Eugen E. Nikolsky

Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non‐quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non‐quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H‐effect) following blockade of skeletal muscle post‐synaptic nicotinic receptors by (+)‐tubocurarine and cholinesterase by armin (diethoxy‐p‐nitrophenyl phosphate). Glutamate was shown to inhibit non‐quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate‐induced decrease of the H‐effect was enhanced by glycine. Glycine alone also lowered the H‐effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N‐methyl‐d‐aspartate (NMDA) receptors with (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzocyclohepten‐5,10‐imine (MK801), dl‐2‐amino‐5‐phosphopentanoic acid (AP5) and 7‐chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate‐induced decrease of the H‐effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG‐nitro‐l‐arginine methyl ester (l‐NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H‐effect. The results suggest that glutamate, acting on post‐synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non‐quantal secretion of ACh from motor nerve terminals. Non‐quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non‐quantal transmitter following muscle denervation triggers the early post‐denervation depolarization of muscle fibres.

Acetylcholine and carbachol prevent muscle depolarization in denervated rat diaphragm

MUSCLE fibres of the rat diaphragm kept in a tissue culture medium became depolarized by 8–10 mV within 3 h after denervation. In the presence of carbachol (CB; 5 × 10−8 M), and acetylcholine (ACh; 5 × 10−8 M, the postdenervation depolarization was reduced. Both drugs were used in concentrations which mimicked the effect of non-quantal release of ACh. (+)Tubocurarine (TC) and ouabain did not prevent the protective action of CB, indicating that this effect is not mediated through ACh nicotinic receptors or the electrogenic Na+,K+pump. Addition of Mg2+, verapamil, diltiazem, nifedipine and Cd2+ in concentrations which block Ca2+ entry virtually inhibited the effect of both cholinomimetics. L-Nitroarginine methylester (NAME), an inhibitor of NO synthase, and haemoglobin, an extracellular scavenger of the NO radical, completely eliminated the protective effect of CB on post-denervation depolarization. The retrograde action of NO produced by cholinomimetics on nerve terminals is postulated.

Effect of N‐acetylaspartylglutamate (NAAG) on non‐quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

N‐Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non‐quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non‐quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H‐effect) caused by inhibition of nicotinic receptors by (+)‐tubocurarine and acetylcholinesterase by armin (diethoxy‐p‐nitrophenyl phosphate). Application of exogenous NAAG decreased the H‐effect in a dose‐dependent manner. The reduction of the H‐effect by NAAG was completely removed when N‐acetyl‐β‐aspartylglutamate (βNAAG) or 2‐(phosphonomethyl)‐pentanedioic acid (2‐PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane‐associated ectoenzyme that hydrolyzes NAAG to glutamate and N‐acetylaspartate. Bath application of glutamate decreased the H‐effect similarly to the action of NAAG but N‐acetylaspartate was without effect. Inhibition of NMDA receptors by dl‐2‐amino‐5‐phosphopentanoic acid, (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzocyclohepten‐5,10‐imine (MK801), and 7‐chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by NG‐nitro‐l‐arginine methyl ester and 3‐bromo‐7‐nitroindazole completely prevented the decrease of the H‐effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath‐applied NAAG, can modulate non‐quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.

End-plate currents evoked by paired stimuli in frog muscle fibres

Using voltage-clamp techniques spontaneously occuring miniature end-plate currents (mepc) and nerve-evoked end-plate currents (epc) were recorded in frog glycerol-treated or cut muscle preparations. Epcs were induced by pairs of stimuli (the delay of the 2nd stimulus, Δt being 6 ms–30 s; one pair was delivered every 60–90 s). The decay time constant of the epc (τepc) was longer, the larger its quantal content despite the presence of active acetylcholinesterase (AChE). After treatment with anticholinesterases (prostigmine or armin, an irreversible inhibitor) this increase in τepc became more pronounced. When AChE was fully active the decay of the 1st epc τ1 was slightly faster than the decay of the 2nd epc τ2 only when the interstimulus interval was rather short (Δt<20 ms). Following treatment with anticholinesterases this difference between τ2 and τ1 could be determined even when Δt was as long as 30 s. In anticholinesterase-treated preparations Δτ was found to be inversely proportional to log Δt: a 50% increase in the decay time-constant of the 2nd epc occurred with Δt=120 ms. During continuous stimulation (10 impulses/s) τepc increased from the 1st to the 5–6th responses, but then decreased in parallel with the fall in the epc amplidude. The phenomenon of postsynaptic potentiation we observed could be readily abolished when quantal content was decreased by the presence of magnesium ions, but it was relatively unaffected when the receptor density was decreased by α-bungarotoxin (α-BuTX).The possible existence is discussed of two kinds of repetitive binding of ACh molecules, first, to free cholinoreceptors (a process which could be inhibited by α-BuTX) and, second, to a complex of the cholinoreceptor plus one molecule of ACh (a process which is less sensitive to α-BuTX blocking action).

Effect of the desensitization-potentiating agent SKF-525A on frog and-plate currents

Abstract SKF-525A (10-6 and 10-5 mol·1-1) enhanced the desensitization of end-plate currents evoked by iontophoretically applied acetylcholine. No changes were found in the amplitude of spontaneous and nerve-evoked end-plate currents in the presence of the drug. SKF-525A has no effect on the time course and potential dependence of natural end-plate currents.

Non‐quantal release of acetylcholine from parasympathetic nerve terminals in the right atrium of rats

Acetylcholinesterase (AChE) inhibitors provoke typical cholinergic effects in the isolated right atrium of the rat due to the accumulation of acetylcholine (ACh). Our study was designed to show that in the absence of vagal impulse activity, ACh is released from the parasympathetic nerve fibres by means of non‐quantal secretion. The conventional microelectrode technique was used to study changes in action potential (AP) configuration in the right atrium preparation of rats during application of AChE inhibitors. Staining with the lipophilic fluorescent dye FM1‐43 was used to demonstrate the presence of endocytosis in cholinergic endings. The AChE inhibitors armin (10−7–10−5 m) and neostigmine (10−7 to 5 × 10−6 m) caused a reduction of AP duration and prolonged the cycle length. These effects were abolished by atropine and were therefore mediated by ACh accumulated in the myocardium during AChE inhibition. Putative block of impulse activity of the postganglionic neurons by tetrodotoxin (5 × 10−7 m) and blockade of ganglionic transmission by hexomethonium (2 × 10−4 m), as well as blockade of all forms of quantal release with Clostridium botulinum type A toxin (50 U ml−1), did not alter the effects of armin. Experiments with FM1‐43 dye confirmed the effective block of exocytosis by botulinum toxin. Selective inhibition of the choline uptake system using hemicholinium III (10−5 m), which blocks non‐quantal release at the neuromuscular junction, suppressed the effects of AChE inhibitors. Thus, accumulation of ACh is likely to be caused by non‐quantal release from cholinergic terminals. We propose that non‐quantal release of ACh, shown previously at the neuromuscular junction, is present in cholinergic postganglionic fibres of the rat heart in addition to quantal release.

Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism.

Nitric oxide (NO), previously demonstrated to participate in the regulation of the resting membrane potential in skeletal muscles via muscarinic receptors, also regulates non‐quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non‐quantal ACh release was estimated by the amplitude of endplate hyperpolarization (H‐effect) following a blockade of skeletal muscle post‐synaptic nicotinic receptors by (+)‐tubocurarine. The muscarinic agonists oxotremorine and muscarine lowered the H‐effect and the M1 antagonist pirenzepine prevented this effect occurring at all. Another muscarinic agonist arecaidine but‐2‐ynyl ester tosylate (ABET), which is more selective for M2 receptors than for M1 receptors and 1,1‐dimethyl‐4‐diphenylacetoxypiperidinium (DAMP), a specific antagonist of M3 cholinergic receptors had no significant effect on the H‐effect. The oxotremorine‐induced decrease in the H‐effect was calcium and calmodulin‐dependent. The decrease was negated when either NO synthase was inhibited by NG‐nitro‐l‐arginine methyl ester or soluble guanylyl cyclase was inhibited by 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one. The target of muscle‐derived NO is apparently nerve terminal guanylyl cyclase, because exogenous hemoglobin, acting as an NO scavenger, prevented the oxotremorine‐induced drop in the H‐effect. These results suggest that oxotremorine (and probably also non‐quantal ACh) selectively inhibit the non‐quantal secretion of ACh from motor nerve terminals acting on post‐synaptic M1 receptors coupled to Ca2+ channels in the sarcolemma to induce sarcoplasmic Ca2+‐dependent synthesis and the release of NO. It seems that a substantial part of the H‐effect can be physiologically regulated by this negative feedback loop, i.e., by NO from muscle fiber; there is apparently also Ca2+‐ and calmodulin‐dependent regulation of ACh non‐quantal release in the nerve terminal itself, as calmidazolium inhibition of the calmodulin led to a doubling of the resting H‐effect.

The effect of acetylcholine and related drugs on currents at the frog motor nerve terminal

Acetylcholine, acetylthiocholine, carbachol, suberyldicholine, propionylcholine, succinylcholine, methylfurmethide and F 2268 were tested on motor nerve ending currents recorded with an extracellular microelectrode. The isolated and transversally cut cutaneous pectoris muscle of frog Rana ridibunda was used. Only acetylcholine and acetylthiocholine affected the spike waveforms in a concentration-dependent manner. Lower concentrations (1-6 x 10(-4) M) prolonged the inward Na+ current and increased the outward K+ current at the proximal and central parts of the nerve terminal. Most remote parts of the terminal were not affected. At 7 x 10(-4) M and higher, both drugs further prolonged the Na+ current and eliminated the K+ component of the spike. The potentiating effect of acetylcholine and acetylthiocholine on the K+ phase of nerve terminal current disappeared after treatment with tetraethylammonium and 4-aminopyridine. The effect also disappeared when synaptic cholinesterase was inhibited by the anticholinesterases or by treatment with collagenase. Reactivation of cholinesterase by dipyroxime restored the facilitating effect of acetylcholine. Choline and slight acidification to pH 6.8 did not mimic the acetylcholine action on the terminal currents. Facilitation of the K+ current by acetylcholine was not calcium-dependent. The results indicate that lower acetylcholine concentrations inhibit the delayed rectifier only, whereas 7 x 10(-4) M and higher concentrations of acetylcholine depress all outward currents of the terminal.

Mechanisms of Cardiac Muscle Insensitivity to a Novel Acetylcholinesterase Inhibitor C-547

We compared the effects of the novel acetylcholinesterase (AChE) inhibitor C-547 on action potential configuration and sinus rhythm in the isolated right atrium preparation of rat with those of armin and neostigmine. Both armin (10−7, 10−6, and 10−5 M) and neostigmine (10−7, 10−6, and 5 × 10−6 M) produced a marked decrease in action potential duration and slowing of sinus rate. These effects were abolished by atropine and are attributable to the accumulation of acetylcholine in the myocardium. The novel selective AChE inhibitor C-547 (10−9 to 10−7 M), an alkylammonium derivative of 6-methyluracil, had no such effects. The inhibition constant of C-547 on cardiac AChE is 40-fold higher than that on extensor digitorum longus muscle AChE. These results suggest that C-547 might be employed to treat diseases such as myasthenia gravis or Alzheimer disease, without having unwanted effects on the heart.

Dependence of miniature endplate current on kinetic parameters of acetylcholine receptors activation: a model study.

Mathematical modeling was applied to study the dependence of miniature endplate current (MEPC) amplitude and temporal parameters on the values of the rate constants of acetylcholine binding to receptors (k+) when cholinesterase was either active or inactive. The simulation was performed under two different sets of parameters describing acetylcholine receptor activation–one with high and another with low probability (Pohigh and Polow) of receptor transition into the open state after double ligand binding. The dependence of model MEPC amplitudes, rise times, and decay times on k+ differs for set Polow and set Pohigh. The main outcome is that for set Pohigh, the rise time is significantly longer at low values of k+ because of the prolongation of ACh diffusion time to the receptor. For the set Polow, the rise time is shorter at low values of k1, which can be explained by the small probability of AChR forward isomerization after ACh binding and faster MEPCs peak formation.