Functional M3 cholinoreceptors are present in pacemaker and working myocardium of murine heart.

The presence of M3 cholinoreceptors and their role in mediation of action potential waveform modulation were determined by immunolabeling of receptor proteins and standard microelectrode technique, respectively. The sinoatrial node (SAN), which was determined as a connexin 43 negative area within the intercaval region, the surrounding atrial tissue, and the working ventricular myocardium exhibited labeling of both M3 and M2 receptors. However, the density of M3 and M2 labeling was about twofold higher in the SAN compared to working myocardium. The stimulation of M3 receptors was obtained by application of nonselective M1 and M3 muscarinic agonist pilocarpine (10(-5) M) in the presence of selective M2 blocker methoctramine (10(-7) M). Stimulation of M3 receptors provoked marked shortening of action potential duration in atrial and ventricular working myocardium. In the SAN, M3 stimulation leads to a significant reduction of sinus rhythm rate accompanied with slowing of diastolic depolarization and increase of action potential upstroke velocity. All electrophysiological effects of selective M3 stimulation were suppressed by specific blocker of M3 receptors 4-DAMP (10(-8) M). We conclude that M3 cholinoreceptors are present in pacemaker and working myocardium of murine heart, where they mediate negative cholinergic effects: slowing of sinus rhythm and shortening of action potentials.

Both neuronal and non-neuronal acetylcholine take part in non-quantal acetylcholine release in the rat atrium.

AIMS: In mammalian myocardium acetylcholine (ACh), neurotransmitter which strikingly affects the cardiomyocytes, can be released from the neurons both via quantal (vesicular) and nonquantal (non-vesicular) mechanism of secretion. Non-quantal release is continuous, independent on vagus activity and provides accumulation of ACh in myocardium in the presence of acetylcholinesterase (AChE) inhibitors. The aim of the present study was to determine the source of non-quantal ACh in isolated atrial myocardium of adult and newborn rats. MAIN METHODS: Standard microelectrode technique was used to determine the cholinergic changes of electrical activity under the action of AChE inhibitor paraoxon, which correlates with the intensity of nonquantal ACh release. KEY FINDINGS: In adult rats selective inhibitor of neuronal choline uptake system hemicholinium III (10(-5) M) decreased all effects of paraoxon (5 × 10(-6) M) more than twofold. Inhibitor of polyspecific 3 organic cation transporters corticosterone (10(-4) M) also significantly decreased effects of paraoxon in adult rats, indicating that non-neuronal ACh, which is synthesized by cardiomyocytes, takes part in accumulation of ACh in the myocardium. When hemicholinium III and corticosterone were applied together, paraoxon effects in adult atrial myocardium were suppressed almost completely. In newborn rats cardiomyocytes do not excrete ACh. In accordance with this fact hemicholinium III completely abolished effects of paraoxon in newborn myocardium, while corticosterone was ineffective. Thus, non-quantal ACh is released both from cholinergic nerves and cardiomyocytes in adult rat myocardium, while it has exclusively neuronal nature in newborns. SIGNIFICANCE: The study demonstrates dual neuronal and non-neuronal nature of non-quantal ACh in the heart.

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 x 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 x 10(7)m) and blockade of ganglionic transmission by hexomethonium (2 x 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.

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 x 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.

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 N(G)-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 Ca(2+) channels in the sarcolemma to induce sarcoplasmic Ca(2+)-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 Ca(2+)- 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.

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-beta-aspartylglutamate (betaNAAG) 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 N(G)-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.

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 k+, which can be explained by the small probability of AChR forward isomerization after ACh binding and faster MEPC's peak formation.

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.