Nitric oxide modulates intensity of non-quantal acetylcholine release in myocardium of the right atrium of rat

The main parasympathetic neurotransmitter acetylcholine (ACh) is released in the myocardium from the intramural postganglionic parasympathetic nerve endings. The mechanism of non-quantal ACh release has been recently demonstrated in these neurons. Non-quantal ACh release does not depend on exocytosis of ACh-containing vesicles in response to nerve impulse activity but is assumed to be mediated by the high-affinity choline uptake system. The intensity of non-quantal ACh release in the myocardium correlates with the degree of manifestation of the effects of acetylcholinesterase inhibitors inducing the accumulation of non-quantal ACh in the myocardium. The present study deals with the influence of putative modulators of non-quantal ACh release: nitric oxide (NO) and ATP, on the intensity of cholinergic effects induced by organophosphorous acetylcholinesterase inhibitor paraoxon. Intracellular registration of bioelectrical activity in isolated right atrium preparations from rats was used. Under normal conditions, paraoxon (10^?7–10^?5 M) induced a marked decrease in the action potential (AP) duration at a level of 50 and 90% repolarization in the working right atrial myocardium and slowed down the sinus rhythm. ATP, which is known to suppress nonquantal ACh release in the neuromuscular junction, did not induce significant reduction or augmentation of the effects of paraoxon (5 × 10^?6 M). The NO donors, sodium nitroprusside (10^?5 M) and SNAP (10^?4 M), significantly reduced the paraoxon-induced AP shortening. Moreover, sodium nitroprusside decreased the negative chronotropic effect of paraoxon by 43.7%. On the contrary, NO synthase inhibitor L-NAME (10^?4 M), which is known to suppress endogenous NO production, augmented the AP shortening caused by paraoxon. It may be deduced that NO is a universal regulator of non-quantal ACh release intensity both in the myocardium and in the neuromuscular junction.     

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.     

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.     

Spontaneous quantal and non-quantal release of acetylcholine at mouse endplate during onset of hypoxia

At 20 (0)C, both quantal and non-quantal spontaneous acetylcholine release (expressed as miniature endplate potential frequency [f-MEPPs] and the H-effect, respectively) increased during the first 30 min of hypoxia in solution with normal extracellular calcium ([Ca(2+)](o) = 2.0 mM). The hypoxia-induced tenfold increase of the f-MEPPs was virtually absent in low calcium solution([Ca(2+)](o) = 0.4 mM) whereas there was still a significant increment of non-quantal release. This indicates that each of these two processes of acetylcholine release is influenced by mechanisms with different oxygen sensitivity. The rise of f-MEPPs during the onset of hypoxia apparently requires Ca(2+) entry into the nerve terminal, whereas the non-quantal release can be increased by another factors such as a lower level of ATP.     

Effects of "non-quantal" acetylcholine on miniature endplate currents in mice

The possibility of postsynaptic potentiation (PSP) and desensitization developing due to nonquantal acetylcholine (ACh) secretion was investigated in mouse diaphragm with reference to time-amplitude relationships of miniature endplate currents (MEPC). The H effect (which characterizes nonquantal secretion (NS) of ACh) fell to zero over 3 h under the action of armine-induced inhibition of acetylcholinesterase (AChE) at a temperature of 20°C. A decline in the decay time constant () of MEPC unaccompanied by observable alteration in MEPC amplitude occurred at the same time. This accelerated decay of MEPC was not observed in the absence of NS (the early stages of denervation). Start of NS did not show any effect on maximum retardation of MEPC decay due to AChE inhibition, indicating that no PSP sets in under the effects of non-quantal secretion. The effect of decline in accelerated with a rise in temperature; it could be reproduced with neostigmine replacing armine, while remained unchanged in the time spells investigated with AChE in its active state. Non-quantal ACh is not thought to produce substantial retardation of MEPC decay, although it does bring about desensitization, signs of which may be partially masked owing to concurrent onset of PSP.S. V. Kurashov Medical Institute, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 507–513, July–August, 1990.     

Effects of "non-quantal" acetylcholine on postsynaptic membrane senstitivity: Effects of ouabain,and mimicry by exogenous acetylcholine

The development of postsynaptic potentiation (PSP) and desensitization due to "non-quantal" acetylcholine that occurs when acetylcholinesterase (AChE) is inhibited was studied using the Na,K-ATPase inhibitor, ouabain, to alter (initially increasing, then decreasing) the level of non-quantal acetylcholine secretion, and exogenous acetylcholine. When ouabain increased non-quantal secretion the time constant () of the miniature end-plate current (MEPC) decay increased, i.e., PSP developed. The later the application of ouabain relative to inhibition of AChE, the greater the degree of PSP. During the next phase when non-quantal secretion was inhibited the MEPC time course shortened more rapidly than in the controls, i.e., desensitization occurred. If ouabain abolished non-quantal secretion before AChE had been inhibited did not change, and neither PSP nor desensitization developed. When AChE was not inhibited ouabain had no effect on . When ACh was continuously applied at 20 nmol·liter^–1, similar to the nonquantal concentration, the shortening of slowed down, and the signal amplitude declined more rapidly than in controls. Addition of exogenous ACh (50 nmol·liter^–1) after acceleration of MEPC decay had developed caused to increase to its initial value. The combined appearance of PSP and desensitization during the action of non-quantal ACh, and the sustained desensitization after removal of released ACh from the synaptic cleft are discussed.S. V. Kurashova Institute of Medicine, Russian Federation Ministry of Public Health, Kzan. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 396–404, July–August, 1992.     

Compensatory mechanisms enhance hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase

Central cholinergic neurotransmission was studied in learning-impaired transgenic mice expressing human acetylcholinesterase (hAChE-Tg). Total catalytic activity of AChE was approximately twofold higher in synaptosomes from hippocampus, striatum and cortex of hAChE-Tg mice as compared with controls (FVB/N mice). Extracellular acetylcholine (ACh) levels in the hippocampus, monitored by microdialysis in the absence or presence of 10(-8)-10(-3) M neostigmine in the perfusion fluid, were indistinguishable in freely moving control and hAChE-Tg mice. Muscarinic receptor functions were unchanged as indicated by similar effects of scopolamine on ACh release and of carbachol on inositol phosphate formation. However, when the mice were anaesthetized with halothane (0.8 vol. %), hippocampal ACh reached significantly lower levels in AChE-Tg mice as compared with controls. Also, the high-affinity choline uptake (HACU) in hippocampal synaptosomes from awake hAChE-Tg mice was accelerated but was reduced by halothane anaesthesia. Moreover, hAChE-Tg mice displayed increased motor activity in novel but not in familiar environment and presented reduced anxiety in the elevated plus-maze test. Systemic application of a low dose of physostigmine (100 microgram/kg i.p.) normalized all of the enhanced parameters in hAChE-Tg mice: spontaneous motor activity, hippocampal ACh efflux and hippocampal HACU, attributing these parameters to the hypocholinergic state due to excessive AChE activity. We conclude that, in hAChE-Tg mice, hippocampal ACh release is up-regulated in response to external stimuli thereby facilitating cholinergic neurotransmission. Such compensatory phenomena most likely play important roles in counteracting functional deficits in mammals with central cholinergic dysfunctions.     

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.     

Structures of functional interest in the myocardium of lower vertebrates

• 1.1. The functional relevance of the diversity in myocardial organization among lower vertebrates has been elucidated by comparing special features of the ventricular wall and the sarcoplasmic reticular network in higher and lower vertebrates.
• 2.2. Temperature effects on the contraction-relaxation cycle in isolated atrial preparations of some poikilotherms (frog, trout, flounder, shark and hagfish) are reported, providing circumstantial evidence for functional adaptations in the mechanisms involved in calcium sequestration and energy production.
• 3.3. It is suggested that the poikilotherm hearts may serve as excellent, experimental models for elucidations of the plasticity of the vertebrate myocardium, notably of its ability to handle necrotic stimuli such as excessive adrenergic stimulation at high pCO₂ and low oxygen.

     

Effects of ambient temperature on brain acetylcholinesterase activity and protein content in three Egyptian vertebrates

Exposure to cold caused an increase in AChE activity of the different brain regions of both Arvicanthis and Columbia and a decrease in the enzyme activity of Scincus midbrain. Heat exposure provoked variable changes in AChE activity of the various brain regions of the three experimental species. The changes in AChE activities may be one of the mechanisms by which birds and mammals tend to acclimatize themselves to various forms of stress. In reptiles, AChE activity varied with changes in ambient temperature and this is probably due to the adaptive significance of thermally directed changes in enzyme substrate affinity.     

Effects of different secretagogues and intracellular messengers on the muscarinic modulation of [3H]acetylcholine release

We have investigated whether muscarinic receptors modulate the release of [³H]ACh elicited by secretagogues that act by different mechanisms in rat cerebral cortical synaptosomes. Oxotremorine (10 M) reduced the calcium-dependent [³H]ACh release induced by mild K⁺-depolarization (10 and 15 mM K⁺), but not that by higher K⁺ concentrations. The ACh-release induced by A23187 (0.2–5 g/ml), liposomes laden with 113 mM CaCl₂, or 4-aminopyridine (1–10 mM) was not modulated by oxotremorine. Ouabain (100 M)-induced release of [³H]ACh was reduced by oxotremorine in normal but not calcium-free KR, indicating that extracellular calcium-uptake but not Na⁺, K⁺-ATPase activity may be necessary for release-modulation. With respect to possible second messenger systems, dibutyrylcyclic AMP (0.1–2 mM), dibutyrylcyclic GMP (0.1–2 mM), forskolin (100 M), and phorbol ester (0.3–3 g/ml) were without effect on release or release-modulation. These results are consistent with an involvement of K⁺-channels and voltage-sensitive calcium-channels in the muscarinic release-inhibition process. They argue against an involvement of Na⁺, K⁺-ATPase, adenylate cyclase, guanylate cyclase, and phosphatidylinositol turnover in the release-modulation process.     

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. Ba²⁺ (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.     

Effects of acetylcholine on thyrotropin-releasing hormone release from the rat caecum in vitro

Effects of acetylcholine on the release of thyrotropin-releasing hormone (TRH) from the rat caecum in vitro were studied. The rat caecum was incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) (medium). The amount of TRH release into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat caecum was enhanced significantly in a dose-related manner with the addition of acetylcholine, but not changed with atropine. The stimulatory effect of acetylcholine on ir-TRH release from the rat caecum was blocked with an addition of atropine. Elution profile of acid-methanol-extracted rat caecum on Sephadex G-10 was identical to that of synthetic TRH. The findings suggest that the cholinergic system stimulates TRH release from the rat caecum in vitro.     

Potency of several oximes to reactivate human acetylcholinesterase and butyrylcholinesterase inhibited by paraoxon in vitro

Organophosphorus pesticides (e.g. chlorpyrifos, malathion, and parathion) and nerve agents (sarin, tabun, and VX) are highly toxic organophosphorus compounds with strong inhibition potency against two key enzymes in the human body—acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8). Subsequent accumulation of acetylcholine at synaptic clefts can result in cholinergic crisis and possible death of intoxicated organism. For the recovery of inhibited AChE, derivatives from the group of pyridinium or bispyridinium aldoximes (called oximes) are used. Their efficacy depends on their chemical structure and also type of organophosphorus inhibitor. In this study, we have tested potency of selected cholinesterase reactivators (pralidoxime, obidoxime, trimedoxime, methoxime and H-oxime HI-6) to reactivate human erythrocyte AChE and human plasma BuChE inhibited by pesticide paraoxon. For this purpose, modified Ellman's method was used and two different concentrations of oximes (10 and 100 μM), attainable in the plasma within antidotal treatment of pesticide intoxication were tested. Results demonstrated that obidoxime (96.8%) and trimedoxime (86%) only reached sufficient reactivation efficacy in case of paraoxon-inhibited AChE. Other oximes evaluated did not surpassed more than 25% of reactivation. In the case of BuChE reactivation, none of tested oximes surpassed 12.5% of reactivation. The highest reactivation efficacy was achieved for trimedoxime (12.4%) at the concentration 100 μM. From the data obtained, it is clear that only two from currently available oximes (obidoxime and trimedoxime) are good reactivators of paraoxon-inhibited AChE. In the case of BuChE, none of these reactivators could be used for its reactivation.