Modulation of the structure‐binding relationships of antagonists for muscarinic acetylcholine receptor subtypes
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1. Membranes from rat cerebral cortex, myocardium and extraorbital lacrimal gland were used as sources of M1, M2 and M3 muscarinic acetylcholine receptors respectively and the affinities of seven antagonists for the three subtypes were examined under different experimental conditions. 2. The affinities for the membrane-bound receptors were measured at different ionic strengths and temperatures and compared with those determined on the receptor solubilised in the neutral detergent digitonin or the zwitterionic detergent, CHAPSO. 3. The range of measured affinity constants of a given antagonist for a specific subtype varied from 2 (atropine at M1 receptors) to 1000 (AF-DX 116 at M2 receptors). 4. As a consequence of these changes in affinity, which were dependent on the drug, the subtype and the experimental conditions, both the structure-binding relationships of a given subtype can be markedly changed as well as the selectivity of a drug for the different subtypes. For example it is possible to change the relative affinities of AF-DX 116 and gallamine at membrane-bound M1 receptors from 50:1 to 1:60. 5. Experimental conditions for the observation of high selectivity of pirenzepine, AF-DX 116, gallamine and hexahydrosiladiphenidol for the three subtypes are given. 6. When the receptors are removed from their membrane environment by solubilisation in detergent, antagonist affinities are changed but the subtypes still retain different structure-binding relationships. 7. In general, AF-DX 116 and the allosteric antagonist, gallamine, behave differently from the other antagonists, suggesting that they bind in different ways to muscarinic receptors. Careful attention should therefore be paid to the experimental conditions in binding assays used to assess the affinities and selectivities of new muscarinic antagonists in order to avoid misleading results. 9. The ability to produce enhanced or attenuated affinities and selectivities of antagonists, resulting from the induction of different conformations of the receptor by a variety of physical, chemical or molecular biological perturbations may lead to a better understanding of the structural basis of drug receptor interactions.Keywords:
Affinities
Competitive antagonist
Muscarinic antagonist
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These studies were undertaken to investigate the selectivity of cortical muscarinic receptor radioligand binding in muscarinic M(1) and M(4) receptor knockout mice and to determine whether a marked decrease in [(3)H]pirenzepine binding in Brodmann's area (BA) 9 from a subset of people with schizophrenia was predictive of decreased muscarinic receptors in other central nervous system (CNS) regions. Our data show that, under the conditions used, [(3)H]pirenzepine binding was highly selective for the muscarinic M(1) receptor whereas both [(3)H]AF-DX 386 and [(3)H]4DAMP had less discriminatory power. In addition, the data suggest that a marked decrease in [(3)H]pirenzepine binding in BA 9 from a subset of people with schizophrenia is predictive of decreases in muscarinic receptors in other CNS regions. However, there were some region-specific decreases in muscarinic receptors in tissue from people with schizophrenia who were outside this subset. These data add to a growing body of evidence suggesting there are widespread decreases in muscarinic receptors in the CNS of some subjects with schizophrenia, as demonstrated by neuroimaging. Our data have implications for understanding the potential clinical utility of drugs directed at the orthosteric and allosteric sites of muscarinic receptors to treat schizophrenia.
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The muscarinic receptor mediating vasodilatation of the rabbit aorta and dog femoral artery has been assessed using muscarinic antagonists. With the exception of pirenzepine, the antagonist affinities were similar to those reported for the ileal receptors and dissimilar to those reported for the atrial receptors. Pirenzepine exhibited an affinity (7.54) intermediate between that reported for the CNS receptors (8.4) and that reported for the ileal receptors (6.77). This value for pirenzepine was confirmed using acetylcholine as the agonist and using the dog femoral artery as the vascular tissue. It is concluded that the muscarinic receptor profile mediating vasodilatation is not easily accommodated into the current receptor classification.
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Methoctramine
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A family of five cholinergic muscarinic receptor genes (m1, m2, m3, m4, and m5) has recently been identified and cloned. In order to investigate the pharmacological properties of the individual muscarinic receptors, we have transfected each of these genes into Chinese hamster ovary cells (CHO-K1) and have established stable cell lines expressing each receptor. In the present study we have examined the antagonist binding properties of each muscarinic receptor. Antagonists were chosen that had previously been proposed to be selective for muscarinic receptor subtypes and included pirenzepine, AF-DX 116, methoctramine, dicyclomine, hexohydrodifenidol, hexahydrosiladifenidol, hexocyclium, and silahexocyclium. m1, m2, and m3 receptors express binding properties similar to those expected of high affinity pirenzepine-type receptors of cerebral cortex ("M1"), low affinity pirenzepine-type receptors of atria ("M2 cardiac type"), and the intermediate affinity pirenzepine-type receptors found in exocrine glands ("M2 glandular type"), respectively. The M1/M2 schema cannot readily accommodate the binding properties of the m4 and m5 receptors. Pirenzepine, methoctramine, and hexahydrosiladifenidol were the most selective agents for the m1, m2, and m3 receptors, respectively. None of the antagonists used in this study were uniquely selective for either the m4 or m5 receptors. The diverse binding profiles of individual cloned receptors and the widespread distribution of m1-m4 mRNAs indicate that radioligand binding studies performed on primary tissues may actually be assessing the composite properties of a heterogeneous mixture of muscarinic receptor subtypes.
Methoctramine
Muscarinic antagonist
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Methoctramine
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This study was undertaken to assess the important muscarinic receptor subtype in acetylcholine (ACh)-induced rat bronchial smooth muscle contraction. Ring smooth muscle strips of the left main bronchus were used. Isometrical contraction was measured in response to ACh in cumulative concentrations (10(-7)-10(-3) M) with and without preincubations with the muscarinic receptor antagonists, pirenzepine (an M1 antagonist), methoctramine (an M2 antagonist), and 4-diphenylacetoxy N-methylpiperidine (4-DAMP; an M1/M3 antagonist). Preincubation with these antagonists resulted in concentration-dependent rightward shifts of the concentration-response curves to ACh. pA2 values (means+/-sem) were 8.80+/-0.10 for 4-DAMP, 7.03+/-0.06 for pirenzepine and 5.91+/-0.36 for methoctramine, indicating that the most important muscarinic receptor mediating ACh-induced contraction of rat bronchial smooth muscle is of the M3 type.
Methoctramine
Muscarinic antagonist
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1. Kinetic, saturation and competition binding studies were conducted on the muscarinic receptor binding site labelled by [3H]-N-methylscopolamine [( 3H]-NMS) in intact PC12 cells and cell membranes. Similar studies were conducted on M1 receptors of rat cortex labelled with [3H]-pirenzepine and M2 and M3 receptors present in rat heart and submaxillary gland respectively, and labelled with [3H]-NMS. 2. The dissociation of [3H]-NMS from muscarinic receptors in PC12 cells was slower than dissociation from both M2 and M3 muscarinic receptors. 3. The Kd of [3H]-NMS in the PC12 cells was significantly lower than that obtained at the M2 and M3 receptor. 4. In competition studies the affinity data for pirenzepine, hexahydroadiphenine and 4-diphenylacetoxy-N-methylpiperidine methiodide were consistent with the presence of an M3 receptor in the PC12 cells. However, for AF-DX 116, cyclohexylphenyl(2-piperidinoethyl)silanol and methoctramine affinity estimates in PC12 cells were 3-6 fold lower than at the M3 receptor. 5. On the basis of these data we conclude that the muscarinic receptor present in the PC12 cells differs from the M1, M2 and M3 subtypes already described.
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Methoctramine
Oxotremorine
Muscarinic agonist
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Methoctramine
Muscarinic antagonist
Muscarinic agonist
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