Introduction

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The G protein-coupled muscarinic acetylcholine receptors are subject to allosteric modulation (Tuek and Proka, 1995; Ellis, 1997; Christopoulos et al., 1998; Holzgrabe and Mohr, 1998). Initial evidence from experiments with isolated contracting heart preparations demonstrated that gallamine (Clark and Mitchelson, 1976) and alkane-bis-ammonium-type compounds (Lüllmann et al., 1969; Mitchelson, 1975) acted as antagonists with a ceiling effect at high concentrations. Subsequent ligand-receptor binding studies with [³H]N-methylscopolamine ([³H]NMS) supported an allosteric mechanism (Stockton et al., 1983; Jepsen et al., 1988; Choo and Mitchelson, 1989). Allosteric agents retard the dissociation of [³H]NMS, indicating formation of a ternary complex with the radioligand-occupied receptor. In addition, muscarinic allosteric agents interact with unliganded receptors and, thereby, inhibit the association of conventional, orthosteric ligands (Kostenis and Mohr, 1996; Schröter et al., 2000). Hence, allosteric modulation of association and dissociation has opposite effects on equilibrium binding of the orthosteric ligand. Whereas gallamine and many alkane-bis-ammonium agents reduce [³H]NMS binding, alcuronium elevates binding of [³H]NMS to cardiac muscarinic M₂ receptors (Tuek et al., 1990) because of pronounced inhibition of [³H]NMS-receptor dissociation (Schröter et al., 2000). These findings prompted a search for allosteric enhancers of acetylcholine binding. Although alcuronium fails to elevate acetylcholine binding at any of the five muscarinic subtypes (Jakubík et al., 1997), brucine derivatives with a structure similar to one-half of the alcuronium molecule enhance the binding and action of acetylcholine (Birdsall et al., 1999). Among the five subtypes, acetylcholine-occupied M₂ receptors seem to be rather insensitive to this effect of the brucine analogs (Gharagozloo et al., 1999).

Muscarinic allosteric actions depend on the allosteric agent, the orthosteric ligand, and the muscarinic receptor subtype (Lee and El-Fakahany, 1988; Ellis et al., 1991). Jakubík et al. (1997) investigated the effects of alcuronium, brucine, and structurally related compounds on the equilibrium binding of 12 agonists in cloned M₁-M₄ receptors. Among the allosteric agents, alcuronium had, by far, the highest affinity for M₂ receptors. When pilocarpine was bound to the M₂ receptors, the affinity of alcuronium was even somewhat increased. The factor of cooperativity (Ehlert, 1988a) between alcuronium and pilocarpine,  = 0.37 (Jakubík et al., 1997), means a 2.7-fold higher affinity of alcuronium for pilocarpine-occupied M₂ receptors compared with free receptors. Therefore, we selected pilocarpine-alcuronium interactions to investigate the functional consequences of ternary complex formation in an intact M₂-containing tissue, i.e., contracting guinea pig atria. Alcuronium unexpectedly suppressed the agonist effects of pilocarpine and even caused pilocarpine to behave like a muscarinic antagonist. We confirmed these unique functional properties of a muscarinic allosteric agent at the level of receptor-G protein coupling by measuring pilocarpine stimulation of [³⁵S]GTPS binding to Chinese hamster ovary (CHO) membranes expressing M₂ receptors. The combined results demonstrate for the first time that a muscarinic allosteric agent can modulate the intrinsic efficacy of an orthosteric muscarinic receptor ligand.

	Materials and Methods

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Organ Bath Experiments. The procedure to measure the actions of muscarinic agents in contracting guinea pig atria has been described previously (Tränkle et al., 1998). Left atria were mounted in organ baths containing 20 ml of oxygenated Tyrode's solution (136.9 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 1.05 mM MgCl₂, 11.9 mM NaHCO₃, 0.21 mM NaH₂PO₄, and 5.5 mM dextrose; 95% O₂/5% CO₂; pH 7.3; 32°C). The preparations were preloaded with 5 millinewton and electrically stimulated via platinum contact electrodes at 3 Hz with rectangular pulses of 5-ms duration. The voltage was 1.5-fold over the threshold of excitation amounting to about 2 V. Contraction force (CF) was recorded isometrically. After an equilibration period of 60 min (CF = 5.5 ± 0.1 millinewton, mean ± standard error, n = 234 atria), a cumulative concentration/effect curve for the negative inotropic action of the agonist under study was recorded. Each agonist concentration was applied for 10 min, a period sufficient to obtain equilibrium effects. Contraction force was expressed as the percentage of the value found at the end of the equilibration period. The agonist was removed from the organ bath over a period of 30 min by replacing the Tyrode's solution with fresh solution every 10 min. Thereafter, the preparation was preincubated with the allosteric test compound for 60 min before another agonist concentration/effect curve was measured in the presence of the allosteric compound. Contraction force was expressed as the percentage of the value at the end of the preincubation period with the allosteric agent. The agonist was washed out using Tyrode's solution still containing the allosteric agent in the concentration as before. After 30 min of washout, the concentration of the allosteric agent was increased, and, after a preincubation phase of 60 min, another concentration/effect curve of the agonist was measured. Modifications of this protocol are mentioned under Results.

Cell Culture and Membrane Preparation. Cell culture and [³⁵S]GTPS binding experiments were carried out in a similar way as described by Burford et al. (1995). A CHO cell line stably transfected with the human gene for the muscarinic M₂ receptor and wild-type CHO cells were a gift of Prof. Dr. G. Lambrecht (Department of Pharmacology, Biocenter Niederursel, University of Frankfurt/Main, Germany). Cells were cultured at 37°C under humidified air supplemented with 5% CO₂ in Ham's F 12 medium containing 10% fetal calf serum, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 1 mM glutamine. For membrane preparation, cells grown to confluence were treated for 24 h with 5 mM Na-butyrate added to the culture medium before harvesting the cells in a buffer of 10 mM HEPES, 154 mM NaCl, and 0.7 mM EDTA, pH 7.4 at room temperature. The subsequent steps were carried out at 4°C. The cell suspension was centrifuged at 185g (Avanti J25, rotor type JS-7.5; Beckman Instruments, Palo Alto, CA) for 5 min, the supernatant was discarded, the pellet was resuspended in homogenization buffer (10 mM HEPES, 10 mM EDTA, 10 mM NaF, and 10 mM Na₂P₂O₇, pH 7.4), and the first centrifugation step was repeated. The pelleted cells were disrupted using a Polytron homogenizer (PT 10-35; Kinematica AG, Littau, Switzerland; level 7, six bursts of 5-s duration and intervals of 30 s in between), and the resulting homogenate was centrifuged at 40,000g (Avanti J25, rotor type JA 25.50) for 17 min. After discarding the supernatant, the pellet was resuspended in storage buffer (10 mM HEPES and 0.1 mM EDTA, pH 7.4) and centrifuged again. The membranes were resuspended in storage buffer and stored at 80°C.

[³⁵S]GTPS Binding Experiments. Membranes (100 µl, final concentration 150 µg of protein/ml) were added to the incubation buffer (900 µl; 10 mM HEPES, 100 mM NaCl, and 10 mM MgCl₂, pH 7.4) containing (final concentrations) 0.07 nM [³⁵S]GTPS (1250 Ci/mmol), 10 µM GDP, and the test compounds at the indicated concentrations. After an incubation period of 60 min at 30°C, the incubation medium was filtered through glass fiber filters (Schleicher & Schüll, Dassel, Germany), and filter-bound radioactivity was measured by liquid scintillation counting. As determined in homologous competition experiments with [³H]N-methylscopolamine ([³H]NMS 0.2 nM, 83.5 Ci/mmol) and increasing concentrations of unlabeled NMS under incubation conditions as described above, the density of M₂ receptors amounted to about 2.5 pmol/mg of membrane protein.

[³H]Oxotremorine M Binding Experiments. M₂ receptor-containing membranes from guinea pig hearts that had been prepared as described previously (e.g., Tränkle et al., 1998) were incubated with 86 Ci/mmol of 1 nM [³H]oxotremorine M in a buffer analogous to that applied by Jakubík et al. (1997) but without 0.5 mM GTP (136 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 0.2 mM KH₂PO₄, 0.8 mM Na₂HPO₄, and 10 mM Na-HEPES, pH 7.4, at 25°C). After time periods appropriate to reach binding equilibrium of up to 3 h, the incubation medium was filtered through glass fiber filters (no. 6, Schleicher & Schüll) followed by two rinses with ice-cold incubation buffer. Filter-bound radioactivity was measured by liquid scintillation counting. Nonspecific binding of [³H]oxotremorine M was determined in the presence of 1 µM atropine and amounted to about 20% of the total binding.

Data Analysis. Indicated are mean values ± standard error. Unless otherwise indicated concentration/effect curves were fitted to the data by nonlinear regression analysis using the following four-parameter logistic equation

(1)

X is the drug concentration; Y is the response. The parameters "Top" and "Bottom" refer to the upper and lower plateau of the sigmoidal curve, respectively. Log EC₅₀ denotes the log X at the inflection point of the sigmoidal curve, and n_H is the slope factor of the curve.

(2)

(3)

(4)

Drugs. Oxotremorine M iodide was obtained from Research Biochemicals International (Natick, MA). Pilocarpine hydrochloride, gallamine triethiodide, (±)-propranolol, hexamethonium bromide, Ham's F12 medium, fetal calf serum, penicillin G, streptomycin, glutamine, and HEPES were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Na-butyrate was from Acros Organics (Geel, Belgium). [³⁵S]GTPS, [³H]oxotremorine M, and [³H]N-methylscopolamine were from PerkinElmer Life Sciences (Homburg, Germany). Alcuronium chloride was generously provided by Hoffmann-La Roche AG (Grenzach Wyhlen, Germany).

	Results

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Drug Effects on the Contraction Force of Guinea Pig Atria. The concentration/effect curve for the negative inotropic action of pilocarpine as obtained under control conditions is shown in Fig. 1A. The inflection point of the curve was (mean ± standard error, n = 13) pEC₅₀ = 6.10 ± 0.03; the slope factor was n_H = 1.28 ± 0.12; and the maximal effect of pilocarpine was indicated by the bottom plateau of the curve at CF_min = 18 ± 2% of the predrug force of contraction. Repeated measurements of pilocarpine concentration/effect curves in control preparations over intervals of up to 300 min after starting with the first curve revealed that the sensitivity of the preparations to pilocarpine remained stable (data not shown). Alcuronium alone (0.1-100 µM) in the preincubation period of 60 min had no effect on the contraction force of the preparations (data not shown). The time course of the negative inotropic action of pilocarpine seemed to be unchanged under the influence of alcuronium. The pilocarpine concentration/effect curve was hardly affected at 0.1 (not shown) and 0.3 µM alcuronium. At higher concentrations of alcuronium (Fig. 1A), the maximal effect of pilocarpine was reduced (one-way ANOVA, P < 0.0001), whereas the slope factors of the curves (P = 0.298) and the pEC₅₀ values (P = 0.801) were not different from the respective control values.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   A, negative inotropic action of pilocarpine in isolated guinea pig left atria (3 Hz) in the absence and in the presence of alcuronium at the indicated concentrations. Ordinate, isometric force of contraction as a percentage of the predrug value. Abscissa, concentration of pilocarpine added to the organ bath in cumulative fashion. Data represent means ± S.E.M. from measurements in n = 3 to 13 preparations. Error bars are not shown when they do not exceed the symbols. Curve fitting based on a four-parameter logistic function. B, loss of the maximal effect of pilocarpine in dependence on the concentration of alcuronium. The bottom plateaus of the concentration/effect curves of A are plotted on the ordinate against the concentration of alcuronium on the abscissa. Curve fitting based on a four-parameter logistic function, for details see text.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   A, negative inotropic action of oxotremorine M in contracting guinea pig left atria in the absence and in the presence of alcuronium at the indicated concentrations. Ordinate, isometric force of contraction as a percentage of the predrug value. Abscissa, concentration of oxotremorine M added to the organ bath in cumulative fashion. Data represent means ± standard error from measurements in n = 2 to 8 preparations. Error bars are not shown when they do not exceed the symbols. Curve fitting based on a four-parameter logistic function. Slope factors and minimal plateaus did not differ significantly and were set constant to nH = 1.44 and CFmin = 8, respectively. B, Schild plot of the oxotremorine M curve shift induced by alcuronium. Ordinate, dose ratio (DR) = oxotremorine M EC50 in the presence of alcuronium divided by oxotremorine M EC50 without alcuronium. Abscissa, log concentration of alcuronium. Indicated are individual shift factors based on complete concentration/effect curves. Linear regression analysis yielded a line with a slope not different from unity.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   A, concentration/effect curves for the negative inotropic action of oxotremorine M under the influence of pilocarpine in the presence of a fixed concentration of alcuronium. Ordinate, isometric force of contraction of the contracting guinea pig left atria as a percentage of the respective value before addition of oxotremorine M. Abscissa, concentration of cumulatively applied oxotremorine M. Data points indicate mean values ± S.E.M. of n = 2 to 10 preparations. Curve fitting was based on a four-parameter logistic function. Slope factors and minimal plateaus did not differ significantly and were set constant with nH = 1.62 and CFmin = 9, respectively. B, Schild plot of the oxotremorine M curve shift induced by pilocarpine in the presence of 10 µM alcuronium. Ordinate, dose ratio (DR) = oxotremorine M EC50 with pilocarpine in the presence of 10 µM alcuronium divided by oxotremorine M EC50 without pilocarpine in the presence 10 µM alcuronium. Abscissa, log concentration of pilocarpine in the presence of 10 µM alcuronium. Indicated are individual shift factors based on complete concentration/effect curves. Linear regression analysis yielded a line with a slope not different from unity.

Drug Effects on M₂ Receptor-Mediated G Protein Activation. We measured the effects of the test compounds on [³⁵S]GTPS binding in membranes of CHO cells stably transfected with the human M₂ receptor gene. To verify that drug effects were mediated via M₂ receptors, control experiments were carried out in membranes of wild-type CHO cells; none of the test compounds in high concentrations affected [³⁵S]GTPS binding (data not shown). In the membranes of CHO cells expressing M₂ receptors, pilocarpine stimulated [³⁵S]GTPS binding concentration dependently (Fig. 4A); E_max was 172 ± 3%, the half-maximal effect occurred at pEC₅₀ = 5.48 ± 0.12 (slope factor n_H = 0.68 ± 0.10, n = 3 experiments in up to quadruplicate determinations). The effect of pilocarpine was then measured in the presence of 3 µM (n = 3 experiments in duplicate determinations) and 10 µM (n = 2 experiments in duplicate determinations; Fig. 4A) alcuronium. Alcuronium alone significantly suppressed basal [³⁵S]GTPS binding (one-way ANOVA, p < 0.0001). In the presence of 3 µM alcuronium, both the inflection point of the pilocarpine curve, pEC₅₀ = 5.43 ± 0.16, and the slope factor of the curve, n_H = 0.85 ± 0.23, were unchanged compared with the pilocarpine curve recorded under control conditions (unpaired t test, two-tailed, p > 0.05). The maximal effect with 10² M pilocarpine, however, was significantly attenuated by alcuronium (one-way ANOVA, p < 0.0001). In the presence of 10 µM alcuronium, pilocarpine was inactive.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effects of pilocarpine and alcuronium alone and in combination on [35S]GTPS binding to membranes of CHO cells expressing muscarinic M2 receptors. Ordinate, [35S]GTPS binding as a percentage of the value obtained in the absence of test compound. Abscissa, concentration of the indicated test compound. A, effect of pilocarpine in the absence and in the presence of the indicated concentrations of alcuronium. B, concentration/effect curves for the actions of alcuronium on the basal binding of [35S]GTPS in the absence of pilocarpine and on the 102 M pilocarpine-stimulated [35S]GTPS binding. Included is the curve of the conventional muscarinic antagonist atropine, stippled line. Data points indicate mean values ± S.E.M. of n = 2 to 3 experiments. Error bars are not shown when they do not exceed the symbols. Curve fitting based on a four-parameter logistic function; point-to-point connection in Fig. 4A for 10 µM alcuronium.

2

View larger version (19K): [in this window] [in a new window]   Fig. 5.   A, concentration/effect curves for the stimulation of [35S]GTPS binding in the absence and in the presence of the indicated concentrations of alcuronium. Ordinate, [35S]GTPS binding as a percentage of the value measured in the absence of a test compound. Abscissa, log concentration of oxotremorine M. Indicated are mean values ± S.E.M. of n = 1 to 3 experiments. Curve fitting based on a four-parameter logistic function; slope factors and maximal effects did not differ significantly and were set constant to nH = 0.77 and Emax = 165%. Included are data (asterisks) obtained in the presence of a combination of 10 µM alcuronium and 100 µM pilocarpine. For details see text. B, Schild plot of the oxotremorine M curve shift induced by alcuronium. Ordinate, dose ratio (DR) = oxotremorine M EC50 in the presence of alcuronium divided by oxotremorine M EC50 without alcuronium. Abscissa, log concentration of alcuronium. Indicated are individual shift factors based on complete concentration/effect curves. Linear regression analysis yielded a line with a slope not different from unity.

Drug Interactions with Labeled Agonist Binding to M₂ Receptors. As mentioned before, the binding experiments of Jakubík et al. (1997) pointed to a modest positive cooperativity between pilocarpine and alcuronium ( = 0.37), whereas our functional experiments did not give evidence for an increased potency of pilocarpine in the presence of alcuronium and vice versa. We carried out radioligand binding experiments that were designed in a stepwise approach analogous to that of Jakubík et al. (1997) with the following main modifications. We used the agonist [³H]oxotremorine M instead of the antagonist [³H]N-methylscopolamine, and we omitted 0.5 mM GTP from the assay medium. First, the displacement of [³H]oxotremorine by unlabeled oxotremorine M (Fig. 6A) yielded the log equilibrium dissociation constant of [³H]oxotremorine M binding, pK_D = 8.82 ± 0.28. Second, the curve for the competition between [³H]oxotremorine and pilocarpine (Fig. 6A) gave the binding constant of pilocarpine, pK_X = 6.77 ± 0.05. Third, the reduction of [³H]oxotremorine M binding by increasing concentrations of alcuronium (Fig. 6B) yielded (eq. 3) the binding constant of alcuronium at free M₂ receptors, pK_A = 6.09 ± 0.06, and its factor of cooperativity with [³H]oxotremorine M,  = 64 ± 10. In accordance, alcuronium has a low affinity for oxotremorine M-occupied M₂ receptors (p[K_A] = 4.28) as seen previously in [³H]oxotremorine M dissociation experiments (Maass and Mohr, 1996). Fourth, parallel experiments carried out in the presence of a fixed concentration of 0.3 µM pilocarpine (Fig. 6B) yielded (eq. 4) the factor of cooperativity between alcuronium and pilocarpine,  = 1.12 ± 0.25. Thus, under the present conditions, there is a nearly neutral cooperativity between alcuronium and pilocarpine, i.e., the affinity of alcuronium for pilocarpine-occupied receptors (p[K_A] = 6.04) is almost the same as in free receptors and vice versa. The parameters characterizing the actions of the test compounds in the three applied assays are compiled in Tables 1 and 2.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   A, inhibition of the specific binding of 1 nM [3H]oxotremorine M to guinea pig heart membranes by unlabeled oxotremorine M (oxo M) and by pilocarpine (piloc.). Ordinate, [3H]oxotremorine M binding as a percentage of the value measured in the absence of a test compound. Abscissa, log concentration of agonist. Indicated are mean values ± S.E.M. of n = 7 experiments in triplicate determinations. Curve fitting based on a four-parameter logistic function; slope factors did not differ significantly from unity (F-test, p > 0.05) and were set constant to nH = 1.00. B, Inhibition of the specific binding of 1 nM [3H]oxotremorine M by alcuronium in the absence and in the presence of 0.3 µM pilocarpine. Ordinate, [3H]oxotremorine M binding as a percentage of the value measured in the absence of a test compound. Abscissa, log concentration of alcuronium. Indicated are mean values ± S.E.M. of n = 3 to 4 experiments in triplicate determinations. Curve fitting based either on eq. 3 for the control (Ehlert, 1988a) or on eq. 4 for the presence of pilocarpine (Jakubík et al., 1997); for details, see text.

View this table: [in this window] [in a new window]   TABLE 1 Concentrations for half-maximal effects (log EC50 values) of the muscarinic agonists pilocarpine and oxotremorine M under the indicated assay conditions.

View this table: [in this window] [in a new window]   TABLE 2 Alcuronium concentrations (log values) for half maximum effects as estimates of the alcuronium binding affinity under the indicated assay conditions.

	Discussion

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The main finding of the present study is that the allosteric modulator alcuronium converts the agonist pilocarpine into an antagonist in intact myocardial tissues. Muscarinic receptors are linked to myocardial contraction force by a complex signaling pathway. To gain insight into the events on the molecular level, we also studied ligand effects on G protein activation in membranes of CHO cells expressing M₂ muscarinic receptors. In both assays, the allosteric agent alcuronium did not affect the potency of the muscarinic agonist pilocarpine but reduced its efficacy. In contrast, with oxotremorine M as the agonist, alcuronium had no effect on the efficacy but reduced the potency. Because both organ bath and in vitro assays yielded corresponding results, we conclude that effects observed in the contracting guinea pig atria seem to result from alcuronium modulation of agonist-mediated G protein activation. Because alcuronium had no effect on G protein activity in wild-type CHO cells but only in CHO cells expressing M₂ receptors, the site of action of alcuronium seems to be the receptor protein.

The distinct effects of alcuronium on pilocarpine and oxotremorine M likely result from structural differences in the respective agonist/receptor-complexes. With respect to the binding of these agonists, the differential interaction of alcuronium with oxotremorine M- and pilocarpine-occupied M₂ receptors has been demonstrated by Jakubík et al. (1997) in radiolabeled antagonist binding experiments. Likewise, the radiolabeled agonist binding experiments of the present study indicate that the affinity of alcuronium for pilocarpine-occupied receptors was markedly higher than for oxotremorine M-occupied receptors (Table 2). Alcuronium binding to pilocarpine-occupied receptors takes place at concentrations that are close to the concentrations in which alcuronium binds to free M₂ receptors. The slight divergence between the cooperativity factors found by Jakubík et al. (1997) and in this study for the interaction of alcuronium and pilocarpine ( = 0.37 and  = 1.12, respectively) may be accounted for by the different assay conditions. Furthermore, Jakubík et al. (1997), using the antagonist [³H]N-methylscopolamine in the presence of GTP, found identical binding affinity for pilocarpine and oxotremorine M (Table 1), whereas we found ~100-fold lower binding affinity of pilocarpine compared with oxotremorine M in the present study, using [³H]oxotremorine M in the absence of GTP (Table 1). Remarkably, the functional experiments in the contracting guinea pig atria and the [³⁵S]GTPS binding experiments (Table 1) yielded a similar ratio. Thus, the [³H]agonist binding assay applied here seems to mimic the conditions of the functional experiments. Taken together, the findings of the binding and functional assays (Table 2) are compatible with the notion that alcuronium, in terms of the ternary complex model of allosteric interactions, shows nearly neutral cooperativity with pilocarpine and pronounced negative cooperativity with oxotremorine M.

In line with the binding data, the pilocarpine concentration/effect curves for the negative inotropic effect and for the G protein activation (Figs. 1A and 4A) remain in the same concentration range when alcuronium is present. In both sets of experiments a shift of the oxotremorine M concentration/effect curve by pilocarpine (in the presence of alcuronium) provides strong evidence that pilocarpine actually is bound to the receptor and acts as an antagonist (Figs. 3 and 5A asterisks). The alcuronium-induced reduction of the maximal effect of pilocarpine, therefore, indicates that the formation of ternary complexes is paralleled by a loss of pilocarpine's intrinsic efficacy to induce G protein activation.

Loss of agonist efficacy by formation of ternary complexes has not yet been described in muscarinic receptors. When self-limiting antagonistic actions were observed in organ-bath experiments with allosteric agents such as gallamine and alkane-bis-ammonium-type compounds and the agonists carbachol, acetylcholine, and oxotremorine (e.g., Lüllmann et al., 1969; Mitchelson, 1975; Clark and Mitchelson, 1976; Tränkle et al., 1998), the formation of ternary complexes was the pivotal event but the maximal agonist effects were maintained. Furthermore, allosteric augmentation of the action of acetylcholine (Birdsall et al., 1999), oxotremorine M, and other agonists (Doleal and Tuek, 1998) has been observed without an impairment of the maximal agonist response. Enhanced agonist binding depended on the formation of ternary complexes, without changes in agonist efficacy. In a report on gallamine inhibition of rat myocardial adenylate cyclase by oxotremorine M and the partial agonist N-methyl-N-(1-methyl-4-pyrrolidino)-2-butynyl acetamide, Ehlert (1988b) observed reduction of the maximal effect of N-methyl-N-(1-methyl-4-pyrrolidino)-2-butynyl acetamide and raised the possibility that gallamine may have influenced the intrinsic efficacy of the partial agonist.

Pilocarpine is known as a partial agonist in M₂ receptors, whereas oxotremorine M is a full agonist (McKinney et al., 1991; Vogel et al., 1997). In line with this, pilocarpine reduced the contraction force to CF_min = 18 ± 2%, whereas the maximal effect of oxotremorine M (CF_min = 7 ± 2%) was more pronounced (p < 0.002, unpaired t test). In the [³⁵S]GTPS binding experiments, the maximal effect of pilocarpine was equal to that of oxotremorine M, but this may be related to the high level of M₂ receptor expression in the transfected CHO cells. Pilocarpine's sensitivity to allosteric modulation of intrinsic efficacy may be accentuated by its partial agonist character. Alcuronium alone had an inverse agonist action in this study similar to that of other muscarinic allosteric agents, e.g., the alkane-bis-ammonium agent hexane-1,6-bis(dimethyl-3'-phthalimidopropyl-ammonium bromide) (Hilf and Jakobs, 1992). Also in cardiomyocytes, alcuronium and gallamine induced an inverse agonist action on G protein activation (Jakubík et al., 1996) similar to that seen with conventional muscarinic antagonists (Jakubík et al., 1995). Taken together, these prototypal allosteric agents suppress basal G protein activity as inverse agonists and, thus, seem to stabilize preferentially inactive conformations of the muscarinic M₂ receptor. This negative intrinsic activity could have caused the conversion of pilocarpine into an antagonist. As depicted in Fig. 4, alcuronium suppressed the efficacy of pilocarpine to stimulate G proteins (Fig. 4B, upper curve) in the same concentration range in which it reduced spontaneous receptor activity (Fig. 4B, lower curve). This finding suggests that alcuronium's binding site, which is located in the entrance of the ligand binding cavity of the receptor protein (Ellis and Seidenberg, 2000; Buller et al., 2002), has a similar conformation in free and pilocarpine-occupied receptors. One may speculate that pilocarpine-mediated receptor activation goes along with a conformationally malleable receptor state, whereas the rigid alcuronium molecule, upon binding to the allosteric site, may imprint an inactive, more rigid conformation on the pilocarpine/receptor complexes. It is intriguing to speculate that muscarinic allosteric modulators could also induce the reverse action, i.e., preferential allosteric stabilization of active agonist/receptor complexes, thereby, increasing the intrinsic efficacy of an agonist. In case of adenosine A₁ receptors, the 2-amino-3-benzoylthiophene derivative PD 81,723 allosterically enhances agonist action and promotes constitutive receptor activity (e.g., Bruns and Fergus, 1990; Kollias-Baker et al., 1997).

The allosteric modulation of muscarinic receptor signaling described here differs from a topological and mechanistic point of view from the findings that Litschig et al. (1999) made with another G protein-coupled receptor, i.e., the metabotropic glutamate receptor hmGluR1b. A noncompetitive antagonist was found to inhibit receptor signaling without affecting glutamate binding. In that case, the antagonist did not influence basal receptor signaling activity, and the antagonist action was seen with a variety of agonists. The metabotropic glutamate receptors belong to a subfamily of G protein-coupled receptors that is characterized by a long N-terminal amino acid chain, which contains the agonist binding domain. Possibly, the noncompetitive antagonist binds between the extracellular glutamate binding domain and the transmembrane domain mediating signal transduction (Litschig et al., 1999). Muscarinic acetylcholine receptors differ such that the agonist binding domain resides in the transmembrane region of the ligand binding cavity and the allosteric site is located above that place, i.e., in the entrance of the ligand binding pocket. Thus, the present study demonstrates another, novel type of interference with G protein-coupled receptor signaling.

A major goal is to identify allosteric agents that modulate agonist and antagonist effects at specific muscarinic receptor subtypes. Here, we demonstrate for the first time that the intrinsic efficacy of muscarinic agonists may be subject to allosteric modulation at the M₂ subtype. Future studies will give more insight in how far this action depends on the type of allosteric agent, the type of orthosteric ligand, and the subtype of muscarinic receptor.