Effects Of Muscarinic Receptor Agonists Research Articles

The Role of T-Tubule Organization in Regulation of Intracellular Ca2+ Handling by Muscarinic Receptor Stimulation in Ventricular Myocytes

Muscarinic receptors expressed in cardiac myocytes play critical role in mediating regulation of heart function by the parasympathetic nervous system. How structural organization of cardiac myocytes affects muscarinic receptor mediated regulation of Ca2+ handling is not well defined. Using confocal Ca2+ imaging and patch-clamp techniques we studied the relationships between t-tubule density and cholinergic regulation of intracellular Ca2+ handling in normal murine ventricular myocytes and in myocytes in which the t-tubule system was disrupted by formamide treatment. Muscarinic receptor agonist CCh (10 μM) significantly inhibited amplitude of Ca2+ transients evoked by electrical field-stimulation in the presence of 100 nM isoproterenol (Iso), a beta-adrenergic agonist, in formamide-treated but not in control ventricular myocytes. Following Iso stimulation, formamide-treated cells exhibited reduced density of the inward Ca2+ current (ICa), decreased sarcoplasmic reticulum (SR) Ca2+ content, and enhanced fractional SR Ca2+ release when compared to control myocytes. CCh did not change SR Ca2+ content in Iso-stimulated formamide-treated myocytes but significantly reduced fractional release. This effect was associated with reduced Ca2+ release at coupled SR Ca2+ release sites and decreased Ca2+ sensitivity of the uncoupled release sites. CCh inhibited peak ICa recorded in a presence of Iso by ∼20 % in both control and detubulated ventricular myocytes. Reducing ICa amplitude by up to 40% by changing voltage step levels from 0 to −25 mV did not affect Ca2+ transients in control myocytes in the presence of Iso. In contrast, in formamide-treated myocytes similar decrease in ICa significantly diminished Ca2+ transients. We conclude that downregulation of the t-tubule system, as often observed in failing hearts, augments the inhibitory effects of muscarinic receptor agonists on Ca2+ transients by reducing functional reserve of excitation-contraction coupling.

Blockade by 4-Aminopyridine of the Muscarinic-receptor-mediated Responses of Guinea-pig Atria and Trachea

The potassium channel blocker 4-aminopyridine (4-AP) has been shown to antagonize the negative inotropic effects of muscarinic receptor agonists on atrial preparations. This is consistent with muscarinic agonists mediating their negative inotropy through the opening of potassium channels. In the present study, the possibility of a direct antagonism of the muscarinic receptor was examined by comparing the effects of 4-AP upon the responses to carbachol of isolated left atria (negative inotropy) and tracheal spirals (contraction) from reserpine pretreated guinea-pigs. The latter response is K+ channel-independent. The concentration-response curve for carbachol on the paced left atria was displaced 520-fold to the right by 4-AP (10 mM). 4-AP alone caused dose-related contractions of the tracheal spirals. Carbachol-induced contractions were, however, superimposed upon the raised tone and there were substantial rightwards shifts of the concentration-response curves of 4.7- and 31.4-fold by 1 and 10 mM of 4-AP, respectively. Thus 4-AP appears to have muscarinic receptor antagonistic blocking properties. The blockade of the atrial responses was, however, substantially greater and could be attributed to an additional blockade of muscarinic receptor-linked potassium channels. The negative inotropic responses of the A1-adenosine receptor agonist L-phenylisopropyladenosine (L-PIA) were also antagonized by 4-AP, but to a lesser extent than for carbachol. After allowing for the muscarinic receptor blocking activity of 4-AP, carbachol was still antagonized more effectively than L-PIA. This could be due to the presence of a K+ channel-independent component in the response to L-PIA which is not susceptible to 4-AP.

Antihyperalgesic effects of the muscarinic receptor ligand vedaclidine in models involving central sensitization in rats

It is well established that muscarinic cholinergic agonists produce antinociceptive effects in a number of acute pain models. However, relatively little is known about the effects of muscarinic receptor agonists in models which involve central sensitization in pain pathways. The purpose of the present studies was to evaluate the effects of vedaclidine, a muscarinic receptor mixed agonist/antagonist across receptor subtypes, in models involving central sensitization. Vedaclidine (0.3–10 mg/kg s.c.) produced dose-related antihyperalgesic effects in the formalin test as well as a dose-related reversal of capsaicin-induced mechanical hyperalgesia in rats. In the carrageenan test, vedaclidine (0.1–30 mg/kg) produced a dose-related reversal of both mechanical and thermal hyperalgesia that were antagonized by the muscarinic receptor antagonist scopolamine. In addition, the antihyperalgesic effects of vedaclidine in the carrageenan test were synergistic with the antihyperalgesic effects of the non-steroidal antiinflammatory drug ketoprofen, as demonstrated by isobolographic analysis. The present studies demonstrate that vedaclidine produces antihyperalgesic effects in models involving central sensitization, suggesting that vedaclidine, and potentially other muscarinic receptor agonists, may have clinical utility in the management of pain states involving central sensitization, such as neuropathic and inflammatory pain states.

Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus.

The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.

Effect of muscarinic receptor agonists on animal models of psychosis

The purpose of this study was to determine the effects of muscarinic receptor agonists on models of psychosis in rats. A battery of three tests, catalepsy (haloperidol-induced) augmentation, amphetamine-induced stereotypy antagonism and conditioned avoidance response were used. Both muscarinic receptor agonists physostigmine and pilocarpine produced similar results. They dose-dependently augmented the haloperidol-induced catalepsy, inhibited amphetamine-induced stereotypy behavior and inhibited the conditioned avoidance response. Results demonstrate that muscarinic receptor agonists have actions similar to those of dopamine receptor antipsychotic drugs in animal models, suggesting that muscarinic receptor agonists may be used as adjuvant drugs in the treatment of psychosis.

The effects of brucine and alcuronium on the inhibition of [3H]acetylcholine release from rat striatum by muscarinic receptor agonists.

1. Radioligand binding experiments indicate that the affinity of muscarinic receptors for their agonists may be enhanced by allosteric modulators. We have now investigated if brucine can enhance the inhibitory effects of muscarinic receptor agonists on the electrically evoked release of [3H]acetylcholine ([3H]ACh) from superfused slices of rat striatum. 2. The evoked release of [3H]ACh was inhibited by all agonists tested (i.e., furmethide, oxotremorine-M, bethanechol and oxotremorine). 3. Brucine enhanced the inhibitory effects of furmethide, oxotremorine-M and bethanechol on the evoked [3H]ACh release without altering the inhibitory effect of oxotremorine. 4. Alcuronium was applied for comparison and found to diminish the inhibitory effect of furmethide on the evoked [3H]ACh release. 5. The results demonstrate that it is possible both to enhance and diminish the functional effects of muscarinic receptor agonists by allosteric modulators. 6. The direction of the observed effects of brucine and alcuronium on [3H]ACh release fully agrees with the effects of these modulators on the affinities of human M4 receptors for furmethide, oxotremorine-M, bethanechol and oxotremorine, as described by Jakubik et al. (1997). This supports the view that the presynaptic muscarinic receptors responsible for the autoinhibition of ACh release in rat striatum belong to the M4 muscarinic receptor subtype.

Cyclic GMP-dependent protein kinase activation in the absence of negative inotropic effects in the rat ventricle.

1. It has been suggested that activation of cyclic GMP-dependent protein kinase (PKG) is a necessary step in the chain of events leading to the production of negative inotropy by muscarinic receptor agonists in mammalian ventricles, and that some cyclic GMP-elevating agents, such as sodium nitroprusside (SNP), fail to exert a negative inotropic effect because they elevate cyclic GMP levels in a pool that does not activate the kinase. This hypothesis was tested in the present study by monitoring the effects of carbachol, SNP and atrial natriuretic peptide (ANP) on contractility, cyclic GMP content and PKG activity in rat intact ventricular preparations and freshly isolated ventricular cardiomyocytes. 2. The presence of PKG in both the intact vehicle and in isolated ventricular cardiomyocytes was confirmed by MonoQ anion exchange chromatography and Western blotting. The elution profile indicated that the conditions of the PKG assay were selective for measuring PKG activity. 3. Carbachol induced a marked negative inotropic effect in intact, perfused hearts and ventricular strips in the presence of isoproterenol. The negative inotropic effect of carbachol was not associated with significant changes in cyclic GMP content or PKG activity in intact ventricular tissue, or in PKG activity in isolated cardiomyocytes. 4. SNP and ANP significantly increased cyclic GMP levels and activated PKG in intact ventricular preparations. Both drugs also activated PKG in isolated cardiomyocytes. However, neither drug had any negative inotropic effect in isoprenaline-stimulated perfused hearts and ANP did not change the contractility of isoprenaline-stimulated isolated cardiomyocytes. 5. The results of this study demonstrate that the negative inotropic effects of muscarinic receptor agonists can occur in the absence of significant activation of PKG. Conversely, marked increases in ventricular cyclic GMP content and PKG activity caused by SNP or ANP were not accompanied by a negative inotropic effect. 6. These results suggest that increases in cyclic GMP levels and activation of PKG do not play important roles in the regulation of rat ventricular contractility by muscarinic receptor agonists.

CGMP elevation does not mediate muscarinic agonist-induced negative inotropy in rat ventricular cardiomyocytes

Guanosine 3',5'-cyclic monophosphate (cGMP) has been suggested to be involved in the negative inotropic effects of muscarinic receptor agonists in beta-adrenergic receptor agonist-stimulated ventricular preparations. To test this hypothesis, changes in contractility induced by acetylcholine or carbachol, or the nitrovasodilator sodium nitroprusside (SNP), in the presence of 1 nM isoproterenol were measured in electrically stimulated rat ventricular cardiomyocytes. In parallel experiments, cardiomyocytes were treated with the same agonists, and cGMP and adenosine 3',5'-cyclic monophosphate (cAMP) levels were estimated. After 2 min, isoproterenol increased the magnitude of cell shortening by 60% and accelerated contraction and relaxation rates. Carbachol (1 and 10 microM) and acetylcholine (1 and 10 microM) inhibited the positive inotropic effects of isoproterenol, whereas SNP (10 and 100 microM) had no inotropic effect. All three agents increased cGMP levels but had no effect on isoproterenol-stimulated cAMP levels. SNP caused the largest elevations in cGMP. These results suggest that the negative inotropic effects of muscarinic agonists observed in isoproterenol-stimulated rat ventricular cardiomyocytes are not mediated by alterations in cGMP and/or cAMP levels.

Effects of muscarinic receptor agonists and anticholinesterase drugs on high voltage spindles and slow waves

The effects of muscarinic agonists (AF102B, pilocarpine, oxotremorine) and anticholinesterases (physostigmine, tetrahydroaminoacridine) were investigated on the incidence of thalamically generated rhythmic high voltage spindles and on scopolamine (0.2 mg/kg)-induced neocortical slow wave activity (i.e. increased sum amplitude value of the 1–20 Hz band in a quantitative electroencephalography (qEEG) analysis). AF102B and pilocarpine decreased high voltage spindles and scopolamine increased sum amplitude values at 3 and 9 mg/kg, but not at 1 mg/kg. Oxotremorine was less potent than AF102B or pilocarpine in suppressing high voltage spindles. Oxotremorine had no effect on the scopolamine-induced qEEG changes. Tetrahydroaminoacridine decreased high voltage spindles at 1, 3 and 9 mg/kg and slow waves at 9 mg/kg. Physostigmine decreased high voltage spindles and slow waves at 0.12 and 0.36 mg/kg. Based on the present results we propose that agonists possessing muscarinic M 1 receptor activity are effective in decreasing high voltage spindles and scopolamine-induced slow wave activity, but agonists showing predominant muscarinic M 2 receptor activity may be less effective in decreasing high voltage spindles and slow waves. Furthermore, tetrahydroaminoacridine decreased high voltage spindles at doses lower than those required to decrease scopolamine-induced slow waves. Physostigmine decreased high voltage spindles and slow waves over the same dose range. This result may indicate that non-cholinergic mechanisms are involved in the tetrahydroaminoacridine-induced decrease in high voltage spindles.

Effects of muscarinic receptor agonists and antagonists on rat brain serotonergic activity.

The effects of some muscarinic M1 and M2 receptor agonists and antagonists on rat brain serotonergic activity was assessed by noting their effects on the levels of 5-hydroxytryptamine (5-HT) and its major metabolite, 5-hydroxyindole acetic acid (5-HIAA), estimated by a high pressure-liquid chromatographic (HPLC) technique. The muscarinic M1 receptor agonists, arecholine and McN-A-343, and the M2 receptor agonists, gallamine and AF-DX 116, induced a dose-related decrease in the concentrations of both 5-HT and 5-HIAA. On the contrary, scopolamine and the selective M1 receptor antagonist, pirenzepine, increased the levels of the amine and its metabolite. The anti-cholinesterase agent, physostigmine, and the putative M2 receptor agonist, carbachol, induced a dose-related dual effect, with the smaller doses decreasing and the higher doses increasing 5-HT and 5-HIAA concentrations. The results indicate that an inverse relationship exists between the cholinergic and serotonergic neurotransmitter systems in the rat brain due to the likely presence of muscarinic heteroreceptors on serotonergic neurones. The data also indicates that though physostigmine and carbachol may function as M2 receptor agonists, they lose their receptor specificity on dose increment.

The effects of muscarinic receptor agonists L-389,660 and AF102B in tests of long term and working memory: G. R. Dawson, P. Bayley, S. Channell and S. D. Iversen. Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, U.K.

The effects of muscarinic receptor agonists L-389,660 and AF102B in tests of long term and working memory: G. R. Dawson, P. Bayley, S. Channell and S. D. Iversen. Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, U.K.

Effects of muscarinic receptor agonists and antagonists on dopamine-mediated behavioural paradigms

The effects of some muscarinic M1 and M2 receptor agonists and antagonists on two dopamine-mediated behavioural paradigms, namely post-swim grooming response and apormorphine-induced climbing behaviour, was investigated in mice. The M1 agonists and M2 receptor antagonists attenuated both paradigms, whereas the M1 receptor antagonists and M2 receptor agonists facilitated both behaviours. However, carbachol and physostigmine, exhibited a similar dose-dependent dual effect with the lower doses augmenting and the higher doses inhibiting both the behaviours, suggesting that the M2 agonist receptor specificity is lost on dose increment. The findings indicate that the central cholinergic muscarinic heteroreceptors, like the other muscarinic receptors, are functionally divisible into M1 and M2 receptor subtypes in their modulation of dopaminergic activity.

Effects of muscarinic receptor agonists and antagonists on swim-stress-induced 'behavioural despair' in rats

The effects of some selective agonists and antagonists of cholinergic muscarinic receptor subtypes on swim-stress-induced immobility (SI) was investigated in rats. This paradigm has been proposed to assess 'behavioural despair' in rodents as a laboratory model for clinical depression. All the rats were pre-treated with atropine ethoiodide i.p. to obviate any peripheral cholinergic influence. SI was only marginally less in intracerebroventricular (i.c.v.) cannulated rats administered artificial cerebrospinal fluid than in uncan nulated rats administered normal saline i.p. The drugs which do not have access across the blood-brain barrier were administered i.c.v. The muscarinic M(1) receptor agonists, arecholine and McN-A-343, and the non-specific muscarinic receptor agonist, oxotremorine, induced dose-related increases in SI, whereas the muscarinic M(1) receptor antagonists, scopolamine and pirenzepine, and the non-specific antagonist, atropine, produced significant decreases in SI. Carbachol, an M(2) receptor agonist, and physostigmine induced a dose- dependent dual effect, with lower doses attenuating and higher doses augmenting SI. The M(2) receptor antagonists, gallamine and AF-DX 116, increased SI. The data may be interpreted to suggest that muscarinic M(1) receptors may function to accentuate depression whereas muscarinic M(2) receptors may exert an inhibitory modulatory effect.

Effects of muscarinic receptor agonists and antagonists on α2-adrenoceptors in rat brain

The specific binding of [ 3H]clonidine to α 2-adrenoceptors on neural membranes isolated from six brain areas was determined with rats treated for various periods of time with the muscarinic agonists, oxotremorine or pilocarpine, or with the muscarinic antagonists atropine, atropine methyl nitrate, scopolamine and scopolamine methyl bromide. Administration of pilocarpine, 10 mg/kg, twice daily i.p. for 1 and 14 days increased markedly the number of α 2-adrenoceptors on neural membranes from all six brain areas. In contrast, oxotremorine, 0.3 mg/kg, twice daily i.p., for 7 days decreased the number of α 2-adrenoceptors on membranes from all brain areas except the brainstem and caudate nucleus. Both atropine and scopolamine increased the density of α 2-adrenoceptors in specific brain areas. Neither atropine methyl nitrate nor scopolamine methyl bromide had an appreciable effect upon the specific binding of [ 3H]clonidine to neural membranes from most brain areas.

Quantitative effects of some muscarinic agonists on evoked surface‐negative field potentials recorded from the guinea‐pig olfactory cortex slice

1. The effects of muscarinic receptor agonists on the electrically-evoked surface-negative field potential (N-wave) were measured in the guinea-pig olfactory cortex slice maintained in vitro. 2. Bath-superfusion of (+/-)-muscarine, acetylcholine (ACh), carbachol (CCh), or methacholine (MCh) (10-200 microM) produced reversible, dose-dependent depressions of the N-wave (ACh and MCh effects were observed in the presence of 10 microM neostigmine). The order of potencies (based on agonist dose causing 50% field depression: IC50) was: ACh greater than or equal to muscarine greater than CCh greater than MCh. All four agonists depressed the field potential by 100% at doses greater than 500 microM. 3. Pilocarpine and bethanechol were weak agonists and only produced measurable effects at high doses (1-2 mM). Neither agonist evoked a maximum response at doses up to 10 mM. 4. The muscarinic ganglion stimulant, McN-A-343 yielded inconsistent results, depressing the field potential in some slices, but having no effect in others. Pre-application of a conditioning dose (100 microM) of McN-A-343 reduced subsequent responses to CCh, suggesting possible partial agonist properties. 5. Oxotremorine (up to 100 microM) did not depress the field potential, but it reversibly antagonized the effects of CCh. 6. It is concluded that reproducible, quantifiable responses to muscarinic agonists can be evoked in the olfactory cortex slice. We suggest this preparation may be useful for conducting pharmacological studies of 'intact' central muscarinic receptors.

Pharmacological evidence for cardiac muscarinic receptor subtypes

The chronotropic and inotropic effects of muscarinic receptor agonists (Acetylcholine, Arecoline, Carbachol, Furtrethonium) and antagonists (Atropine, N-methyl and N-butyl scopolammonium, pirenzepine) on isolated guinea-pig atria were studied. All had a greater affinity constants for muscarinic receptors as assessed in terms of inotropic effects than in terms of chronotropic effects. This difference, well correlated with the pharmacological effect, suggests the occurrence of cardiac muscarinic receptor subtypes, one mediating heart rate and the other contractile force. The ratio of chronotropic to inotropic potencies for each agent shows that the physiological mediator, Acetylcholine, differentiates best between the two subtypes, while atropine is the least discriminatory.