Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation

Jakubík, Ján; Tuček, Stanislav

doi:10.1111/j.1476-5381.1994.tb17170.x

Cited by 24 publications

(10 citation statements)

References 23 publications

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“…Radioligand Binding Experiments-Measurements of radioligand binding were performed essentially as described (15,22,23). Membranes corresponding to 600,000 cells were incubated at 25°C in a final incubation volume of 0.8 ml.…”

Section: For Details)mentioning

confidence: 99%

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

Jakubík

El‐Fakahany

Tuček

2000

Journal of Biological Chemistry

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Section: For Details)mentioning

confidence: 99%

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

Jakubík

El‐Fakahany

Tuček

2000

Journal of Biological Chemistry

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“…Radioligand binding and its changes in the presence of competitive and allosteric ligands were examined essentially as previously described (6,(18)(19)(20). Membranes corresponding to 600,000 cells were incubated at 25°in a final volume of 0.8 ml for the time periods indicated below.…”

Section: Reagents [mentioning

confidence: 99%

“…The incubation was terminated by filtration on Whatman GF/C glass-fiber filters in a Brandel cell harvester. (21)], were computed by nonlinear regression as previously described (6,18,19). Allosteric interaction between the binding of the allosteric ligands (A) and the unlabeled agonists (L) was evaluated from changes in the binding of [ 3 H]NMS occurring in the presence of a fixed concentration of the agonist and of increasing concentrations of the allosteric ligand; it was characterized by the cooperativity coefficient ␤.…”

Section: Reagents [mentioning

confidence: 99%

Positive Cooperativity of Acetylcholine and Other Agonists with Allosteric Ligands on Muscarinic Acetylcholine Receptors

Bačáková²,

et al. 1997

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SUMMARYIt is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M 1 -M 4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [ 3 H]Nmethylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (Ϫ)-eburnamonine on the M 2 and M 4 receptors and by brucine on the M 1 and M 3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M 1 , M 3 , and M 4 receptors, for pentylthio-TZTP on the M 2 receptors, and for arecoline on the M 3 receptors. (Ϫ)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M 2 receptors and for pilocarpine on the M 4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (Ϫ)-eburnamonine and pilocarpine and (Ϫ)-eburnamonine and oxotremorine-M on the M 2 receptors (25-and 7-fold increases in affinity, respectively) and between brucine and pentylthio-TZTP on the M 2 and brucine and carbachol on the M 1 receptors (8-fold increases in affinity). The discovery that it is possible to increase the affinity of muscarinic receptors for their agonists by allosteric modulators offers a new way to subtype-specific pharmacological enhancement of transmission at cholinergic (muscarinic) synapses.It has long been known that the affinity of muscarinic receptors for their agonists and antagonists can be diminished by compounds acting allosterically (for reviews, see Refs. 1-3). It was discovered, however, that the affinity of cardiac muscarinic receptors for the muscarinic antagonist NMS can be increased by the neuromuscular blocker alcuronium (4, 5). Subsequently, it was shown that the positive allosteric action of alcuronium on the binding of NMS is specific for the M 2 and M 4 muscarinic receptor subtypes (Ref.6, but see Refs. 7 and 8 for conflicting data concerning the M 3 and M 4 subtypes) and that it also applies to the binding ...

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“…The maximum effect (Emax) which a muscarinic ligand had on the measured parameter (the synthesis of cyclic [3H]AMP or of [3H]inositol phosphates) and the values of ECs0 (concentration of the ligand producing one half of the maximum effect) and nH (Hill slope factor) were computed by fitting the following equation [24,25] to the data:…”

Section: Data Analysis'mentioning

confidence: 99%

Constitutive activity of the M₁–M₄ subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists

et al. 1995

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We investigated whether muscarinic receptors of the MrM 4 receptor subtypes are constitutively active. We have found that the synthesis of cyclic AMP was enhanced by the muscarinic antagonists atropine and N-methylscopolamine (NMS) in Chinese hamster ovary (CHO) cells stably transfected with human m2 and m4 muscarinic receptor genes and in rat cardiomyocytes expressing the M2 receptor subtype, and that the production of inositul phosphates was inhibited by atropine and NVIS in CHO cells stably transfected with human ml and m3 and with rat ml muscarinic receptor genes. The muscarinic antagonists quinuclidinyl benzilate and AF-DX 116 had no effect in some cases and acted like atropine and NMS in others. We conclude that the M~-M 4 subtypes of muscarinic receptors are constitutively active in the CHO cell lines expressing them and in cardiomyocytes and that atropine and NMS act as negative antagonists on these receptor subtypes by stabilizing them in the inactive conformation.antagonists permitted the demonstration of the constitutive activity of ~ opioid receptors [7], c~2-adrenoceptors [9], fl2-adrenoceptors [10][11][12], 5-hydroxytryptaminezc receptors [13] and bradykinin receptors [14].The present report describes the effects of four muscarinic antagonists on two processes which are controlled by muscarinic receptors, namely the synthesis of cyclic AMP (normally inhibited by the M 2 and M 4 subtypes of muscarinic receptors) and the hydrolysis of phosphoinositides (normally activated by the Ml, M3 and M5 subtypes of muscarinic receptors -review [15]). No reports were available on the action of muscarinic antagonists applied alone on these processes by the time our experiments were started. Recently, however, Burstein et al.[16] described an inhibitory effect of atropine and pirenzepine on the hydrolysis of phosphoinositides in cells equipped with M3 muscarinic receptors and excess Gq protein.

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Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation

Cited by 24 publications

References 23 publications

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

Positive Cooperativity of Acetylcholine and Other Agonists with Allosteric Ligands on Muscarinic Acetylcholine Receptors

Constitutive activity of the M₁–M₄ subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists

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Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation

Cited by 24 publications

References 23 publications

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

Positive Cooperativity of Acetylcholine and Other Agonists with Allosteric Ligands on Muscarinic Acetylcholine Receptors

Constitutive activity of the M1–M4 subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists

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Constitutive activity of the M₁–M₄ subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists