Comparison of Functional Antagonism Between Isoproterenol and M2 Muscarinic Receptors in Guinea Pig Ileum and Trachea

The ability of the M2 muscarinic receptor to mediate an inhibition of the relaxant effects of forskolin and isoproterenol was investigated in guinea pig ileum and trachea. In some experiments, trachea was first treated with 4-diphenylacetoxy-Nmethylpiperidine (4-DAMP) mustard to inactivate M3 receptors. The contractile response to oxotremorine-M was measured subsequently in the presence of both histamine (10 mM) and isoproterenol (10 nM). Under these conditions, [[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b]- [1,4]benzodiazepine-6-one (AF-DX 116) antagonized the contractile response to oxotremorine-M in a manner consistent with an M3 mechanism. However, when the same experiment was repeated using forskolin (4 mM) instead of isoproterenol, the response to oxotremorine-M exhibited greater potency and was antagonized by AF-DX 116 in a manner consistent with an M2 mechanism. We also measured the effects of pertussis toxin treatment on the ability of isoproterenol to inhibit the contraction elicited by a single concentration of either histamine (0.3 mM) or oxotremorine-M (40 nM) in both the ileum and trachea. Pertussis toxin treatment had no significant effect on the potency of isoproterenol for inhibiting histamine-induced contractions in the ileum and trachea. In contrast, pertussis toxin treatment enhanced the relaxant potency of isoproterenol against oxotremorine-M-induced contractions in the ileum but not in the trachea. Also, pertussis toxin treatment enhanced the relaxant potency of forskolin against oxotremorine-M-induced contractions in the ileum and trachea. We investigated the relaxant potency of isoproterenol when very low, equi-effective (i.e., 20–34% of maximal response) concentrations of either histamine or oxotremorine-M were used to elicit contraction. Under these conditions, isoproterenol exhibited greater relaxant potency against histamine in the ileum but exhibited similar relaxant potencies against histamine and oxotremorine-M in the trachea. Following 4-DAMP mustard treatment, a low concentration of oxotremorine-M (10 nM) had no contractile effect in either the ileum or trachea. Nevertheless, in 4-DAMP mustard- treated tissue, oxotremorine-M (10 nM) reduced the relaxant potency of isoproterenol against histamine-induced contractions in the ileum, but not in the trachea. We conclude that in the trachea the M2 receptor mediates an inhibition of the relaxant effects of forskolin, but not isoproterenol, and the decreased relaxant potency of isoproterenol against contractions elicited by a muscarinic agonist relative to histamine is not due to activation of M2 receptors but rather to the greater contractile stimulus mediated by the M3 receptor compared with the H1 histamine receptor

Analysis of Ligand Bias in Functional Studies Involving the Allosteric Modulation of G Protein- Coupled Receptors

Introduction The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its capacity to activate downstream signaling events. In this report, we describe a reverse-engineering strategy for estimating these microscopic constants. Methods Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein-coupled receptor under conditions of allosteric modulation and reduced receptor expression or partial receptor inactivation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands – including inverse agonists – and the efficacy of the unoccupied receptor (i.e., constitutive activity). Results and discussion We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor-signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe-dependent allosteric interactions and the selectivity of agonists for different signaling pathways. Knowing the isomerization constant and sensitivity constant of a signaling pathway in a given cell line or tissue preparation enables future investigators to estimate the affinity constants of agonists for receptor states simply through analysis of their concentration–response curves. Our approach also provides a means of validating in silico estimates of ligand affinity for crystal structures of active and inactive states of the receptor

Analysis of Agonism and Inverse Agonism in Functional Assays with Constitutive Activity: Estimation of Orthosteric Ligand Affinity Constants for Active and Inactive Receptor States

We describe a modification of receptor theory for the estimation of observed affinities (Kobs) and relative efficacies of orthosteric ligands in functional assays that exhibit constitutive activity. Our theory includes parameters for the fractions of the occupied receptor population in the active (intrinsic efficacy, ε) and inactive (εi) states and analogous parameters for the fractions of the free receptor population in the active (εsys) and inactive (εi-sys) states. The total stimulus represents the summation of the active states of the free and occupied receptor populations. A modified operational model is developed that expresses the response as a logistic function of the total stimulus. This function includes the standard parameters related to affinity and efficacy (Kobs and τ) as well as a parameter proportional to the activity of the free receptor complex, τsys. Two related parameters are proportional to the fraction of the free (τi-sys) and occupied (τi) receptor populations in the inactive state. We show that the estimates of the affinity constants of orthosteric ligands for the active (Kb) and inactive (Ka) states of the receptor are equivalent to τKobs/τsys and τiKobs/τi-sys, respectively. We verify our method with computer simulation techniques and apply it to the analysis of M2 and M3 muscarinic receptors. Our method is applicable in the analysis of ligand bias in drug discovery programs

Analysis of Functional Responses at G Protein-Coupled Receptors: Estimation of Relative Affinity Constants for the Inactive Receptor Sstate

We describe a modification of receptor theory that enables the estimation of relative affinity constants for the inactive state of a G protein-coupled receptor. Our approach includes the traditional parameters of observed affinity (Kobs) and efficacy (fraction of ligand-receptor complex in the active state, ε) and introduces the concept of the fraction of the ligand-receptor complex in the inactive state (intrinsic inactivity, εi). The relationship between receptor activation and the ligand concentration is known as the stimulus, and the operational model expresses the response as a logistic function of the stimulus. The latter function includes Kobs and the parameter τ, which is proportional to ε. We introduce the parameter τi, which is proportional to εi. We have previously shown that the product, Kobsτ, of one agonist, expressed relative to that of another (intrinsic relative activity, RAi), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, Kobsτi, of one agonist, expressed relative to that of another (intrinsic relative inactivity, RIi), is a relative measure of the affinity constant for the inactive state of the receptor. We use computer simulation techniques to verify our analysis and apply our method to the analysis of published data on agonist activity at the M3 muscarinic receptor. Our method should have widespread application in the analysis of agonist bias in drug discovery programs and in the estimation of a more fundamental relative measure of efficacy (RAi/RIi)

Contractile Roles of the M 2 and M 3 Muscarinic Receptors in the Guinea Pig Colon 1

ABSTRACT The contractile roles of the M 2 and M 3 muscarinic receptors were investigated in guinea pig longitudinal colonic smooth muscle. Prior treatment of the colon with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) (40 nM) in combination with [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3b][1,4]benzodiazepine-6-one (AF-DX 116) (1.0 M) caused a subsequent, irreversible inhibition of oxotremorine-M-induced contractions when measured after extensive washing. The estimate of the degree of receptor inactivation after 2 hr (97%) was not much greater than that measured after 1 hr (95%), which suggests that both 4-DAMP mustard-sensitive and -insensitive muscarinic subtypes contribute to the contractile response. Pertussis toxin treatment had no significant inhibitory effect on the control contractile response to oxotremorine-M, but caused an 8.8-fold increase in the EC 50 value measured after a 2-hr treatment with 4-DAMP mustard. These results suggest that, after elimination of most of the M 3 receptors with 4-DAMP mustard, the contractile response can be mediated by the pertussis toxin-sensitive M 2 receptor. After pertussis toxin treatment, the kinetics of alkylation of muscarinic receptors in the colon were consistent with a single, 4-DAMP mustard-sensitive, M 3 receptor subtype mediating the contractile response. When measured after a 2-hr treatment with 4-DAMP mustard and in the presence of histamine (0.30 M) and either forskolin (10 M) or isoproterenol (0.60 M), the contractile responses to oxotremorine-M were pertussis toxin-sensitive and potently antagonized by the M 2 selective antagonist, AF-DX 116. Collectively, our results indicate that the M 2 receptor elicits contraction through two mechanisms, a direct contraction and an indirect contraction by preventing the relaxant effects of cAMP-generating agents. Muscarinic receptors are expressed abundantly in smooth muscle throughout the gastrointestinal tract in a manner that approximates a three-to-one mixture of the M 2 and M 3 subtypes (see The M 2 muscarinic receptor has been shown to mediate a pertussis toxin-sensitive inhibition of adenylyl cyclase activity in the ileum and colon Muscarinic receptors have also been shown to induce a nonselective cation conductance in the longitudinal smooth muscle of the guinea pig ileu

A Simple Method for Estimation of Agonist Activity at Receptor Subtypes: Comparison of Native and Cloned M3 Muscarinic Receptors in Guinea Pig Ileum and Transfected Cells

We describe a simple method for calculating the pharmacological activity of an agonist (A) relative to a standard agonist (S) using only the concentration-response curves of the two agonists. In most situations, we show that the product of the ratios of maximal responses (E max − A/E max − S) and potencies (EC50 − S/EC50 − A) is equivalent to the product of the affinity and intrinsic efficacy of A expressed relative to that of S. We refer to this term as the IRA value of A. In a cooperative system where the concentration-response curve of the standard agonist is steep and that of the test agonist is flatter with a lower maximal response, the simple calculation of IRA described above underestimates agonist activity; however, we also describe a means of correcting the IRA in this situation. We have validated our analysis with modeling techniques and have shown experimentally that the IRA values of muscarinic agonists for stimulating contractions in the guinea pig ileum (M3 response) are in excellent agreement with those measured in the phosphoinositide assay on Chinese hamster ovary cells expressing the M3 muscarinic receptor

Muscarinic Agonist-Mediated Heterologous Desensitization in Isolated Ileum Requires Activation of Both Muscarinic M2 and M3 Receptors

We investigated the subtypes of the muscarinic receptor mediating short-term heterologous desensitization in the isolated ileum. Treatment of the ileum from C57BL/6 mice with acetylcholine (30 μM) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F2α (PGF2α) and the muscarinic agonist, oxotremorine-M. This subsensitivity was characterized by 7- and 3-fold increases in the EC50 values of the agonists, respectively, with no significant effect on the maximal response. The subsensitivity to PGF2α was prevented in both M2 and M3 muscarinic receptor knockout mice. Similarly, the subsensitivity to oxotremorine-M was prevented in M2 knockout mice. Acetylcholine-mediated desensitization of histamine-induced contractions in the guinea pig ileum was inhibited by both M2- and M3-selective muscarinic antagonists with high potency, although careful analysis of the data suggested behavior more consistent with an M2 antagonistic profile. Modeling studies showed that the competitive antagonism of response contingent upon activation of two receptor subtypes should exhibit a pharmacological profile similar to that of the least sensitive signaling pathway. Our results demonstrate that muscarinic agonist-mediated short-term heterologous desensitization of intestinal smooth muscle is contingent upon activation of both M2 and M3 muscarinic receptors and that activation of either receptor by itself is insufficient to cause desensitization

〔研究ノート〕 ムスカリン性アセチルコリン受容体の細胞膜への トラフィッキングの観察

　　G protein-coupled receptors are cell-surface receptors, many of which have conserved motif F（x）6LL in their C-terminal intracellular region. The motif is known to function when the G protein-coupled receptors are exported from the endoplasmic reticulum to the cell surface. We reported previously that the amino acid mutations of the conserved leucines of M1 muscarinic acetylcholine receptor caused significant decrease in the cell-surface expression and significant increase in the endoplasmic reticulum expression of the mutant receptor, and that in the presence of antagonist atropine, the mutant receptor showed cell-surface expression, similar to the expression of the wild-type receptor. In this study, we investigated the export trafficking of the mutant receptor by the measurements of membrane-impermeable antagonist ［3H］N-methylscopolamine binding to the cell surface, and indicated that the cell-surface expression of the mutant receptor in the presence of atropine decreased to 20％ by the depletion of atropine for 3 days, and recovered to the same amount as before by the second addition of atropine. We also investigated the export trafficking of the mutant receptor using the total internal reflection fluorescence microscope, and indicated that the mutant receptor expressed time-dependently to the cell surface by the second addition of atropine