Analysis of muscarinic cholinoceptors mediating phosphoinositide hydrolysis in guinea pig cardiac muscle

doi:10.1016/0922-4106(92)90089-E

European Journal of Pharmacology: Molecular Pharmacology

Volume 225, Issue 2, 13 February 1992, Pages 105-112

https://doi.org/10.1016/0922-4106(92)90089-E Get rights and content

Abstract

The muscarinic receptor mediating stimulation of PI hydrolysis in guinea pig atria and ventricles has been studied. The non-selective muscarinic agonist (+)-cis-dioxolane elicited this response, concentration-dependently, with a potency indicative of a low receptor reserve. The potency of a novel, M₂-selective agonist, L-660, 863 (−log EC₅₀ = 6.3, atria; 6.0, ventricles) was observed to be lower than its apparent affinity (−log K_A = 7.6) for M₂ receptors, indicating an action probably mediated by a population distinct from that producing negative inotropy in the same tissue. The inhibition of the response to (+)-cis-dioxolane by several muscarinic antagonists (atropine, pirenzepine, AF-DX 116, methoctramine, HHSiD and pFHHSiD) generated an affinity profile for this receptor also dissimilar to that described for the receptor mediating the classical cardiac M₂ response. Although no other muscarinic receptor mRNA has been detected in this tissue, these data suggest the presence of a second population of muscarinic sites, which may signify an M₂ receptor diversity.

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A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M₁ receptor were assessed functionally by a microphysiometer apparatus, while M₂, M₃, and M₄ receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M₁ and M₃ subtypes, while a general increase in this parameter is obtained at M₂ subtype. Among the agonists 2–18, compound 4 behaves as a full agonist with a preference for M₃ subtype. Moreover, compound 12 is inactive at M₁ and M₄ receptors while it displays a full agonist activity at M₂ and M₃ subtypes. Since demox displays a variable response on cardiac M₂ receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M₂ receptors. In presence of 10⁻¹¹ and 10⁻¹⁰ M demox, the binding of [³H]-NMS was increased by ≈ 30% as a consequence of an increase of the association of [³H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [³H]-NMS. Higher concentrations of demox decreased the binding of [³H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M₂ muscarinic receptor.
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In the mammalian heart, cardiac function is under the control of the sympathetic and parasympathetic nervous system. All regions of the mammalian heart are innervated by parasympathetic (vagal) nerves, although the supraventricular tissues are more densely innervated than the ventricles. Vagal activation causes stimulation of cardiac muscarinic acetylcholine receptors (M-ChR) that modulate pacemaker activity via I_fand I_K.ACh, atrioventricular conduction, and directly (in atrium) or indirectly (in ventricles) force of contraction. However, the functional response elicited by M-ChR-activation depends on species, age, anatomic structure investigated, and M-ChR-agonist concentration used. Among the five M-ChR-subtypes M₂-ChR is the predominant isoform present in the mammalian heart, while in the coronary circulation M₃-ChR have been identified. In addition, evidence for a possible existence of an additional, not M₂-ChR in the heart has been presented. M-ChR are subject to regulation by G-protein-coupled-receptor kinase. Alterations of cardiac M₂-ChR in age and various kinds of disease are discussed.
Role of nitric oxide production through M<inf>2</inf>-cholinergic receptors in cultured rat ventricular myocytes
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We previously reported that carbachol (CCh) caused the enhancement of NO production coinciding with negative chronotropy in cultured rat ventricular myocytes. In this study, we examined which subtype of muscarinic cholinergic receptor mediated these effects of CCh by measuring the NOx production with an HPLC-Griess reaction system and monitoring the beating with a Fotonic Sensor. The enhancement of NO production and negative chronotropy by 10⁻⁴M CCh stimulation were significantly inhibited by 10⁻⁶M atropine, 10⁻⁶M methoctramine, 3 × 10⁻⁴M L-NMMA, and 10⁻⁵M methylene blue. On the other hand, 10⁻⁶M pirenzepine and 10⁻⁶M HHSiD had no influence on the negative chronotropy by 10⁻⁴M CCh stimulation. Both 10⁻⁶M pirenzepine and 10⁻⁶M HHSiD suppressed the enhancement of NO production by 10⁻⁴M CCh stimulation slightly though not statistically. In addition, the m2 cholinergic receptor gene was expressed in our cell preparations, as demonstrated by reverse-transcriptase/PCR analysis. We concluded that M₂-cholinergic receptor-mediated negative chronotropy may be due in part to activation of the NO-signaling pathway in cultured rat ventricular myocytes.
Characterization of the positive and negative inotropic effects of acetylcholine in the human myocardium
1995, European Journal of Pharmacology
In the human isolated myocardium, acetylcholine (10⁻⁹ to 10⁻³ M) elicited a biphasic inotropic effect (a decrease in the lower and an increase in the higher concentration range) in atrial and a positive inotropic effect in ventricular trabeculae. However, under conditions of raised contractility achieved by exposure to noradrenaline (10⁻⁵ M), only negative inotropic effects were observed in both atria and ventricles. Atropine (10⁻⁶ M), but not propranolol (10⁻⁶ M), antagonized both positive and negative inotropic effects of acetylcholine, thus showing that the responses were mediated by muscarinic acetylcholine receptors. The use of subtype selective muscarinic receptor antagonists (10⁻⁷ to 10⁻⁵ M), pirenzepine (M₁ > M₃ > M₂), AF-DX 116 (11-({2-[(diethylamino)-methyl]-1-piperidyl}acetyl)-5,11-dihydro-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one base; M₂ > M₁ > M and HHSiD (p-fluorohexahydro-siladifenidol hydrochloride; M₃ ≥ M₁ ⪢ M₂) revealed that the negative inotropic effect of acetylcholine in atrial as well as the positive inotropic effect in ventricular trabeculae were best antagonized by AF-DX 116 and not by pirenzepine, suggesting the involvement of the muscarinic M₂ receptor subtype, possibly linked to different second messenger systems. On the other hand, the positive inotropic effect of acetylcholine (10⁻⁶ to 10⁻³ M) in the atrial tissue, observed only in preparation with depressed contractility, was not effectively antagonized by either AF-DX 116 or HHSiD, but was significantly reduced by pirenzepine. Furthermore, the selective muscarinic M₁ receptor agonist McN-A-343 (4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethyl ammonium chloride; 10⁻⁹ to 10⁻³ M), which failed to significantly change the baseline contractility in either atrial or ventricular trabeculae, produced a positive inotropic effect in atrial preparations when contractility had been depressed by prior treatment with acetylcholine (10⁻⁹ to 10⁻⁷ M). This effect of McN-A-343 was effectively antagonized by pirenzepine (10⁻⁵ M). These data show that, besides the muscarinic M₂ receptor mediating both negative (atria) and positive (ventricle) inotropic effects, muscarinic M₁ receptors, capable of reversing depressed atrial contractility, are present in the human heart.
Cholinergic contraction of the guinea pig lung strip is mediated by muscarinic M<inf>2</inf>-like receptors
1993, European Journal of Pharmacology
The muscarinic receptor subtype mediating contraction of the guinea pig lung strip preparation was investigated and compared with that in guinea pig tracheal and human peripheral airway (small bronchi) smooth muscle preparations, using a number of subtype selective muscarinic receptor antagonists. It was found that guinea pig lung strip contraction was not mediated by a homogenous class of muscarinic M₃ receptors, in contrast to guinea pig tracheal and human peripheral airway smooth muscle. The affinities of the M₁- and M₃/M₂-selective muscarinic receptor antagonists on the guinea pig lung strip were between 0.35 and 1.94 log units lower than in the M₃ receptor tissues (respective pA₂ values on guinea pig lung strip and trachea: pirenzepine 6.36/6.71, AF-DX 474 6.39/7.11, AQ-RA 721 6.93/7.96, DAU 5884 6.78/8.72, UH-AH 371 7.04/8.20), whereas the affinities of the M₂/M₃-selective antagonists were between 0.63 and 1.97 log units higher (AF-DX 116 6.63/6.00, AQ-RA 741 7.48/6.63, gallamine 5.44/3.47, methoctramine 7.30/5.38). As a result, a good correlation was obtained when pA₂ values from guinea pig lung strip were compared to pK_i values towards bovine cardiac muscarinic M₂ receptors, though it was noticed that pirenzepine and the M₃/M₂-selective antagonists showed a closer relationship than the M₂-selective compounds. These results suggest that cholinergic contraction of the guinea pig lung strip is mediated by muscarinic M₂-like receptors, possibly representing a novel subtype or a mixture of M₂ (cardiac) and M₃ (or M₄) subtypes. It remains to be established, however, on what structure in the lung these contractile M₂-like receptors are located and also by which transduction mechanism they produce contraction.
The interaction of McN-A-343 with muscarine receptors in cardiac and smooth muscle
1993, Biochemical Pharmacology
The interaction of the muscarine receptor partial agonist (4-m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) was investigated at muscarine receptors in the atria and taenia caeci of the guinea-pig to compare its interaction at the muscarine M₂ receptor in the two tissues. In the smooth muscle, the muscarine M₃ receptor subtype is responsible for the contractile response but the major subtype detected in binding or antibody experiments is the M₂ subtype. In guinea pig atria the dissociation constant of McN-A-343 at muscarine receptors was 15.2 μM determined in functional experiments on left atria in McEwen's solution or 14.8 μM in binding experiments with [³H]-(−)-quinuclidinyl benzilate ([³H]QNB) in the same medium containing 5′-guanylylimidodiphosphate (50 μM). In the taenia caeci, the dissociation constant estimated for McN-A-343 at the M₃ receptor from functional experiments based on the contractile response to the agonist in McEwen's solution was 4.6 μM. This value was similar to the dissociation constant (6.2 μM) estimated from binding studies versus [³H]QNB conducted in the same medium although studies with 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine 6-one (AF-DX 116) versus $[^{3} H]-(−)-N- methylscopolamine$ suggested that 70% of the receptors were the M₂ subtype. The presence of the M₂ subtype in the taenia caeci was also confirmed by the ability of exotremorine to inhibit the increase in cAMP produced by isoprenaline (10 μM) since apparent pK_B values for AF-DX 116 and hexahydrosiladiphenidol were 6.95 and 6.75, respectively. McN-A-343 (100 μM) failed to inhibit the response to isoprenaline and did not antagonize the inhibitory response to oxotremorine. It is concluded that the apparent affinity of McN-A-343 for muscarine M₂ receptors in the atria and the taenia caeci differs and a number of explanations are discussed.

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Analysis of muscarinic cholinoceptors mediating phosphoinositide hydrolysis in guinea pig cardiac muscle

Abstract

Eur. J. Pharmacol.

Life Sci.

Life Sci.

Eur. J. Pharmacol.

FEBS Lett.

Eur. J. Pharmacol.

FEBS Lett.

FEBS Lett.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

An M2 receptor subtype coupled to both adenylyl cyclase and phospho-inositide turnover

Science

Muscarinic cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells

Biochem. J.

Inositol phospholipid hydrolysis in rat cerebral cortical slices. I. Receptor characterisation

J. Neurochem.

Effects of inositol phosphates on Ca2+-sensitivity in skinned porcine cardiac muscle fibers

Naunyn-Schmied. Arch. Pharmacol.

Relationship between the inhibition constant (Ki) and the concentration of an inhibitor which caused 50 per cent inhibition (I50) of an enzymatic reaction

Biochem. Pharmacol.

Selectivity of muscarinic antagonists in radioligand and in vivo experiments for the putative M1, M2 and M3 receptors

J. Pharmacol. Exp. Ther.

Antagonist binding profiles of five cloned human muscarinic receptor subtypes

J. Pharmacol. Exp. Ther.

Muscarinic activity of McN-A-343 and its value in muscarinic receptor classification

Br. J. Pharmacol.

Negative and positive inotropic responses to muscarinic agonists in guinea pig and rat atria in vitro

J. Pharmacol. Exp. Ther.

A novel series of non-quaternary oxadiazoles acting as full agonists at muscarinic receptors

Br. J. Pharmacol.

Comparative studies of the postfunctional activities of some very potent muscarinic agonists

Naunyn-Schmied. Arch. Pharmacol.

Determination of dissociation constants and relative efficacies of some potent muscarinic agonists at postjunctional muscarinic receptors

Naunyn-Schmied. Arch. Pharmacol.

Characterization of the action of L-660,863 (3-(3-amino-1,2,4-oxadiazol-5-yl)-quinuclidine) at muscarinic receptors in vitro

Br. J. Pharmacol.

Muscarinic receptor subtypes

Annu. Rev. Pharmacol. Toxicol.

An M₂ receptor subtype coupled to both adenylyl cyclase and phospho-inositide turnover

Effects of inositol phosphates on Ca²⁺-sensitivity in skinned porcine cardiac muscle fibers

Relationship between the inhibition constant (K_i) and the concentration of an inhibitor which caused 50 per cent inhibition (I₅₀) of an enzymatic reaction

Selectivity of muscarinic antagonists in radioligand and in vivo experiments for the putative M₁, M₂ and M₃ receptors