologic inhibition of twin-pulse facilitation of release of transmitter quanta at the mouse neuromuscular junction

• 1.1. The frequency (F, s⁻¹) of miniature endplate potentials and the quantal content (m) of endplate potentials were simultaneously measured intracellularly at mouse diaphragm endplates in a bath solution that contained 0.6 mM Ca²⁺ ions and 5 mM Mg²⁺ ions.
• 2.2. Twin pulses at 4-ms intervals gave the quantal contents of the first (m1) and second (m2) responses. The ratio of m2/ml was taken as an indicator of the temporal facilitation of the release of transmitter.
• 3.3. Lead ions (Pb²⁺; 10 μM), bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA; loaded for 60 min at 200 μM), and chlortetracycline (CTC; loaded for 30 min at 80 μM) reduced the values of F and m2/ml. Pb²⁺ ions and CTC reduced the value of m, whereas BAPTA did not. ω-Agatoxin (ωAGT; 10 ng/ml) reduced the value of m without affecting F or m2/m1.
• 4.4. These results suggest that synaptic facilitation is modifiable by agents that can affect systems which buffer intracellular levels of Ca²⁺ ions.

     

Presynaptic acetylcholine action at the locust neuromuscular junction

The application, in the bathing medium, of ACh (10⁻⁵ M) to the extensor tibiae nerve-muscle preparation of the locust caused a dosedependent increase in both the spontaneous and evoked release of transmitter and also modified the statistical characteristics of the spontaneous release process. ACh had no postsynaptic action. The effects of ACh on spontaneous transmitter release were dependent on extracellular calcium concentration and could be mimicked by carbachol, nicotine and acetyl-β-methylcholine and antagonised by D-tubocurarine and decamethonium. The results suggest the presence of cholinergic receptors on the nerve terminal of the locust neuromuscular junction.     

Effects of some natural extracts on the acetylcholine release at the mouse neuromuscular junction.

Natural extracts have been proved to be useful in different human pathological conditions. The scientific consideration of the therapeutic potential of plant extracts is still inappropriate due to the lack of both pharmacological and epidemiological basic studies. Here, we started from an electrophysiological point of view, a study on the effects of two extracts on the acetylcholine (ACh) release at the neuromuscular junction. The extracts purified from Sugar cane (policosanol) and Psidium guajava (quercetin) have been submitted to this study. The wide epidemiology of these agents suggests therapeutic potentials not yet well outlined at the basic level. Our data demonstrate some interactions in the modulation of the ACh release at the mouse neuro-muscular junction, which are well correlated with the suggested molecular mechanisms. Policosanol enhances to a small extent either the spontaneous or the evoked ACh release. Furthermore, an increase of the rate of the conformational change induced at the nicotinic receptor-channel complex by ACh is also observed. Quercetin induced a reduction of the ACh evoked release. The possibility that this effect could be ascribed to some interaction with presynaptic calcium channel is noteworthy. The results are discussed in terms of a possible interference with acetylcholinesterase by policosanol and of a presynaptic molecular action of quercetin modulating the cytosolic calcium concentration.     

Increase of evoked release of acetylcholine at the neuromuscular junction by a fraction from the venom of the eastern green mamba snake (Dendroaspis angusticeps)

The effects of a fraction (DA3) isolated from the venom of the Eastern green mamba, Dendroaspis angusticeps, were tested in the toad sartorius muscle-sciatic nerve preparation using intracellular recording techniques. In magnesium-paralysed preparations, DA3 (0·5μg/ml and above) caused a slow, progressive increase in the quantal content of the endplate potential (e.p.p.); the largest increase measuring 40 times the control value. DA3 also altered the spontaneous release of acetylcholine, firstly reducing the frequency of miniature e.p.p.s (m.e.p.p.s) to 20–50% of control and then increasing the frequency to about four times control. In the presence of the toxin, very large spontaneous e.p.p.s were seen. Such potentials could be 10 times the amplitude of control m.e.p.p.s although their time course was similar to that of control m.e.p.p.s. In the absence of external Ca²⁺ ions and in the presence of 1 mM EGTA, DA3 still produced the biphasic change in m.e.p.p. frequency and the appearance of large spontaneous e.p.p.s. We conclude that DA3 increases the amount of acetylcholine released by each nerve stimulation, possibly by inducing the clumping of synaptic vesicles.     

Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

Background and Purpose

The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved.

Experimental Approach

End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques.

Key Results

Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A₃ receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A₃ receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd²⁺, the removal of extracellular Ca²⁺ and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the G_i/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62.

Conclusion and Implications

Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A₃ receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A₃ receptors appear to be coupled to G_i/o protein. PKC and calmodulin may be involved in these effects of inosine.     

Effects of diacylglycerol and phorbol ester on acetylcholine release and action at the neuromuscular junction in mice.

Miniature endplate potentials and nerve-evoked endplate potentials were studied in a phrenic nerve-diaphragm preparation of the mouse. A phorbol ester, phorbol-12 beta, 13 alpha-dibutyrate, and a diacylglycerol, 1-oleyl-2-acetyl-sn-glycerol, both increased the frequency of miniature endplate potentials. The effect of the phorbol ester on the frequency was still significant when the preparation was incubated in a calcium-deficient solution. The phorbol ester, but not the diacylglycerol, caused a significant increase in the amplitude of the miniature potentials. The phorbol ester also increased the amplitude of the endplate potentials in the presence of (+)-tubocurarine. The mechanisms of action of phorbol ester and diacylglycerol are discussed in relation to the control of acetylcholine release and action at the neuromuscular junction.     

Adenosine inhibits N-type calcium channels at the rat neuromuscular junction

1. In earlier studies, it has been reported that under in vitro conditions transmitter release at the rat neuromuscular junction is normally suppressed due to the effect of adenosine release from the isolated tissue. In the present study we wanted to determine whether this action may involve the inhibition of calcium influx through adenosine-sensitive calcium channels. 2. In order to test this hypothesis, we examined the role of N-type calcium channels in regulating nerve-evoked transmitter release by using the N-type calcium channel-specific blocker omega-conotoxin GVIA (CTX). In order to control the inhibitory action of adenosine, we also used the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). We tested the effect of blocking N-type calcium channels with CTX in the presence and absence of DPCPX. We examined the effects of these drugs on quantal transmitter release in the transected preparation of the phrenic nerve-hemidiaphragm of the rat using intracellular recording techniques. 3. At 10 nmol/L, CTX alone had no effect on nerve-evoked transmitter release; however, in the presence of 0.1 micro mol/L DPCPX, CTX significantly depressed nerve-evoked transmitter release. 4. These data support the view that adenosine inhibits nerve-evoked transmitter release by inhibiting N-type calcium channels on nerve terminals.     

Changes in neurotransmitter release at a neuromuscular junction of the lobster caused by cannabinoids

In vitro intracellular recording techniques were used on an excitatory neuromuscular junction of a walking-limb stretcher muscle of the lobster in order to define the synaptic pharmacology of delta-9-tetrahydrocannabinol (THC), 11-hydroxy-THC and cannabidiol. Delta-9-tetrahydrocannabinol and 11-hydroxy-THC, in relatively small concentrations, increased the amplitude of the excitatory junctional potential and the mean quantum content of a muscle fiber, whereas larger concentrations produced depression. In contrast, cannabidiol reduced the excitatory junctional potential and the mean quantum content. All three cannabinoids, however, depressed the amplitude of the spontaneous miniature junctional potential. The changes in mean quantum content point to a presynaptic site of action for the drug, while the reduction of the amplitude of the miniature junctional potential presumes a postsynaptic site. Such findings suggest synaptic mechanisms and sites of action for the central excitatory and depressant properties of the cannabinoids.     

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.     

Inositol derivatives modulate spontaneous transmitter release at the frog neuromuscular junction

One of the consequences of G-protein-coupled receptor activation is stimulation of phosphoinositol metabolism, leading to the generation of IP3 and its metabolites 1,3,4,5-tetrakisphosphate (IP4) and inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Previous reports indicate that high inositol polyphosphates (IP4 and IP6) are involved in clathrin-coated vesicular recycling. In this study, we examined the effects of IP4 and IP6 on spontaneous transmitter release in the form of miniature endplate potentials (MEPP) and on enhanced vesicular recycling by high K+ at frog motor nerve endings. In resting conditions, IP4 and IP6 delivered intracellularly via liposomes, caused concentration-dependent increases in MEPP frequency and amplitude. Pretreatment with the protein kinase A (PKA) inhibitor H-89 or KT 5720 reduced the IP4-mediated MEPP frequency increase by 60% and abolished the IP6-mediated MEPP frequency increases as well as the enhancement in MEPP amplitude. Pretreatment with antibodies against phosphatidylinositol 3-kinase (PI 3-K), enzyme also associated with clathrin-coated vesicular recycling, did not alter the IP4 and IP6-mediated MEPP frequency increases, but reduced the MEPP amplitude increase by 50%. In our previous reports, IP3, but not other second messengers releasing Ca2+ from internal Ca2+ stores, is able to enhance the MEPP amplitude. In order to dissociate the effect of Ca2+ release vs. metabolism to IP4 and IP6, we evaluated the effects of 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate (3F-IP3), which is not converted to IP4 or IP6. 3F-IP3 produced an increase then decrease in MEPP frequency and a decrease in MEPP amplitude. In elevated vesicle recycling induced by high K+-Ringer solution, IP4 and IP6 have similar effects, except decreasing MEPP frequency at a higher concentration (10(-4) M). We conclude that (1) high inositol polyphosphates may represent a link between IP3 and cAMP pathways; (2) the IP3-induced increase of MEPP amplitude is likely to be due to its high inositol metabolites; (3) PI 3-K is not involved in the IP4 and IP6-mediated MEPP frequency increases, but may be involved in MEPP size.