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)

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

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

Diurnal pattern of QTc interval: How long is prolonged? Possible relation to circadian triggers of cardiovascular events

Objectives.This study sought to evaluate the range and variability of the QT and corrected QT (QTc) intervals over 24 h and to assess their pattern and relation to heart rate variability.Background.Recent Holter monitoring data have revealed a high degree of daily variability in the QTc interval. The pattern of this variability and its relation to heart rate variability remain poorly characterized.Methods.We developed and validated a new method for continuous measurement of QT intervals from three-channel, 24-h Holter recordings. Average RR, QT, QTc and heart rate variability were measured from 5-min segments of data from 21 healthy subjects.Results.Measurement of 6,048 segments showed mean (±SD) RR, QT and QTc intervals of 830 ± 100, 407 ± 23 and 445 ± 16 ms, respectively (mean QTc interval for men 434 ± 12 ms, 457 ± 10 ms for women, p < 0.0001). The average maximal QTc interval was 495 ± 21 ms and the average QTc range 95 ± 20 ms. The maximal QTc interval was ≥500 ms in 6 subjects and ≥490 ms in 13. The 95% upper confidence limit for the mean 24-h QTc interval was 452 ms (men 439 ms, women 461 ms). The RR, QT and QTc intervals and the high frequency component of heart rate variability were greater during sleep. Both the QTc interval and the variability between hourly minimal and maximal QTc intervals reached their circadian peak shortly after awakening, before declining to daytime levels.Conclusions.The maximal QTc interval over 24 h in normal subjects is longer than heretofore thought. Both QT and QTc intervals are longer during sleep. The QTc interval and QTc variability reach a peak shortly after awakening, which may reflect increased autonomic instability during early waking hours, and the time of the peak value corresponds in time to the period of reported increased vulnerability to ventricular tachycardia and sudden cardiac death. These findings have implications regarding the definition of QT prolongation and its use in predicting arrhythmias and sudden death