We have recently developed methods for estimating the active (Kact) and inactive (Kinact) receptor‐state affinity constants of ligands in functional studies on G protein‐coupled receptors. Our approach is valid for in vitro responses... more
We have recently developed methods for estimating the active (Kact) and inactive (Kinact) receptor‐state affinity constants of ligands in functional studies on G protein‐coupled receptors. Our approach is valid for in vitro responses measured downstream from receptor activation, such as second messenger signaling in cell lines and responses in isolated tissues. The data required for our analysis include agonist‐mediated responses measured in the absence and presence of reduced receptor expression and modulation with an allosteric agonist (J Pharmacol Toxicol Methods 69:253–279, 2014). We also described an analogous technique that employs a constitutively active receptor mutant (J Pharmacol Toxicol Methods 83:94–106, 2017) instead of an allosteric agonist. In this study, we sought to validate our functional determination of Kact by using an independent biophysical method for estimating Kact. It is well known that muscarinic agonists exhibit high affinity for a subset of M2 muscarinic...
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A functional role for the M2 muscarinic receptor in smooth muscle contraction was investigated in isolated guinea pig ileum. Contractile responses to the muscarinic agonist oxotremorine-M (oxo-M) were measured in isolated ilea that had... more
A functional role for the M2 muscarinic receptor in smooth muscle contraction was investigated in isolated guinea pig ileum. Contractile responses to the muscarinic agonist oxotremorine-M (oxo-M) were measured in isolated ilea that had been pretreated with histamine (0.32 microM) and isoproterenol (0.64 microM) to achieve conditions of elevated cAMP. The resulting concentration-effect curve was biphasic, consisting of high (0-50 nM) and low (> 50 nM) potency components. The reversible M2-selective antagonist AF-DX 116 ([[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one) (1 and 10 microM) shifted this curve in a manner that was inconsistent with competitive antagonism at a single receptor site; the high affinity component was significantly blocked, whereas there was little effect on the low affinity portion of the curve. To inactivate the M3 muscarinic receptors selectively, ilea were incubated with the irreversible M1/M3-selective muscarinic antagonist 4-DAMP mustard [N-(2-chloroethyl)-4-piperidinyldiphenylacetate] (40 nM) for 1 hr in the presence of AF-DX 116 (1 microM) and were then washed extensively. Under these conditions, the contractile responses to oxo-M, in the presence of histamine and isoproterenol or forskolin, were antagonized by AF-DX 116 (1 microM) in a manner consistent with that mediated by an M2 receptor. AF-DX 116 caused 6.6- and 11-fold increases in the EC50 value for oxo-M for ilea pretreated with isoproterenol and forskolin, respectively, and a significant increase in the Hill coefficient in both cases. Under basal conditions, AF-DX 116 caused only a 1.34-fold increase in the EC50 value and no change in the Hill coefficient. In addition, under basal conditions 4-DAMP mustard treatment shifted the oxo-M contractile response curve to the right approximately 20-fold. However, when histamine was present in combination with isoproterenol or forskolin 4-DAMP mustard treatment shifted the concentration-effect curves for oxo-M to the right only about 3.5-fold. Oxo-M produced an M3-mediated stimulation of phosphoinositide hydrolysis in the longitudinal muscle of rat ileum with an EC50 value of 30 microM. 4-DAMP mustard (10 nM; 1 hr) prevented this response, resulting in a 6.6-fold increase in the EC50 value with a 65% reduction of the maximal response. In contrast, this treatment blocked M2-mediated inhibition of isoproterenol-stimulated adenylate cyclase with only a 2-fold increase in EC50, without affecting maximum inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)
Research Interests: Endocrinology, Chemistry, Molecular Pharmacology, Medicine, Histamine, and 15 moreInternal Medicine, cyclic AMP, Animals, Male, Antagonist, Parasympatholytics, Muscle contraction, Hydrolysis, Neurosciences, Piperidines, Agonist, Guinea pigs, Pharmacology and pharmaceutical sciences, Muscarinic Agonist, and In Vitro Techniques
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The M2 muscarinic receptor inhibits the development of streptozotocin-induced neuropathy in mouse urinary bladder
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Research Interests: Medicine and Anxiolytic
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Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [50]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously... more
Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [50]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously expressed in the human body and are therefore attractive targets for many disorders. Functionally, M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins, whilst M2 and M4 mAChRs predominantly couple to Gi/o proteins. Both agonists and antagonists of mAChRs are clinically approved drugs, including pilocarpine for the treatment of elevated intra-ocular pressure and glaucoma, and atropine for the treatment of bradycardia and poisoning by muscarinic agents such as organophosphates.
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Muscarinic acetylcholine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [45]) are GPCRs of the Class A, rhodopsin-like family where the endogenous agonist is acetylcholine. In... more
Muscarinic acetylcholine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [45]) are GPCRs of the Class A, rhodopsin-like family where the endogenous agonist is acetylcholine. In addition to the agents listed in the table, AC-42, its structural analogues AC-260584 and 77-LH-28-1, N-desmethylclozapine, TBPB and LuAE51090 have been described as functionally selective agonists of the M1 receptor subtype via binding in a mode distinct from that utilized by non-selective agonists [243, 242, 253, 155, 154, 181, 137, 11, 230]. There are two pharmacologically characterised allosteric sites on muscarinic receptors, one defined by it binding gallamine, strychnine and brucine, and the other defined by the binding of KT 5720, WIN 62,577, WIN 51,708 and staurosporine [161, 162].
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The specific binding of [3H] antagonists to muscarinic receptors from neuronal tissue has been demonstrated by several investigators (6,19,24,28,32,37). In each case, the binding of [3H] antagonists is consistent with the law of mass... more
The specific binding of [3H] antagonists to muscarinic receptors from neuronal tissue has been demonstrated by several investigators (6,19,24,28,32,37). In each case, the binding of [3H] antagonists is consistent with the law of mass action for a single class of independent receptors. Similarly, the results of antagonist/ [3H] antagonist competition experiments are also consistent with the law of mass action (23). Perhaps the most convincing evidence demonstrating that the binding of [3H] antagonists represents a specific interaction with the muscarinic receptor is the excellent quantitative agreement between the values of dissociation constants determined by binding experiments and by antagonism of agonist induced contractions of the guinea pig ileum (8,10).
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A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)] of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) was synthesized, and its conversion to an... more
A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)] of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) was synthesized, and its conversion to an aziridinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4 and 37 degrees, 4-DAMP mustard released an equivalent amount of chloride. The release of chloride was consistent with a first-order process having a half-time of 5.7 min. The aziridinium ion reached a peak concentration at 32 min, corresponding to 75% of the initial concentration of 4-DAMP mustard. When homogenates of rat brain, heart, and submaxillary gland were incubated with 4-DAMP mustard (9 nM) for 1 hr, washed extensively, and then assayed for muscarinic receptor binding properties, a 56% decrease in the binding capacity of N-[3H]methylscopolamine in the heart and brain and a 71% decrease in the gland were observed, without a sign...
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Analysis of agonism and inverse agonism in functional assays with constitutive activity: Estimation of orthosteric ligand affinity constants for active and inactive receptor states
jpet.aspetjournals.org D ow nloaded from JPET #145219 2 Running title: Agonist activity at muscarinic receptor subtypes To whom correspondence should be addressed:
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We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a... more
We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a constitutively active mutant of it under control conditions and after partial receptor inactivation or reduced receptor expression. Our subsequent analysis is based on the assumption that the activating mutation increases receptor isomerization into the active state without affecting the affinities of ligands for receptor states. A means of confirming this assumption is provided. Global nonlinear regression analysis yields estimates of 1) the active (Kact) and inactive (Kinact) receptor-state affinity constants, 2) the isomerization constant of the unoccupied receptor (Kq-obs), and 3) the sensitivity constant of the signaling pathway (KE-obs). The latter two parameters define the output response of the receptor, and hence, their ratio (Kq-obs/KE) is a useful m...
Research Interests: Chemistry, Nonlinear dynamics, Medicine, Signal Transduction, Humans, and 13 moreComputer Simulation, Mutation, G protein-coupled receptors, Regression Analysis, Receptor, Structure activity Relationship, Protein Binding, Ligands, Monte Carlo Method, Agonist, binding sites, Functional selectivity, and Pharmacology and pharmaceutical sciences
ABSTRACT
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4-DAMP mustard (N-2-chloroethyl)-4-piperidinyl diphenylacetate) has been shown to selectively and irreversibly inhibit muscarinic receptors. In an attempt to increase the rate of formation and peak concentration of the reactive... more
4-DAMP mustard (N-2-chloroethyl)-4-piperidinyl diphenylacetate) has been shown to selectively and irreversibly inhibit muscarinic receptors. In an attempt to increase the rate of formation and peak concentration of the reactive intermediate, an analog [N-(2-bromoethyl)-4-piperidinyl diphenylacetate (4-DAMP bromo mustard)] was synthesized and the molecular formula confirmed by mass analysis. The 4-DAMP bromo mustard was shown to cyclize in phosphate buffer (pH 7.4) to the corresponding aziridinium ion with a first-order rate constant (k1) of 0.071 min-1 at 0 degrees C. At 25 degrees C and 37 degrees C, the formation of the aziridinium ion was nearly instantaneous (100% cyclized within 15 sec) at neutral pH. The rate constants (k2) for the hydrolysis of the aziridinium ion at 25 degrees C and 37 degrees C (pH 7.4) were 0.0027 and 0.010 min-1, respectively, in excellent agreement with the published rate constants for the hydrolysis of the aziridinium ion formed from 4-DAMP mustard. In ...
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Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the... more
Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M(1) and M(2) muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M(1) (Asp105) and M(2) (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M(2) D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M(1) D105N muta...
Research Interests: Chemistry, Molecular Pharmacology, Medicine, Humans, Animals, and 14 moreStereochemistry, Nitrogen, Acetylcholine, Alkylation, Base Sequence, Muscarinic Receptor, Protein Binding, Site-directed Mutagenesis, Neurosciences, Molecular Sequence Data, binding sites, CHO cells, Cricetinae, and Pharmacology and pharmaceutical sciences
A 3-chloropropylamine derivative (N-(3-chloropropyl)-4-piperidinyl diphenylactate) of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate was synthesized and its conversion to a stable azetidinium ion and... more
A 3-chloropropylamine derivative (N-(3-chloropropyl)-4-piperidinyl diphenylactate) of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate was synthesized and its conversion to a stable azetidinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4, N-(3-chloropropyl)-4-piperidinyl diphenylactate formed a stable azetidinium ion with a half-time of approximately 3.6 hr. The selectivity of the azetidinium ion for native M1, M2 and M3 subtypes of the muscarinic receptors was investigated in competitive binding experiments on the hippocampus, heart and submaxillary gland of rats, respectively, using N-[3H]methylscopolamine as the radioligand. The azetidinium ion exhibited equivalent high affinities for the M1 and M3 mucarinic receptor subtypes (KD = approximately 5 nM), but 10-fold lower affinity for the M2 muscarinic receptor subtype (KD = 44 nM). Similar competitive binding experiments were carried out...
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The effects of pertussis toxin on muscarinic receptor-induced contractions of the isolated guinea pig ileum were investigated. In control tissues, isoproterenol (1 microM) only slightly inhibited contractions in response to... more
The effects of pertussis toxin on muscarinic receptor-induced contractions of the isolated guinea pig ileum were investigated. In control tissues, isoproterenol (1 microM) only slightly inhibited contractions in response to oxotremorine-M; however, in pertussis toxin-treated tissues, isoproterenol exhibited an enhanced inhibition of the concentration-effect curve. Under basal conditions, pertussis toxin had no dampening effect on oxotremorine-M-induced contractions. When ilea were precontracted with histamine (1 microM) and then relaxed back to base line using forskolin (10 microM) before contractile responses to oxotremorine-M were measured, pertussis toxin shifted the concentration-effect curve to oxotremorine-M by a 6.1-fold increase in the EC50 value. Ilea were then incubated with [N-(2-chloroethyl)-4-piperidinyl diphenylacetate] (40 nM; 1 hr) in the presence of [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (1 microM) to ...
Research Interests: Endocrinology, Chemistry, Medicine, Signal Transduction, Internal Medicine, and 13 morecyclic AMP, Animals, Male, Experimental Pharmacology, Guanosine Triphosphate, Muscle contraction, Hydrolysis, Ileum, Isoproterenol, Pirenzepine, Guinea pigs, Pharmacology and pharmaceutical sciences, and In Vitro Techniques
Research Interests: Chemistry, Medicine, Animals, Male, In Vivo, and 6 moreRats, Parasympatholytics, Receptor, Piperidines, Pirenzepine, and Damp
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Research Interests: Pharmacology, Endocrinology, Chemistry, Cell Biology, Medicine, and 15 moreInternal Medicine, Muscle, Animals, Male, Antagonist, Cattle, Muscle contraction, Atropine, Hydrolysis, Piperidines, Ileum, Guinea pigs, Pharmacology and pharmaceutical sciences, In Vitro Techniques, and histamine antagonists
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... the most divergent regions are the extracellular loops and the amino-and carboxyl-terminals (Fig.1). Based on results of pharmacological investigations, δ-, κ-, and μ-opioid receptors have been further subdivided into receptor... more
... the most divergent regions are the extracellular loops and the amino-and carboxyl-terminals (Fig.1). Based on results of pharmacological investigations, δ-, κ-, and μ-opioid receptors have been further subdivided into receptor subtypes (Satoh and Minami, 1995); however, the ...
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The involvement of the M2 muscarinic receptor in contractile responses of the guinea pig trachea, guinea pig esophagus, and rat fundus was investigated. In the standard assay, oxotremorine-M elicited contractions of the trachea with an... more
The involvement of the M2 muscarinic receptor in contractile responses of the guinea pig trachea, guinea pig esophagus, and rat fundus was investigated. In the standard assay, oxotremorine-M elicited contractions of the trachea with an EC50 value of approximately 73 nanoM.--2- -(Diethylamino)methyl- -1-piperidinyl-acetyl--5,11- dihydro-6H-pyrido-2,3-b--1,4- benzodiazepine-6-one (AF-DX 116) at 1 and 10 microM antagonized these contractions by 2.1- and 9.0-fold increases in the EC50 value for oxotremorine-M. These effects are consistent with antagonism of an M3-mediated contractile response. In subsequent experiments, the M3 receptors were first inactivated selectively by incubation with N-(2-chloroethyl)-4- piperidinyl diphenylacetate (4-DAMP mustard) (40 nanoM) for 1 hr in the presence of AF-DX 116 (1 microM) followed by extensive washing. In 4-DAMP mustard treated trachea, oxotremorine-M elicited contractions with an EC50 value of 0.31 microM in the presence of histamine (10 microM) and forskolin (4 microM). Under these conditions, AF-DX 116 at 1 and 10 microM antagonized contractions to oxotremorine-M by 8- and 59-fold increases in the EC50, respectively, while para- fluorohexahydrosiladiphenidol(p-F-HHSiD) (0.1 microM) had no effect. These effects are consistent with a contraction being mediated by an M2 receptor. In the guinea pig esophagus and rat fundus, AF-DX 116 and p-F-HHSiD blocked contractions measured under similar conditions with magnitudes intermediate between what would be expected from an M2 and an M3 receptor, suggesting that perhaps both subtypes contribute to the overall contractile response under these conditions. In addition, contractions of the guinea pig trachea measured in the presence of histamine and forskolin were pertussis toxin sensitive. These results that, in the trachea, M2 receptors can dominate the contractile response after a majority of the M3 receptors have been inactivated, whereas in the guinea pig esophagus and rat fundus, M2 receptors may contribute to, but do not play a dominant role in the overall response.
Research Interests: Endocrinology, Biology, Medicine, Histamine, In Vitro, and 15 moreInternal Medicine, cyclic AMP, Animals, Male, Esophagus, Rat, Guinea Pig, Muscle contraction, BIOCHEMICAL PHARMACOLOGY, Muscarinic Receptor, Isoproterenol, Pirenzepine, Guinea pigs, Pharmacology and pharmaceutical sciences, and In Vitro Techniques
During the last decade, the availability of muscarinic cholinergic drugs of high specific activity has enabled a direct characterization of the way in which acetylcholine and other drugs interact with the muscarinic receptor. As might be... more
During the last decade, the availability of muscarinic cholinergic drugs of high specific activity has enabled a direct characterization of the way in which acetylcholine and other drugs interact with the muscarinic receptor. As might be anticipated, the advent of a new experimental method of scientific inquiry is often followed by a surge of information, and such is the relationship between the development of receptor binding techniques and muscarinic receptor Pharmacology. In general, the recent knowledge derived from studies of the binding of muscarinic agonists and antagonists is consistent with classical theories of agonist-receptor interaction and competitive inhibition which were developed primarily from studies of the interaction of drugs with isolated smooth muscle preparations such as the guinea pig ileum. However, additional complicated ligand-receptor interactions have been detected by receptor binding methods which could not have been realized from the results of classical Pharmacological assays on whole tissue preparations.
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Muscarinic agonists elicit contraction in the standard guinea pig ileum bioassay through activation of M3 muscarinic receptors that are also linked to phosphoinositide hydrolysis. Surprisingly, the most abundant muscarinic receptor in the... more
Muscarinic agonists elicit contraction in the standard guinea pig ileum bioassay through activation of M3 muscarinic receptors that are also linked to phosphoinositide hydrolysis. Surprisingly, the most abundant muscarinic receptor in the ileum is the M2 which causes a specific inhibition of cyclic AMP accumulation elicited by the beta-adrenergic receptor. After most of the M3 receptors are inactivated, the ileum still retains high sensitivity to muscarinic agonists provided that the contractile responses are measured in the presence of histamine and forskolin, which together, have no effect on contraction. Under these conditions, the potencies of antagonists for blocking the contractile response are consistent with those expected for an M2 response. Moreover, the muscarinic contractile response measured in the presence of histamine and forskolin after inactivation of M3 receptors is pertussis toxin sensitive. In contrast, muscarinic contractions in the standard bioassay are pertussis toxin insensitive. These results demonstrate that the M2 muscarinic receptor can cause an indirect contraction of the guinea pig ileum by preventing the relaxing effect of agents that increase cAMP.
Research Interests: Endocrinology, Chemistry, Life Sciences, Medicine, Signal Transduction, and 15 moreHistamine, Internal Medicine, cyclic AMP, Animals, Heart, Rats, Guinea Pig, Muscle contraction, High Sensitivity, Muscarinic Receptor, Biochemistry and cell biology, Piperidines, Ileum, Guinea pigs, and Pharmacology and pharmaceutical sciences
... There was essentially no agreement between the antagonist binding affinities measured in the duodenum and those measured in the cerebral cortex (M,), submandibular gland (M3), and the values published by Dorje et al. 25 for the cloned... more
... There was essentially no agreement between the antagonist binding affinities measured in the duodenum and those measured in the cerebral cortex (M,), submandibular gland (M3), and the values published by Dorje et al. 25 for the cloned human M4 receptor. ...
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The identity and distribution of muscarinic cholinergic receptor subtypes and associated signal transduction mechanisms was characterized for the cerebral circulation using correlated functional and biochemical investigations. Subtypes... more
The identity and distribution of muscarinic cholinergic receptor subtypes and associated signal transduction mechanisms was characterized for the cerebral circulation using correlated functional and biochemical investigations. Subtypes were distinguished by the relative affinities of a panel of muscarinic antagonists, pirenzepine, AF-DX 116 [11-2-[[2-[diethylaminomethyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one], hexahydrosiladifenidol, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methobromide, dicyclomine, para-fluoro-hexahydrosiladifenidol and atropine. Muscarinic receptors characterized by inhibition of [3H]quinuclidinylbenzilate binding in membranes of bovine pial arteries were of the M2 subtype. In contrast pharmacological analysis of [3H]-quinuclidinylbenzilate binding in bovine intracerebral microvessels suggests the presence of an M4 subtype. Receptors mediating endothelium-dependent vasodilation in rabbit pial arteries were of the M3 subtype, whereas muscarinic receptors stimulating endothelium-independent phosphoinositide hydrolysis in bovine pial arteries were of the M1 subtype. These findings suggest that characteristics of muscarinic receptors in cerebral blood vessels vary depending on the type of vessel, cellular location and function mediated.
Research Interests: Pharmacology, Kinetics, Autonomic Nervous System, Female, Animals, and 15 moreMale, Cattle, Cardiovascular system, Radioligand Assay, Chemical Composition, Rabbits, Membrane Protein, Reaction Kinetics, Muscarinic Receptor, Blood Vessel, Organic Compound, Correlation function, binding sites, Domestic animal, and Pharmacology and pharmaceutical sciences
Both M(2) and M(3) muscarinic receptors are expressed in smooth muscle and influence contraction through distinct signaling pathways. M(3) receptors interact with G(q) to trigger phosphoinositide hydrolysis, Ca(2+) mobilization and a... more
Both M(2) and M(3) muscarinic receptors are expressed in smooth muscle and influence contraction through distinct signaling pathways. M(3) receptors interact with G(q) to trigger phosphoinositide hydrolysis, Ca(2+) mobilization and a direct contractile response. In contrast, M(2) receptors interact with G(i) and G(o) to inhibit adenylyl cyclase and Ca(2+)-activated K(+) channels and to potentiate a Ca(2+)-dependent, nonselective cation conductance. Ultimately, these mechanisms lead to the prediction that the influence of the M(2) receptor on contraction should be conditional upon mobilization of Ca(2+) by another receptor such as the M(3). Mathematical modeling studies of these mechanisms show that the competitive antagonism of a muscarinic response mediated through activation of both M(2) and M(3) receptors should resemble the profile of the directly acting receptor (i.e., the M(3)) and not that of the conditionally acting receptor (i.e., the M(2)). Using a combination of pharmacological and genetic approaches, we have identified two mechanisms for the M(2) receptor in contraction: 1) a high potency inhibition of the relaxation elicited by agents that increase cytosolic cAMP and 2) a low potency potentiation of contractions elicited by the M(3) receptor. The latter mechanism may be involved in muscarinic agonist-mediated heterologous desensitization of smooth muscle, which requires activation of both M(2) and M(3) receptors.