K+ACh channel activation with carbachol increases atrial ANP release

Although it has been known that atrial natriuretic peptide (ANP) release is regulated through muscarinic acetylcholine receptors (mAChR), the mechanism by which this neurotransmitter regulates atrial ANP release is largely unknown. This study tested the hypothesis that K(+)(ACh) channels mediate the action of mAChR on atrial myocyte ANP release. Experiments were performed in perfused beating rabbit atria. Carbachol (CCh), an agonist of cardiac mAChR, increased atrial myocyte ANP release concomitantly with a decrease in stroke volume and intra-atrial pulse pressure in a concentration-dependent manner. Isoproterenol, a beta-adrenoceptor agonist, decreased ANP release concomitantly with an increase in cAMP and mechanical dynamics. In the presence of isoproterenol, the CCh-induced increase in ANP release and decrease in cAMP efflux levels and mechanical dynamics were able to be repeated. The CCh-induced changes were blocked by selective M(2) mAChR antagonists. Tertiapin, a selective G-protein-gated K(+)(ACh) channel blocker, attenuated the CCh-induced increase in ANP release and decrease in mechanical dynamics in a concentration-dependent manner, but without a significant effect on the CCh-induced decrease in cAMP efflux levels. The CCh-induced changes in ANP release and atrial dynamics were inhibited in the atria from pertussis toxin-pretreated rabbits. These findings demonstrate that G-protein-gated K(+)(ACh) channels regulate atrial myocyte ANP release. The present study also shows that mAChR and adrenoceptors have opposing roles in the regulation of ANP release.     

Purinergic Modulation of Hippocampal Acetylcholine Release Involves α-Dendrotoxin-Sensitive Potassium Channels

Modulation of acetylcholine (ACh) release from superfused hippocampal slices was examined when the release of ACh was stimulated by exposure of slices to elevated K+ concentration. Evoked release was not sensitive to inhibition by 0.1 microM tetrodotoxin, but it could be inhibited in a dose-dependent manner by a muscarinic agonist (10-100 nM oxotremorine) and a purinergic agonist (10-100 nM 2-chloroadenosine). The alpha-dendrotoxin (100 nM), which selectively blocks voltage-gated inactivating K+ channels in nerve endings, did not affect the release of ACh under resting or depolarized conditions. However, alpha-dendrotoxin reduced the 2-chloroadenosine-induced inhibition of release, but did not alter the oxotremorine-induced inhibition. These results suggest that an alpha-dendrotoxin-sensitive K+ channel may be activated as an obligatory step in the modulation of ACh release by presynaptic purinergic receptor activation, but not in the modulation by presynaptic muscarinic receptors.     

Differential inhibition of the release of endogenous and newly synthesized acetylcholine from Torpedo synaptosomes by presynaptic muscarinic receptors

Activation of Torpedo presynaptic muscarinic acetylcholine (ACh) receptors with the agonist oxotremorine (20 μM) results in the inhibition of Ca²⁺-dependent release of endogenous ACh from Torpedo synaptosomes. This effect is reversed by the muscarinic antagonist atropine (1 μM) which, by itself, has no effect. In contrast, under the same conditions the amount of newly synthesized radiolabeled [³H]ACh released is not affected by muscarinic ligands. These findings suggest that presynaptic muscarinic inhibition in the Torpedo is due to interference with the mobilization of ACh from a storage pool.     

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (V_max) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (B_max) and affinity (K_d) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.     

Newly Synthesized and Preformed Acetylcholine Are Released from Torpedo Synaptosomes by Different Pathways

In this study, we investigated the mechanisms underlying the release of preformed and of newly synthesized acetylcholine (ACh) from isolated Torpedo nerve terminals (synaptosomes). This was pursued by examining and comparing the effects of anticytoskeletal and anticalmodulin drugs and of activating the presynaptic muscarinic ACh receptors on the release of preformed endogenous ACh and of newly synthesized radiolabeled ACh. The anticytoskeletal drugs vinblastine, cytochalasin B, and colchicine inhibit the Ca2+-dependent K+-mediated release of newly synthesized radiolabeled ACh, but have no effect on the release of preformed ACh. By contrast, the muscarinic agonist oxotremorine markedly inhibits the release of preformed ACh, but has little effect on the release of newly formed ACh. Treatment of the synaptosomes with the calmodulin antagonist trifluoperazine inhibits the release of both ACh pools concomitantly. These findings show that preformed and newly synthesized ACh are released by different routes and suggest that their secretion is mediated by converging pathways. The significance of these results in view of the previously demonstrated preferential release of newly synthesized ACh is discussed.     

Possible existence of dopaminergic receptors on cholinergic nerve terminals in the guinea-pig Auerbach's plexus.

In the Auerbach's plexus of guinea-pig ileum lower concentrations of oxotremorine (Oxo-T) produced an increase, whereas higher concentrations of Oxo-T caused inhibition of evoked acetylcholine (ACh) release, measured by bioassay using dorsal muscle of leech. Dopamine inhibited the increase in evoked release of ACh induced by Oxo-T as a function of its concentration and this inhibitory effect was nullified in presence of pimozide, the dopamine receptor antagonist. The results demonstrate existence of presynaptic dopamine receptors having inhibitory influence on excitatory presynaptic muscarinic receptors on regulation of ACh release. However, no physiological role of dopamine could be observed on ACh release in this preparation.     

Acetylcholine stimulates cyclic ADP-ribose formation via M1 muscarinic receptors in rat superior cervical ganglion

The role of cyclic ADP-ribose (cADPR) as the downstream signal of neuronal muscarinic acetylcholine receptors (mAChRs) and the enzyme responsible for its synthesis, ADP-ribosyl cyclase, were examined in the rat superior cervical ganglion (SCG). Application of acetylcholine or other mAChR agonists increased the ADP-ribosyl cyclase activity by about 250-300% in crude membrane fractions from the SCG of 14-day-old rats. This effect was inhibited by atropine or by the M1-mAChR antagonist, pirenzepine, and was mimicked by GTP. These results indicate that the M1 mAChRs couple to the membrane-bound form of ADP-ribosyl cyclase and suggest that cADPR is a second messenger of M1 mAChR signaling in nervous tissue.     

Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors.

The role of muscarinic receptors in the down-regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10(-6) M) and atropine (10(-8) to 10(-6) M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10(-6) M) caused a slight depression in the amplitude of responses to ACh pressure-applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA-immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10(-6) M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10(-6) M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10(-6) M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse.     

Studies on the Characterization of the Subtype(S) of Muscarinic Receptor Involved in Prostacyclin Synthesis in Rabbit Cardiomyocytes

Abstract

The present study was conducted to localize and characterize the subtype(s) of muscarinic receptor involved in prostacyclin (PGI₂) production elicited by the cholinergic transmitter acetylcholine (ACh) in various cell types in the rabbit heart. ACh increased PGI₂ synthesis measured as 6-keto-PGF1α, in cultured coronary endothelial cells and freshly dissociated ventricular myocytes in a dose dependent manner but not in cultured coronary smooth muscle cells of rabbit heart. McN-A-343, a partially selective M₁ muscarinic ACh receptor (mAChR) agonist, did not alter 6-keto-PGF1α synthesis in these cell types. ACh induced 6-keto-PGF1α synthesis in coronary endothelial cells and ventricular myocytes was not altered by a low concentration (10^?8 M) of pirenzipine, an M₁ mAChR antagonist but was reduced by a higher concentration (10^?6 M). In coronary endothelial cells ACh induced 6-keto-PGF1α production was reduced by hexahydro-sila-difendial (HHSiD), an M₃ mAChR antagonist, and in ventricular myocytes by both 11-(2-[(di-ethylamino) methyl]-1-piperidinyl]acetyl-5,11-dihydro-6-H-pyrido-[2,3-b]-benzodiazepine-6 one] (AF-DX 116), an M₂ receptor antagonist, and HHSiD. The decrease by ACh of isoporterenol stimulated cAMP accumulation was minimized by AF-DX 116 but not by HHSiD or pirenzipine. Pertussis toxin treatment minimized ACh induced decrease in isoproterenol stimulated rise in cAMP and ATP release, but not ACh induced 6-keto-PGF1α synthesis. These data suggest that ACh stimulates prostacyclin production in coronary endothelial cells via M₃ mAChR and in ventricular myocytes M₂ and M₃ mAChR. Moreover, ACh induced decrease in cAMP, but not the increase in 6-keto-PGF1α production, is mediated by pertussis toxin sensitive Gαi proteins in these cells.     

The effects of muscarinic receptor blockers on the turnover rate of acetylcholine in various regions of the rat brain.

The actions of antimuscarinic agents (benztropine, trihexyphenidyl, and scopolamine) on the dynamics of acetylcholine (ACh) in central cholinergic neurons were examined in various rat brain areas. It was found that the pattern of changes in ACh turnover (TRACh) elicited by these drugs exhibited marked regional variations. After administration of the anticholinergic drugs, the TRACh in hippocampus and thalamus was increased, in cortex it was decreased, and in striatum it was unchanged. ACh concentration in the cortex and striatum was decreased while in hippocampus and thalamus ACh levels were unaltered. Further analysis of the cholinergic septo-hippocampal pathway using lesions of the fimbria-fornix and local drug injections into the septum argue against an in vivo action of these drugs on presynaptic or cell body muscarinic autoreceptors. Moreover, the data suggest that muscarinic receptor blockers cause an increased TRACh only in those areas where a feedback loop is operative, possibly by inhibiting a neuronal feedback loop involving at least one noncholinergic interneuron.     

Determination of Acetylcholine Release in the Striatum of Anesthetized Rats Using In Vivo Microdialysis and a Radioimmunoassay

A vertical-type in vivo microdialysis probe and a sensitive, specific radioimmunoassay (RIA) were used to study the mechanism of acetylcholine (ACh) release in the striatum of anesthetized rats. Without the use of physostigmine, a cholinesterase inhibitor, our RIA could still detect the amount of ACh present in the perfusate (5.6 +/- 0.6 fmol/min, n = 16). Tetrodotoxin (1 microM) produced a significant decrease in the amount of ACh collected in the perfusate, suggesting that basal ACh determined under the present experimental conditions was related to cholinergic neural activity. Atropine (0.1-1 microM) applied topically via the dialysis probe did not affect the amount of ACh recovered in the perfusate in the absence of physostigmine. Addition of physostigmine (10 microM) to the perfusion fluid produced about a 100-fold increase in the amount of ACh collected. In the presence of physostigmine, topical administration of atropine and pirenzepine (0.01-1 microM) through a dialysis probe produced a further three- to fourfold increase in ACh output, whereas a slight increase was produced by AF-DX 116 at the highest concentration (1 microM). These results indicate that presynaptic modulation of ACh release in the striatum does not occur under basal conditions, and that presynaptic M1 muscarinic receptors are involved in the modulation of ACh release when the ACh concentration is raised under certain conditions.     

The lymphocytic cholinergic system and its biological function

Lymphocytes are now known to possess the essential components for a non-neuronal cholinergic system. These include acetylcholine (ACh); choline acetyltransferase (ChAT), its synthesizing enzyme; and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Stimulating lymphocytes with phytohemagglutinin, a T-cell activator; Staphylococcus aureus Cowan I, a B-cell activator; or cell surface molecules enhances the synthesis and release of ACh and up-regulates expression of ChAT and M(5) mAChR mRNAs. Activation of mAChRs and nAChRs on lymphocytes elicits increases in the intracellular Ca(2+) concentration and stimulates c-fos gene expression and nitric oxide synthesis. On the other hand, long-term exposure to nicotine down-regulates expression of nAChR mRNA. Abnormalities in the lymphocytic cholinergic system have been detected in spontaneously hypertensive rats and MRL-lpr mice, two animal models of immune disorders. Taken together, these data present a compelling picture in which immune function is, at least in part, under the control of an independent non-neuronal lymphocytic cholinergic system.     

Human tenocytes are stimulated to proliferate by acetylcholine through an EGFR signalling pathway

Studies of human patellar and Achilles tendons have shown that primary tendon fibroblasts (tenocytes) not only have the capacity to produce acetylcholine (ACh) but also express muscarinic ACh receptors (mAChRs) through which ACh can exert its effects. In patients with tendinopathy (chronic tendon pain) with tendinosis, the tendon tissue is characterised by hypercellularity and angiogenesis, both of which might be influenced by ACh. In this study, we have tested the hypothesis that ACh increases the proliferation rate of tenocytes through mAChR stimulation and have examined whether this mechanism operates via the extracellular activation of the epidermal growth factor receptor (EGFR), as shown in other fibroblastic cells. By use of primary human tendon cell cultures, we identified cells expressing vimentin, tenomodulin and scleraxis and found that these cells also contained enzymes related to ACh synthesis and release (choline acetyltransferase and vesicular acetylcholine transporter). The cells furthermore expressed mAChRs of several subtypes. Exogenously administered ACh stimulated proliferation and increased the viability of tenocytes in vitro. When the cells were exposed to atropine (an mAChR antagonist) or the EGFR inhibitor AG1478, the proliferative effect of ACh decreased. Western blot revealed increased phosphorylation, after ACh stimulation, for both EGFR and the extracellular-signal-regulated kinases 1 and 2. Given that tenocytes have been shown to produce ACh and express mAChRs, this study provides evidence of a possible autocrine loop that might contribute to the hypercellularity seen in tendinosis tendon tissue.     

Determination of acetylcholine by inhibition of the specific binding of tritiated cis-methyldioxolane: application to the study of the effects of acetylcholine accumulation in a murine neuroblastoma culture

The radioreceptor assay for acetylcholine (ACh) is based on the ability of the ACh to compete with the specific binding of [3H] cis-methyldioxolane to muscarinic receptors of rat cerebral cortex membranes. The technique described was used to measure ACh levels in NS 20 cells treated with an organophosphorus compound. The down regulation of muscarinic acetylcholine receptors of neuroblastoma cells observed is probably related to ACh accumulation.     

The release of acetylcholine: from a cellular towards a molecular mechanism

The isolation of synaptic vesicles rich in acetylcholine (ACh) from the electric organ of Torpedo has indeed strengthened the hypothesis of transmitter exocytosis, but soon after it was found that non-vesicular free ACh was released and renewed upon stimulation. In contrast, vesicular ACh and the number of vesicles remained stable during physiological stimulations. In addition free ACh variations (representing the cytoplasmic pool) were correlated to the release kinetics as measured by the electroplaque discharge. Consequently, the mechanism releasing ACh from the cytoplasm in a packet form was searched at the presynaptic membrane itself. With synaptosomes isolated from the electric organ of Torpedo, it became possible to freeze them rapidly at the peak of ACh release and study their membrane and contents after cryofracture. A statistical analysis showed that the main structural change was the occurrence of large intramembrane particles at the peak of ACh release and under all release conditions. This impressive change contrasted with the stability in the number of vesicles. Another role for the vesicle was envisaged during intense stimulations when the cytoplasmic ACh and ATP pools become exhausted. The decrease in ATP leads to an increase in calcium and protons in the cytoplasm; this signals the depletion of vesicular ACh and ATP stores in the cytoplasm. Release can go on, while ATP promotes the uptake of calcium by vesicles. At the end of its cycle the vesicle will be full of calcium and will perhaps release it. As far as the mechanism of ACh release is concerned it probably depends on a membrane component (perhaps the large particles) activated by calcium and able to translocate ACh in a quantal or subquantal form. In most recent work we showed that if a lyophilized presynaptic membrane was used to make proteoliposomes filled with ACh, they released ACh upon calcium action.     

Effects of atropine on the release of newly synthesized acetylcholine from rat striatal slices at various concentrations of calcium ions

The release of acetylcholine (ACh) from brain tissue is known to be inhibited by muscarinic autoreceptors on cholinergic nerve terminals but the mechanism of the inhibition is not understood. Atropine brings about an increase of ACh release by removing the inhibitory action of autoreceptors. We investigated whether the effect of atropine on the release of [¹⁴C]ACh newly synthesized during incubations from [U-¹⁴C] glucose depends on the concentration of Ca²⁺ in the medium. In rat striatal slices incubated in the presence of an inhibitor of cholinesterases and of 30 mmol/l K⁺, significant increases in the release of [¹⁴C]ACh elicited by atropine were only observed during incubations with very low concentrations of Ca²⁺. This finding supports the view that the activation of presynaptic muscarinic autoreceptors in the brain affects the release of ACh by reducing the availability of Ca²⁺ that is required for transmitter liberation.     

Intracellular calcium release mediated by two muscarinic receptor subtypes

Four subtypes of muscarinic acetylcholine receptor (mAChR) were stably expressed in neuroblastoma-glioma hybrid cells (NG108-15). By combining fluorescent indicator dye (fura-2) studies with electrophysiological measurements it is shown that stimulation of mAChR I and mAChR III readily leads to release of calcium from intracellular stores and to associated conductance changes, whereas stimulation of mAChR II and mAChR IV exerts no such effect. Dose-response curves describing the amplitude or the delay of the calcium rise induced by acetylcholine suggest that the apparent affinity of mAChR III for its agonist is higher by about one order of magnitude than that of mAChR I. Ionic substitution experiments and current fluctuation analysis indicate that calcium activates a K⁺-specific conductance of ‘small’ single-channel amplitude similar to the SK type [1]. Furthermore, an outward current (M current) suppressed by activation of mAChR I and mAChR III has a single-channel amplitude corresponding to a conductance of approximately 3 pS.     

Simultaneous Release of Acetylcholine and ATP from Stimulated Cholinergic Synaptosomes

Abstract: The release of acetylcholine (ACh) and ATP from pure cholinergic synaptosomes isolated from the electric organ of Torpedo was studied in the same perfused sample. A presynaptic ATP release was demonstrated either by depolarization with KCl or after the action of a venom extracted from the annelid Glycera convoluta (GV). The release of ATP exhibited similar kinetics to that of ACh release and was therefore probably closely related to the latter. The ACh/ATP ratio in perfusates after KCl depolarization was 45; this was much higher than the ACh/ATP ratio in cholinergic synaptic vesicles, which was 5. The ACh/ATP ratio released after the action of GV was also higher than that of synaptic vesicles. These differences are discussed. The stoichiometry of ACh and ATP release is not consistent with the view that the whole synaptic vesicle content is released by exocytosis after KCl depolarization, as is the case for chromatin cells in the adrenal medulla.     

Depolarization affects the binding properties of muscarinic acetylcholine receptors and their interaction with proteins of the exocytic apparatus.

Membrane depolarization is the signal that triggers release of neurotransmitter from nerve terminals. As a result of depolarization, voltage-dependent Ca(2+) channels open, level of intracellular Ca(2+) increases. and release of neurotransmitter commences. Previous study had shown that in rat brain synaptosomes, muscarinic acetylcholine (ACh) receptors (mAChRs) interact with soluble NSF attachment protein receptor proteins of the exocytic machinery in a voltage-dependent manner. It was suggested that this interaction might control the rapid, synchronous release of acetylcholine. The present study investigates the mechanism for such a voltage-dependent interaction. Here we show that depolarization shifts mAChRs, specifically the m2 receptor subtype, to a low affinity state toward its agonists. At resting potential, mAChRs are in a high affinity state (K(d) of approximately 20 nM) and they shift to a low affinity state (K(d) of tens of microM) upon membrane depolarization. In addition, interaction between m2 receptor subtype and the exocytic machinery increases with receptor occupancy. Both phenomena are independent of Ca(2+) influx. We propose that these results may explain control of ACh release from nerve terminals. At resting potential the exocytic machinery is clamped due to its interaction with the occupied mAChR and depolarization relieves this interaction. This, together with Ca(2+) influx, enables release of ACh to commence.