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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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Articles by Frederick J. Ehlert in JoVE

 JoVE General

Quantifying Agonist Activity at G Protein-coupled Receptors


JoVE 3179 12/26/2011

1, 2, 3

1Department of Pharmacology, University of California, Irvine, 2Department of Pharmacology, University of California, 3Schmid College of Science, Chapman University

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

Other articles by Frederick J. Ehlert on PubMed

Identification and Molecular Characterization of Two Closely Related G Protein-coupled Receptors Activated by Prokineticins/endocrine Gland Vascular Endothelial Growth Factor

The Journal of Biological Chemistry. May, 2002  |  Pubmed ID: 11886876

We previously described two mammalian secreted proteins, prokineticin 1 and prokineticin 2, that potently contract gastrointestinal smooth muscle. Prokineticin 1 has also been shown to promote angiogenesis by stimulating proliferation, migration, and fenestration of endocrine organ-derived endothelial cells. Here we report the cloning and characterization of two closely related G protein-coupled receptors as receptors for prokineticins. Expression of prokineticin receptors in heterologous systems shows that these receptors bind to and are activated by nanomolar concentrations of recombinant prokineticins. Activation of prokineticin receptors leads to mobilization of calcium, stimulation of phosphoinositide turnover, and activation of p44/p42 MAPK signaling pathways that are consistent with the effects of prokineticins on smooth muscle contraction and angiogenesis. mRNA expression analysis reveals that prokineticin receptors are expressed in gastrointestinal organs, endocrine glands, and other tissues.

Increased Relaxant Action of Forskolin and Isoproterenol Against Muscarinic Agonist-induced Contractions in Smooth Muscle from M2 Receptor Knockout Mice

The Journal of Pharmacology and Experimental Therapeutics. Apr, 2003  |  Pubmed ID: 12649358

The ability of forskolin and isoproterenol to inhibit the contractile action of the muscarinic agonist, oxotremorine-M, was investigated in smooth muscle from wild-type and M(2) muscarinic receptor knockout mice. Forskolin (5.0 micro M) caused a significant reduction in the contractile activity of oxotremorine-M in ileum, trachea, and urinary bladder from both wild-type and M(2) muscarinic receptor knockout mice. This reduction in contractile activity was characterized by decreases in potency or maximal response, but not always both. Similar results were obtained with isoproterenol (1.0 micro M). The relaxant effects of forskolin in ileum, trachea, and urinary bladder from M(2) receptor knockout mice were approximately 3- to 9-fold greater than those observed in the same tissues from wild-type mice. Similar results were obtained with isoproterenol in ileum and urinary bladder, although the differences between wild-type and M(2) receptor knockout tissues were less than those observed with forskolin. In contrast, there was no significant difference between the relaxant effect of isoproterenol in trachea from wild-type and M(2) receptor knockout mice. In contrast to the results observed with oxotremorine-M as the contractile agent, forskolin and isoproterenol did not exhibit greater relaxant activity against KCl-induced contractions in M(2) receptor knockout mice compared with wild-type mice. These results suggest that a component of the contractile response to muscarinic agonists in smooth muscle involves an M(2) muscarinic receptor-mediated inhibition of the relaxant effects of agents that increase cAMP levels.

Comparison of the Pharmacological Antagonism of M2 and M3 Muscarinic Receptors Expressed in Isolation and in Combination

Biochemical Pharmacology. Apr, 2003  |  Pubmed ID: 12694864

We compared the binding properties of selective muscarinic antagonists with their potencies for antagonizing muscarinic responses in Chinese hamster ovary (CHO) cells expressing M(2) and M(3) muscarinic receptors in combination and in isolation. When measured by the competitive displacement of [3H]N-methylscopolamine binding to CHO cells expressing both M(2) and M(3) muscarinic receptors (CHO M(2)+M(3) cells), the competition curves of the subtype-selective muscarinic antagonists were consistent with a two-site model. One site exhibited binding properties identical to those of CHO M(2) cells, whereas the other site exhibited properties like those of CHO M(3) cells. Oxotremorine-M, a muscarinic agonist, elicited a robust, pertussis toxin-insensitive stimulation of phosphoinositide hydrolysis in both CHO M(3) and CHO M(2)+M(3) cells, but not in CHO M(2) cells. The pharmacological antagonism of the phosphoinositide response exhibited similar properties in both CHO M(3) and CHO M(2)+M(3) cells. Oxotremorine-M elicited a pertussis toxin-sensitive, robust inhibition of forskolin-stimulated cyclic AMP (cAMP) accumulation in both CHO M(2) and CHO M(2)+M(3) cells and a less robust inhibition in CHO M(3) cells. At higher concentrations, oxotremorine-M elicited an increase in cAMP accumulation over the maximal inhibition noted at lower concentrations in both CHO M(3) and CHO M(2)+M(3) cells. Following pertussis toxin treatment, only the stimulatory phase of the cAMP response to oxotremorine-M was observed in CHO M(2), CHO M(3), and CHO M(2)+M(3) cells. The pharmacological antagonism of the cAMP response in CHO M(2)+M(3) cells resembled that expected for a response mediated independently by both M(2) and M(3) receptors.

Pharmacological Analysis of the Contractile Role of M2 and M3 Muscarinic Receptors in Smooth Muscle

Receptors & Channels. 2003  |  Pubmed ID: 12893538

Muscarinic receptors expressed on smooth muscle cells are primarily of the M(2) and M(3) subtypes. The M(3) subtype triggers contraction through an interaction with G(q) proteins to stimulate phosphoinositide hydrolysis and mobilize Ca(2+). In contrast, activation of M(2) receptors modulates contraction by preventing relaxation or by potentiating M(3) receptor-mediated contractions, which enhances heterologous desensitization. These effects can be explained by the coupling of M(2) receptors to G(i) proteins that mediate an inhibition of adenylyl cyclase and calcium-activated potassium channels. The pharmacological antagonism of a response mediated through an interaction between M(2) and M(3) receptors has been shown to resemble the profile of the directly acting receptor (M(3)), primarily, and not that of the conditional receptor (M(2)). Evidence for a contractile role of the M(2) receptor has been obtained by inactivating its signaling pathway with pertussis toxin or by measuring contractile effects of muscarinic agonists after M(3) receptors have been covalently inactivated. Under these conditions, M(2) receptors have been shown to mediate an inhibition of the relaxant effects of agents, like isoproterenol, on the contractile effects of nonmuscarinic spasmogens. Muscarinic M(2) and M(3) receptor knockout mice are useful tools for exploring interactions between these receptors in smooth muscle.

Contractile Role of M2 and M3 Muscarinic Receptors in Gastrointestinal, Airway and Urinary Bladder Smooth Muscle

Life Sciences. Dec, 2003  |  Pubmed ID: 14607264

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.

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

The Journal of Pharmacology and Experimental Therapeutics. Jan, 2004  |  Pubmed ID: 14563784

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 microM) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F2alpha (PGF2alpha) 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 PGF2alpha 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.

Functional Analysis of Muscarinic Acetylcholine Receptors Using Knockout Mice

Life Sciences. Nov, 2004  |  Pubmed ID: 15474550

Because of the low selectivity of available ligands, pharmacological approaches to elucidate the functional difference among muscarinic acetylcholine receptor (mAChR) subtypes have been problematic. As an alternative approach, we have established a series of mutant mouse lines deficient in each mAChR subtype (mAChR KO mice). The systematic analyses of these mice have been useful in revealing the functional difference among mAChR subtypes. Here, we review our prior research on these mutant mice and also some notable findings reported by other research groups.

The M2 Muscarinic Receptor Mediates Contraction Through Indirect Mechanisms in Mouse Urinary Bladder

The Journal of Pharmacology and Experimental Therapeutics. Apr, 2005  |  Pubmed ID: 15608083

We investigated the contractile role of M2 muscarinic receptors in mouse urinary bladder. When measured in the absence of other agents, contractions elicited to the muscarinic agonist oxotremorine-M exhibited properties consistent with that expected for an M3 response in urinary bladder from wild-type and M2 knockout (KO) mice. Evidence for a minor M2 receptor-mediated contraction was revealed by a comparison of responses in M3 knockout and M2/M3 double knockout mice. Treatment of wild-type and M2 knockout urinary bladder with N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard) caused a large inhibition of the muscarinic contractile response. The residual contractions were much smaller in M2 knockout bladder compared with wild type, suggesting that M2 receptors rescue the muscarinic contractile response in wild-type bladder following inactivation of M3 receptors with 4-DAMP mustard. When measured in the presence of prostaglandin F2alpha and isoproterenol or forskolin, oxotremorine-M mediated a potent contractile response in urinary bladder from M3 KO mice. This response exhibited an M2 profile in competitive antagonism studies and was completely absent in M2/M3 KO mice. Following 4-DAMP mustard treatment, oxotremorine-M elicited a contractile response in wild-type urinary bladder in the presence of KCl and isoproterenol or forskolin, and this response was diminished in M2 KO mice. Our results show that the M2 receptor mediates contractions indirectly in the urinary bladder by enhancing M3 receptor-mediated contractions and inhibiting relaxation. We also show that it is difficult to detect M2 receptor function in competitive antagonism studies under conditions where a simultaneous activation of M2 and M3 receptors occurs.

Analysis of Allosterism in Functional Assays

The Journal of Pharmacology and Experimental Therapeutics. Nov, 2005  |  Pubmed ID: 16046613

The theoretical basis for analyzing the effects of an allosteric modulator on the response to an agonist is described. The effects of an allosteric modulator on the concentration-response curve to an agonist can be attributed to changes in the observed dissociation constant and intrinsic efficacy of the agonist-receptor complex. Each of these two changes can be represented by a coefficient or factor. It is possible to estimate the ratio of the coefficient of change in agonist efficacy divided by that for the agonist dissociation constant. This ratio is designated as the relative activity (RA) of the agonist in the presence of the allosteric modulator. The RA value can be estimated for each concentration of allosteric modulator by nonlinear regression analysis, regardless of the shape of the concentration-response curve. Regression analysis of the RA values against the concentration of allosteric modulator yields estimates of the dissociation constant (K(A)) of the allosteric modulator and the maximal RA value. If the concentration-response curve to the agonist obeys a logistic function and the allosteric modulator influences the maximal response, it is possible to distinguish between the maximal change in affinity from that of efficacy. If the agonist concentration-response curve obeys a logistic equation with a Hill slope of 1, the RA values can be estimated easily from the agonist EC(50) and E(max) values. This analysis illustrates the utility of the RA value in quantifying allosteric effects.

Comparison of the Antimuscarinic Action of P-fluorohexahydrosiladifenidol in Ileal and Tracheal Smooth Muscle

The Journal of Pharmacology and Experimental Therapeutics. Feb, 2005  |  Pubmed ID: 15456838

We investigated the ability of the muscarinic antagonist p-fluorohexahydrosiladifenidol to inhibit muscarinic agonist-induced contractions and phosphoinositide hydrolysis in the guinea pig ileum and trachea. This antagonist displayed higher potency at blocking oxotremorine-M-induced contractions of the ileum compared with those of the trachea. When estimated using a simple model for competitive antagonism, the observed dissociation constant of p-fluorohexahydrosiladifenidol exhibited approximately 12-fold higher potency in the ileum compared with the trachea. We also investigated the ability of p-fluorohexahydrosiladifenidol to affect the inhibition of contraction caused by the known competitive muscarinic antagonist atropine. Using resultant analysis to analyze this interaction, we found that the true dissociation constant of p-fluorohexahydrosiladifenidol for competitively antagonizing oxotremorine-M-induced contractions in the ileum exhibited significantly lower potency than when calculated assuming a simple competitive model. In contrast, resultant analysis showed little difference between the true and observed potencies of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced contractions in the trachea. Using a simple competitive model, we found little difference in the observed dissociation constant of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced phosphoinositide hydrolysis in guinea pig ileum and bovine trachea. We also noted that p-fluorohexahydrosiladifenidol (0.3-1.0 microM) moderately inhibited histamine-induced contractions of ileum but not of trachea. Our results suggest that p-fluorohexahydrosiladifenidol does not discriminate markedly between M(3) muscarinic receptors in the ileum and trachea and that it may posses a more potent, nonmuscarinic inhibitory effect on contraction in the ileum.

Determination of the Rate of Muscarinic M1 Receptor Plasma Membrane Delivery Using a Regulated Secretion/aggregation System

Journal of Pharmacological and Toxicological Methods. May-Jun, 2006  |  Pubmed ID: 16213169

In this study, we used the regulated secretion/aggregation technology (RPD) to determine the rate of human muscarinic M1 (hM1) receptor plasma membrane delivery.

Differential Coupling of Muscarinic M1, M2, and M3 Receptors to Phosphoinositide Hydrolysis in Urinary Bladder and Longitudinal Muscle of the Ileum of the Mouse

The Journal of Pharmacology and Experimental Therapeutics. Aug, 2006  |  Pubmed ID: 16675640

We investigated the coupling of muscarinic receptor (M) subtypes to phosphoinositide hydrolysis in ileum and urinary bladder using muscarinic receptor knockout mice. In urinary bladder from wild-type mice, the muscarinic agonist oxotremorine-M, elicited a robust phosphoinositide response characterized by an EC50 value of 0.22 microM and a maximal response (Emax) of 32.8% conversion of [3H]inositol-labeled phosphoinositides into [3H]inositol phosphates. A similar response was observed in urinary bladder from M2 knockout mice, whereas no measurable response was observed in urinary bladder from M3 and M2/M3 knockout mice. In ilea from wild-type and M2 knockout mice, substantial phosphoinositide responses to oxotremorine-M were measured, characterized by EC50 values of 0.37 and 0.52 microM and Emax values of 35.8 and 34.7%, respectively. Oxotremorine-M also elicited phosphoinositide hydrolysis in ilea from M3 and M2/M3 knockout mice, although these responses were less sensitive (EC50 values of 1.6 and 1.4 microM; Emax values of 31.2 and 20.8%, respectively). The response in ileum from the M2/M3 knockout was significantly smaller than that from the M3 knockout. The muscarinic phosphoinositide response in ilea from M2/M3 knockout mice originated in the smooth muscle and exhibited a profile for competitive antagonism consistent with an M1 mechanism. These data suggest a major role for the M3 receptor in eliciting phosphoinositide hydrolysis in the ileum and urinary bladder and minor roles for the M1 and M2 in ileum.

Estimation of Agonist Activity at G Protein-coupled Receptors: Analysis of M2 Muscarinic Receptor Signaling Through Gi/o,Gs, and G15

The Journal of Pharmacology and Experimental Therapeutics. Jun, 2007  |  Pubmed ID: 17392404

We developed novel methods for analyzing the concentration-response curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA(i)), is most applicable for the analysis of responses at G protein-coupled receptors. RA(i) is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M(2) receptor, including stimulation of phosphoinositide hydrolysis through G(alpha15) in HEK 293T cells, inhibition of cAMP accumulation through G(i) in Chinese hamster ovary (CHO) cells, and stimulation of cAMP accumulation through G(s) in CHO cells treated with pertussis toxin. The RA(i) values of carbachol, oxotremorine-M, and the enantiomers of aceclidine were approximately the same in the three assay systems. In contrast, the activity of 4-[[N-[3-chlorophenyl]carbamoy]oxy-2-butynyl]trimethylammonium chloride (McN-A-343) was approximately 10-fold greater at M(2) receptors coupled to G(alpha15) in HEK 293T cells compared with M(2) receptors coupled to G(i) in the same cells or in CHO cells. Our results show that the RA(i) estimate is a useful measure for quantifying agonist activity across different assay systems and for detecting agonist directed signaling.

Neuronally Released Acetylcholine Acts on the M2 Muscarinic Receptor to Oppose the Relaxant Effect of Isoproterenol on Cholinergic Contractions in Mouse Urinary Bladder

The Journal of Pharmacology and Experimental Therapeutics. Aug, 2007  |  Pubmed ID: 17473176

We investigated whether M(2) muscarinic receptor activation opposes isoproterenol-induced relaxation in mouse urinary bladder and whether endogenous acetylcholine acts through a similar M(2) mechanism. When measured in urinary bladder from M(3) receptor knockout mice, the muscarinic agonist oxotremorine-M elicited only very weak contractions. In the presence of alpha,beta-methylene ATP (30 microM) and isoproterenol (1 microM), however, oxotremorine-M elicited a robust contractile response. This response was completely absent in bladder from M(2)/M(3) double knockout mice, indicating that activation of the M(2) receptor inhibits the relaxant effect of isoproterenol on the contraction to alpha,beta-methylene ATP. Similar results were obtained when prostaglandin F(2alpha) (5 microM) was used as the contractile agent but not when serotonin was used. Electrical field stimulation of the urinary bladder from wild-type mouse elicited contractions that were inhibited 20% by atropine and 40% by desensitization with alpha,beta-methylene ATP. When measured in the presence of alpha,beta-methylene ATP to desensitize the purinergic component of contraction, isoproterenol exhibited moderately greater relaxant activity in field-stimulated bladder from the M(2) knockout mouse compared with that observed in wild-type bladder. This differential relaxant effect of isoproterenol was greatly increased in the presence of physostigmine. In contrast, no differential effects were noted for isoproterenol in similar experiments on bladders from M(3) knockout and M(2)/M(3) double knockout mice in the presence of physostigmine. Our results suggest that neuronally released acetylcholine acts on the M(2) muscarinic receptor to inhibit the relaxant effect of isoproterenol on the minor, cholinergic component of contraction in the field-stimulated mouse urinary bladder.

Cysteine Pairs in the Third Intracellular Loop of the Muscarinic M1 Acetylcholine Receptor Play a Role in Agonist-induced Internalization

The Journal of Pharmacology and Experimental Therapeutics. Jan, 2008  |  Pubmed ID: 17540859

We determined the functional role of a small domain in the third intracellular loop of the human muscarinic M(1) (hM(1)) receptor. Using site-directed mutagenesis, several mutant hM(1) receptors were made possessing either a deletion or point mutations within the third intracellular loop domain (252)PETPPGRCCRCC(263). Wild-type and mutant hM(1) receptors were transiently expressed in Chinese hamster ovary cells, and the effects of each mutation on radioligand binding, agonist-mediated phosphoinositide hydrolysis, and agonist-induced internalization were determined. The mutant receptors exhibited a modest reduction in affinity for [(3)H]N-methylscopolamine (pK(D) = approximately 9.0) and a moderately increased binding capacity relative to the wild-type receptor. This moderate increase in binding capacity was associated with small increases in the maximal response and potency of carbachol for eliciting phosphoinositide hydrolysis through the mutant receptors (pEC(50) = approximately 5.5) relative to wild-type (pEC(50) = 5.35 +/- 0.05). In contrast, carbachol-induced internalization of mutant hM(1) receptors possessing either C259A/C260A or C262A/C263A or both double point mutations was significantly reduced compared to the wild-type hM(1) receptor. Of the hM(1) receptor mutants tested, those possessing a C262D/C263D double point mutation had the least carbachol-induced internalization. The desensitization and down-regulation of receptors possessing either Cys/Ala or Cys/Asp double point mutations were similar to those observed for the wild-type hM(1) receptor. Collectively, these observations suggest that Cys pairs Cys259/Cys260 and Cys262/Cys263 play an important role in the agonist-induced internalization of hM(1) receptors.

On the Analysis of Ligand-directed Signaling at G Protein-coupled Receptors

Naunyn-Schmiedeberg's Archives of Pharmacology. Jun, 2008  |  Pubmed ID: 18253722

The phenomenon of "ligand-directed signaling" is often considered to be inconsistent with the traditional receptor theory. In this review, I show how the mathematics of the receptor theory can be used to measure the observed affinity and relative efficacy of protean ligands at G protein-coupled receptors. The basis of this analysis rests on the assumption that the fraction of agonist bound in the form of the active receptor-G protein-guanine nucleotide complex is the biochemical equivalent of the pharmacological stimulus. Consequently, this stimulus function is analogous to the current response of a ligand-gated ion channel. Because guanosine triphosphate (GTP) greatly inhibits the formation of the active quaternary complex, even the most efficacious agonists probably only elicit partial receptor activation, and it seems likely that the ceiling of 100% receptor activation is not reached in the intact cell with high intracellular concentrations of GTP. Under these conditions, the maximum of the stimulus function is proportional to the ratio of microscopic affinity constants of the agonist for ground and active states. Ligand-directed signaling depends on the existence of different active states of the receptor with different selectivities for different G proteins or other effectors. This phenomenon can be characterized using classic pharmacological methods. Although not widely appreciated, it is possible to estimate the product of observed affinity and intrinsic efficacy expressed relative to that of another agonist (intrinsic relative activity) through the analysis of the concentration-response curves. No other information is required. This approach should be useful in quantifying agonist activity and in converting the two disparate parameters of potency and maximal response into a single parameter dependent only on the agonist-receptor-effector complex.

Two-state Models and the Analysis of the Allosteric Effect of Gallamine at the M2 Muscarinic Receptor

The Journal of Pharmacology and Experimental Therapeutics. Jun, 2008  |  Pubmed ID: 18305010

We measured the influence of gallamine on the functional responses and binding properties of selected agonists at the M(2) muscarinic receptor and analyzed the data within the context of the allosteric ternary complex model. Our analysis showed that gallamine modified agonist affinity without influencing efficacy. To explain this behavior, we investigated the allosteric ternary complex model at a deeper level of analysis to assess allosterism in terms of the differential affinity of gallamine for ground and active states of the receptor. Our simulations showed that two-state models based on a single orthosteric site for the agonist linked to an allosteric site for gallamine could not account for affinity-only modulation, even if multiple conformations of ground and active states were considered. We also expanded the tandem two-site model (J Biol Chem 275:18836-18844, 2000) within the context of the allosteric ternary complex model and analyzed the resulting hybrid model at the level of receptor states. This model posits that the agonist first binds to a relay site and then shuttles to the activation site to turn on the receptor. If it is assumed that allosterism occurs at the relay site and not the activation site, then this model can account for affinity-only modulation in a manner consistent with the allosteric ternary complex model.

Use of Acetylcholine Mustard to Study Allosteric Interactions at the M(2) Muscarinic Receptor

The Journal of Pharmacology and Experimental Therapeutics. Nov, 2008  |  Pubmed ID: 18682569

We explored the interaction of a nitrogen mustard derivative of acetylcholine with the human M(2) muscarinic receptor expressed in Chinese hamster ovary cells using the muscarinic radioligand, [3H]N-methylscopolamine (NMS). Acetylcholine mustard caused a concentration-dependent, first-order loss of [3H]NMS binding at 37 degrees C, with the half-maximal rate constant occurring at 24 microM and a maximal rate constant of 0.16 min(-1). We examined the effects of various ligands on the rate of alkylation of M(2) receptors by acetylcholine mustard. N-methylscopolamine and 4-(trimethylamino)-2-butynyl-(3-chlorophenyl)carbamate (McN-A-343) competitively slowed the rate of alkylation, whereas the inhibition by gallamine reached a plateau at high concentrations, indicating allosteric inhibition. In contrast, 17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-beta]-pyrimido[1,2-alpha]benzimidazole (WIN 51708) had no effect. We also measured the inhibition of [3H]NMS binding by acetylcholine mustard at 0 degrees C, conditions under which there is little or no detectable covalent binding. In these experiments, the dissociation constant of the aziridinium ion of acetylcholine mustard was estimated to be 12.3 microM. In contrast, the parent mustard and alcoholic hydrolysis product of acetylcholine mustard were without effect. Our results show that measurement of the effects of ligands on the rate of inactivation of the orthosteric site by a small site-directed electrophile is a powerful method for discriminating competitive inhibition from allosterism.

Selectivity of Agonists for the Active State of M1 to M4 Muscarinic Receptor Subtypes

The Journal of Pharmacology and Experimental Therapeutics. Jan, 2009  |  Pubmed ID: 18824613

We measured the intrinsic relative activity (RA(i)) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA(i) is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. First, we estimated RA(i) values for a panel of agonists acting at the M(4) muscarinic receptor coupled to three distinct G-protein pathways: G(i) inhibition of cAMP accumulation, G(s) stimulation of cAMP accumulation, and G alpha(15) stimulation of phosphoinositide hydrolysis. Our results show similar RA(i) values for each agonist, suggesting that the same active state of the M(4) receptor triggers the activation of the three G proteins. We also estimated RA(i) values for agonists across M(1) to M(4) muscarinic subtypes stably transfected in Chinese hamster ovary cells. Our results show selectivity of McN-A-343 [4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride] for the M(1) and M(4) subtypes and selectivity of pilocarpine for the M(1) and M(3) subtypes. The other agonists tested lacked marked selectivity among M(1) to M(4) receptors. Finally, we estimated RA(i) values from published literature on M(1), M(2), and M(3) muscarinic responses and obtained results consistent with our own studies. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity.

Estimation of Relative Microscopic Affinity Constants of Agonists for the Active State of the Receptor in Functional Studies on M2 and M3 Muscarinic Receptors

Molecular Pharmacology. Feb, 2009  |  Pubmed ID: 18996972

In prior work, we have shown that it is possible to estimate the product of observed affinity and intrinsic efficacy of an agonist expressed relative to that of a standard agonist simply through the analysis of their respective concentration-response curves. In this report, we show analytically and through mathematical modeling that this product, termed intrinsic relative activity (RA(i)), is equivalent to the ratio of microscopic affinity constants of the agonists for the active state of the receptor. We also compared the RA(i) estimates of selected muscarinic agonists with a relative estimate of the product of observed affinity and intrinsic efficacy determined independently through the method of partial receptor inactivation. There was good agreement between these two estimates when agonist-mediated inhibition of forskolin-stimulated cAMP accumulation was measured in Chinese hamster ovary cells stably expressing the human M(2) muscarinic receptor. Likewise, there was good agreement between the two estimates when agonist activity was measured on the ileum from M(2) muscarinic receptor knockout mice, a convenient assay for M(3) receptor activity. The RA(i) estimates of agonists in the mouse ileum were similar to those estimated at the human M(3) receptor with the exception of 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), which is known to be an M(1)- and M(4)-selective muscarinic agonist. Additional experiments showed that the response to McN-A-343 in the mouse ileum included a non-M(3) muscarinic receptor component. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity and ligand-directed signaling.

The Guinea Pig Ileum Lacks the Direct, High-potency, M(2)-muscarinic, Contractile Mechanism Characteristic of the Mouse Ileum

Naunyn-Schmiedeberg's Archives of Pharmacology. Oct, 2009  |  Pubmed ID: 19582435

We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M(2) receptor knockout, and M(3) receptor knockout mice characterized by negative log EC(50) (pEC (50)) values +/- SEM of 6.75 +/- 0.03, 6.26 +/- 0.05, and 6.99 +/- 0.08, respectively. The corresponding E (max) values in wild-type and M(2) receptor knockout mice were approximately the same, but that in the M(3) receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M(3) responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M(3) knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response. The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

A Conserved Motif in the Membrane Proximal C-terminal Tail of Human Muscarinic M1 Acetylcholine Receptors Affects Plasma Membrane Expression

The Journal of Pharmacology and Experimental Therapeutics. Jan, 2010  |  Pubmed ID: 19841475

We investigated the functional role of a conserved motif, F(x)(6)LL, in the membrane proximal C-tail of the human muscarinic M(1) (hM(1)) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence (423)FRDTFRLLL(431). Wild-type and mutant hM(1) receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [(3)H]N-methylscopolamine binding assays. The plasma membrane expression of hM(1) receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM(1) receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L(430) and L(431) play a role in folding hM(1) receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V(46) and L(47), that, when mutated, reduce the plasma membrane expression of hM(1) receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM(1) receptors to achieve a transport-competent state.

Investigating the Interaction of McN-A-343 with the M2 Muscarinic Receptor Using Its Nitrogen Mustard Derivative

Biochemical Pharmacology. Apr, 2010  |  Pubmed ID: 19913516

We investigated whether the aziridinium ion formed from a nitrogen mustard derivative (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate; BR384) structurally related to McN-A-343 (4-(trimethyl-amino)-2-butynyl N-(3-chlorophenyl)carbamate) interacts allosterically or orthosterically with the M(2) muscarinic receptor. Chinese hamster ovary cells expressing the human M(2) muscarinic receptor were incubated with the aziridinium ion of BR384 in combination with McN-A-343 or other known orthosteric and allosteric ligands for various incubation times. After removing unreacted ligands, we measured the binding of [(3)H]N-methylscopolamine to residual unalkylated receptors. Affinity constants, rate constants for alkylation, and cooperativity constants were estimated for the interacting ligands using a mathematical model. Receptor alkylation by BR384 was consistent with a two-step process. After rapidly equilibrating with the receptor (step one), the aziridinium ion-receptor complex became covalently linked with a first order rate constant of about 0.95min(-1) (step two). McN-A-343, acetylcholine and N-methylscopolamine competitively protected the M(2) receptor from irreversible alkylation by BR384. In contrast, the allosteric modulators, gallamine and WIN 51,708 (17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-beta]pyrimido[1,2-alpha]benzimidazole), allosterically inhibited or had no effect on, respectively, receptor alkylation by BR384. There was good agreement between affinity constants estimated from the kinetics of receptor alkylation and by displacement of [(3)H]N-methylscopolamine binding. Our results suggest that BR384 covalently binds to the orthosteric site of the M(2) receptor and that McN-A-343 binds reversibly at the same locus. Our method of analyzing allosteric interactions does not suffer from the limitations of more conventional approaches and can be adapted to detect allosteric interactions at receptors other than the muscarinic subtypes.

Mutagenesis of Nucleophilic Residues Near the Orthosteric Binding Pocket of M1 and M2 Muscarinic Receptors: Effect on the Binding of Nitrogen Mustard Analogs of Acetylcholine and McN-A-343

Molecular Pharmacology. Oct, 2010  |  Pubmed ID: 20643905

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 mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M(2) D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M(2) D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M(2) receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M(1) and M(2) receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.

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

The Journal of Pharmacology and Experimental Therapeutics. Aug, 2011  |  Pubmed ID: 21576379

We describe a modification of receptor theory for the estimation of observed affinities (K(obs)) 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 (ε(i)) states and analogous parameters for the fractions of the free receptor population in the active (ε(sys)) and inactive (ε(i-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 (K(obs) and τ) as well as a parameter proportional to the activity of the free receptor complex, τ(sys). Two related parameters are proportional to the fraction of the free (τ(i-sys)) and occupied (τ(i)) receptor populations in the inactive state. We show that the estimates of the affinity constants of orthosteric ligands for the active (K(b)) and inactive (K(a)) states of the receptor are equivalent to τK(obs)/τ(sys) and τ(i)K(obs)/τ(i-sys), respectively. We verify our method with computer simulation techniques and apply it to the analysis of M(2) and M(3) 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 State

The Journal of Pharmacology and Experimental Therapeutics. Aug, 2011  |  Pubmed ID: 21576380

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 (K(obs)) 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, ε(i)). 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 K(obs) and the parameter τ, which is proportional to ε. We introduce the parameter τ(i), which is proportional to ε(i). We have previously shown that the product, K(obs)τ, of one agonist, expressed relative to that of another (intrinsic relative activity, RA(i)), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, K(obs)τ(i), of one agonist, expressed relative to that of another (intrinsic relative inactivity, RI(i)), 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 M(3) 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 (RA(i)/RI(i)).

Characterization of Muscarinic Receptors in the Human Bladder Mucosa: Direct Quantification of Subtypes Using 4-DAMP Mustard

Urology. Sep, 2011  |  Pubmed ID: 21777958

To characterize pharmacologically relevant muscarinic receptors in the human bladder mucosa and detrusor muscle using radioligand binding assays with [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) and 4-DAMP mustard.

Muscarinic Agonists and Antagonists: Effects on Gastrointestinal Function

Handbook of Experimental Pharmacology. 2012  |  Pubmed ID: 22222706

Muscarinic agonists and antagonists are used to treat a handful of gastrointestinal (GI) conditions associated with impaired salivary secretion or altered motility of GI smooth muscle. With regard to exocrine secretion, the major muscarinic receptor expressed in salivary, gastric, and pancreatic glands is the M(3) with a small contribution of the M(1) receptor. In GI smooth muscle, the major muscarinic receptors expressed are the M(2) and M(3) with the M(2) outnumbering the M(3) by a ratio of at least four to one. The antagonism of both smooth muscle contraction and exocrine secretion is usually consistent with an M(3) receptor mechanism despite the major presence of the M(2) receptor in smooth muscle. These results are consistent with the conditional role of the M(2) receptor in smooth muscle. That is, the contractile role of the M(2) receptor depends on that of the M(3) so that antagonism of the M(3) receptor eliminates the response of the M(2). The physiological roles of muscarinic receptors in the GI tract are consistent with their known signaling mechanisms. Some so-called tissue-selective M(3) antagonists may owe their selectivity to a highly potent interaction with a nonmuscarinic receptor target.

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