Non-quantal release of acetylcholine in rat atrial myocardium is inhibited by noradrenaline.

In the mammalian myocardium, ACh, which is the main neurotransmitter of cardiac parasympathetic postganglionic fibres, can be released via both quantal (vesicular) and non-quantal (non-vesicular) mechanisms of secretion. Non-quantal release is continuous and independent of vagus activity and exocytosis of ACh-containing vesicles. During the incubation of myocardium in the presence of acetylcholinesterase (AChE) inhibitors, non-quantal ACh release leads to accumulation of ACh in the myocardium and cholinergic effects, which are proportional to the intensity of non-quantal secretion. The aim of the present study was to reveal whether non-quantal release of ACh can be modulated by another major cardioregulator, noradrenaline, or whether it represents uncontrolled leakage of ACh from cholinergic fibres. Cholinergic changes of electrical activity induced by the AChE inhibitor paraoxon (5 × 10(-6) M) in isolated rat right atrial preparations were determined by means of a standard microlectrode technique and used as a measure of the intensity of non-quantal release. Noradrenaline (10(-7) and 10(-6) M) substantially suppressed, but did not abolish, effects of paraoxon via stimulation of α-adrenoceptors, because all experiments were conducted in the presence of the ß-blocker propranolol (5 × 10(-6) M). A blocker of ganglionic transmission, hexamethonium bromide (10(-4) M), failed to alter the inhibitory effect of noradrenaline, indicating that only non-quantal ACh release is suppressed by this neurotransmitter. The effects of noradrenaline could be reduced by the α2-antagonist yohimbine (10(-6) M). However, both the α1-agonist phenylephrine (10(-6) M) and the α2-agonist clonidine (10(-6) M) significantly inhibited the cholinergic effects of paraoxon, indicating the possible involvement of both α-adrenoceptor subtypes in mediation of the adrenergic inhibition of non-quantal ACh release. Thus, cardiac non-quantal ACh release can be negatively regulated by noradrenaline, providing another facet of sympathetic-parasympathetic interaction in the heart.

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.

Bioelectrical activity in the heart of the lugworm Arenicola marina.

Standard microelectrode technique was used to study electrical activity of the isolated heart of the polychaete annelid, Arenicola marina. Typical pacemaker activity with slow diastolic depolarization was observed in all recordings. The average maximum diastolic potential (-58.4 +/- 3.2 mV), the average amplitude of the action potential (28.7 +/- 4.7 mV) and the average total duration of the action potential (2,434 +/- 430 ms) were determined. There has been no gradient of automaticity observed in our studies, which suggests that all regions of the Arenicola heart could possess pacemaker functions. Acetylcholine (ACh) produced a concentration dependent (5 x 10(-8)-5 x 10(-5) M) increase of the beating rate via increase in the rate of the diastolic depolarization. ACh (5 x 10(-5) M) increased beating rate by 2.5-fold compared to the control rate. A stronger action of ACh resulted in depolarization, block of action potential generation and contracture of the heart. The non-hydrolysable ACh analog carbacholine (10(-8)-10(-6) M) produced similar effects. All effects of ACh and carbacholine were abolished by 5 x 10(-6) M atropine. D-Tubocurarine (5 x 10(-5) M) did not significantly alter effects of ACh or carbacholine. Epinephrine (10(-8)-10(-6) M) caused the slowing of pacemaker activity and marked decrease of action potential duration. 10(-6) M epinephrine produced complete cardiac arrest. The effects of epinephrine were not significantly altered by the beta-blocker propranolol (5 x 10(-6) M). The beta-agonist isoproterenol (10(-7)-10(-5) M) and the alpha-agonist xylometazoline (10(-6)-10(-5) M) did not produce significant effects. Thus, cholinergic effects in the Arenicola heart are likely to be mediated via muscarinic receptors, while the nature of adrenergic effects needs further investigation.

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.

Cholinergic modulation of activation sequence in the atrial myocardium of non-mammalian vertebrates.

Cholinergic changes of electric activity were studied in isolated atrium preparations from fishes (cod and carp), amphibians (frog) and reptilians (lizard) using the microelectrode technique and high-resolution optical mapping. Perfusion of isolated atrium with acetylcholine (10(-6)-5.10(-5) M) caused gradual suppression of action potential generation and, eventually, completely blocked the excitation in a part of the preparation. Other regions of atrium, situated close to the sinoatrial and atrioventricular junctions, remained excitable. Such cholinergic suppression of electric activity was observed in the atrial myocardium of frog and in both fish species, but not in reptilians. Ba(2+) (10(-4) M), which blocks the acetylcholine-dependent potassium current (I(KACh)), prevented cholinergic reduction of action potential amplitude. In several preparations of frog atrium, cholinergic suppression of excitation coincided with episodes of atrial fibrillation. We conclude that the phenomenon of cholinergic suppression of electric activity is typical for atria of fishes and amphibians. It is likely to be caused by I(KACh) activation and may be important for initiation of atrial arrhythmias.