Introduction

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Both xanomeline (Shannon et al., 1994) and sabcomeline (Loudon et al., 1997) have been identified and developed as functionally selective muscarinic agonists for the M₁ receptor. Such agents have been proposed to enhance cognitive function and certain other behaviors associated with Alzheimer's disease (Bodick et al., 1997; Hatcher et al., 1998). Although xanomeline and sabcomeline are both reported to be selective agonists at M₁ receptors based on measurement of several in vitro and in vivo M₁-mediated functional responses (Shannon et al., 1994; Loudon et al., 1997), both compounds show relatively nonselective affinities for all five cloned muscarinic receptors (Table 1) with xanomeline possessing approximately 10- to 40-fold higher affinity than sabcomeline at each of the muscarinic receptors.

The high affinity of xanomeline and sabcomeline for M₂ receptors, the important role of M₂ receptors in mediating bradycardia (Stengel et al., 2000), and the lack of direct comparative information on the effects of these selective M₁ agonists at atrial receptors have prompted the present studies. Previous studies examining the effects of these agents at cardiac M₂ receptors were independently conducted in vivo in conscious (Shannon et al., 1994) and anesthetized (Loudon et al., 1997) rats. Interestingly, intravenous administration of sabcomeline produced a bradycardic effect (Loudon et al., 1997), whereas subcutaneous administration of xanomeline resulted in a dose-dependent increase in heart rate, an effect attributed to activation of M₁ receptors in sympathetic ganglia (Shannon et al., 1994). Thus, although these agents have high M₂ receptor affinities, the relatively high M₂ receptor affinities did not consistently translate into marked bradycardic effects. Recently, data are emerging to suggest that carbamylcholine can interact with both a high- and low-affinity state of the M₂ receptor in heart (Gies and Landry, 1988; Dawson and Poretski, 1990; Fryer et al., 1990; Haddad et al., 1990; Daeffler et al., 1999) and other preparations (Gies and Landry, 1988; Dawson and Poretski, 1990; Fryer et al., 1990; Haddad et al., 1990; Daeffler et al., 1999). Thus, in addition to comparing the functionally selective M₁ receptor agonists sabcomeline and xanomeline to the nonselective muscarinic agonist carbamylcholine at M₂ receptors mediating atrial rate, we also explored the potential role of the high and low M₂ receptor affinity states in atrial bradycardia.

	Materials and Methods

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Animals. Mice (129SvEv/CF-1 hybrids) were housed in polycarbonate ventilated cages. The animal room was maintained at 22-24°C with a relative humidity of 35 to 70% and daily light/dark cycle (6:00 AM-6:00 PM light). Food (Laboratory Rodent Diet, 5001; PMI Feed, Inc., St. Louis, MO) and water were supplied ad libitum. Experimental protocols and procedures were approved by the Eli Lilly and Company Animal Care and Use Committee.

Atrial Preparation. Mice (29-49 g) (Taconic Farms, Inc., Germantown, NY) were killed by cervical dislocation and the heart was quickly excised and placed in modified Krebs-bicarbonate buffer solution of the following composition: 4.6 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 118.2 mM NaCl, 10.0 mM glucose, 1.6 mM CaCl₂·2H₂O, and 24.8 mM NaHCO₃. Spontaneously beating left and right atria were dissected from ventricles and the left atrium was attached with thread to a stationary glass rod, whereas the right atrium was tied with thread to a force displacement transducer. The atria were placed in organ baths containing 10 ml of Krebs-bicarbonate buffer (see above for composition). The organ bath solution was maintained at 37°C and aerated with a 95:5% mixture of O₂:CO₂. The spontaneously beating left and right atria were placed under an initial force of 0.5 g and equilibrated for 20 min during which time the tissues were washed at 5-min intervals. Atrial rate in beats per minute was measured with Sensotec transducers (model MBL55140-02; Columbus, OH) that were coupled to a Compaq Deskpro-compatible data acquisition system (BIOPAC Systems, Inc., Goleta, CA).

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Determination of Receptor Occupancy for Carbamylcholine, Sabcomeline, and Xanomeline. Noncumulative bradycardic concentration-response curves to carbamylcholine, sabcomeline, or xanomeline were obtained as indicated above. EC₅₀ values were taken as the concentration of agonist that produced half-maximal bradycardia. For carbamylcholine and xanomeline we assumed a maximal response of 100% inhibition of heart rate.

Determination of Apparent Antagonist Dissociation Constants for Atropine and Sabcomeline. Atria were incubated with appropriate concentrations of vehicle, sabcomeline, xanomeline, or atropine for 30 min and bradycardic responses to carbamylcholine or sabcomeline were determined. Only one concentration of antagonist was studied in each tissue. Calculation of the antagonist dissociation constants assumed that the M₂ receptor was the predominant receptor involved in atrial bradycardia (Stengel et al., 2000), that inhibition involved competitive equilibrium antagonism, and that the compounds were not appreciably metabolized by the tissue.

Statistical Analyses. Results were expressed as the mean ± S.E.M. of isolated atria obtained from 3 to 10 animals as indicated in parentheses in the figures. Agonist concentration-response curves were analyzed by a three-parameter logistic nonlinear model (De Lean et al., 1978). The three modeled parameters included the maximal response of the tissue, the EC₅₀, and the slope of the curves. Each curve was fitted using SAS (SAS Institute Inc., Cary, NC) on a Compaq (Deskpro 5133; Compaq, Houston, TX) personal computer. One-way analysis of variance was used to compare changes in atrial rate (at 1, 3, 5, 10, 15, 20, 25, and 30 min) and mean pEC₅₀ (the negative logarithm of the EC₅₀) values between vehicle- and compound-treated groups. Dunnett's test for multiple comparisons versus a single control group was performed when appropriate. Analyses were run using SigmaStat for Windows (version 2.03; SPSS Science Inc., Chicago, IL) on the Compaq personal computer. Comparisons were considered significant for P values of 0.05 or less.

Drugs. Xanomeline (LY246708 hydrochloride) and sabcomeline were provided by the Lilly Research Laboratories (Indianapolis, IN). Carbamylcholine chloride and atropine sulfate were purchased from Sigma Chemical Co. (St. Louis, MO).

	Results

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Agonist Effects in Mouse Atria. Carbamylcholine (3.0 × 10⁷-3.0 × 10⁶ M) produced a marked concentration-dependent bradycardia in isolated mouse atria (Fig. 1, top). For all three effective concentrations, the reduction in atrial rate was maximal within 5 min and was maintained for the 30-min duration of the experiment. Carbamylcholine (3.0 × 10⁷ M) lowered atrial rate by approximately 20% (Fig. 1, top). A higher concentration of carbamylcholine (10⁵ M) virtually stopped atrial beating (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Time course for the bradycardia produced by increasing concentrations of carbamylcholine, sabcomeline, and xanomeline in mouse atria. Points are mean values and vertical bars represent the standard error of the mean for the number of tissues indicated in parentheses. Asterisks indicate those values that significantly differ from vehicle (P < 0.05).

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View larger version (19K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves for bradycardia (measured at 30 min) produced by sabcomeline, carbamylcholine, and xanomeline in mouse atria. Points are mean values and vertical bars represent the standard error of the mean for the number of tissues indicated in parentheses.

Receptor Occupancy versus Response Characteristics of Carbamylcholine, Sabcomeline, and Xanomeline. Assuming that the bradycardia produced by carbamylcholine, sabcomeline, and xanomeline was mediated by activation of M₂ atrial receptors, the fractional receptor occupancy was calculated for each agonist concentration (Fig. 3, top). This analysis indicated that carbamylcholine was a full agonist requiring less than 10% of the receptors to be occupied for greater than 50% response only when carbamylcholine was considered to interact with the low-affinity state of the M₂ receptor. When the affinity of carbamylcholine at the high-affinity state of the M₂ receptor was used, the receptor occupancy calculation was not consistent with classical receptor theory regarding receptor occupancy for a full agonist (Fig. 3, top). For example, if carbamylcholine were interacting with the high-affinity state of the M₂ receptor, then over 50% of the receptors must be occupied for a 20% response, an unlikely situation for a full agonist. Thus, carbamylcholine-induced bradycardia must be associated with activation of the low- not high-affinity state of the cardiac M₂ receptor.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Bradycardia produced by carbamylcholine in mouse atria (top) and by carbamylcholine, xanomeline, and sabcomeline as a function of the percentage of receptors occupied ([RA]/[RT] × 100). Curves for carbamylcholine (top) were generated using the apparent dissociation constants determined for interaction with the high- and low-affinity states of the M2 receptor (Table 2). Curves for sabcomeline and xanomeline (bottom) were generated using the apparent dissociation constants reported for interactions with M2 receptors (Table 1).

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Antagonism of Carbamylcholine-Induced Bradycardia by Sabcomeline and Xanomeline. Sabcomeline (3.0 × 10⁷ M and 10⁵ M) produced a marked inhibition of carbamylcholine-induced bradycardia (Fig. 4, top) with a K_B value at M₂ atrial receptors of 3.0 × 10⁸ M (pK_B = 7.5), close to the antagonist dissociation constant reported for M₂ receptors (pK_i = 6.69) (Loudon et al., 1997). In contrast, xanomeline (3.0 × 10⁷-10⁵ M) did not significantly alter carbamylcholine-induced bradycardia (Fig. 4, bottom). Thus, sabcomeline was a partial agonist at M₂ receptors, whereas xanomeline was not interacting with M₂ receptors in mouse atria.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effect of sabcomeline (top) and xanomeline (bottom) to antagonize carbamylcholine-induced bradycardia in mouse atria. Points are mean values and vertical bars represent the standard error of the mean for the number of preparations indicated in parentheses.

Effect of Atropine to Antagonize Carbamylcholine-, Sabcomeline-, and Xanomeline-Induced Bradycardia. The fact that sabcomeline was a partial M₂ receptor agonist, whereas xanomeline was a weak bradycardic agonist without M₂ receptor antagonist activity in mouse atria, led us to examine further whether the bradycardia produced by these M₁ receptor-selective agonists was mediated by muscarinic receptors. Atropine (3.0 × 10⁸ M) markedly inhibited carbamylcholine- and sabcomeline-induced bradycardia with pK_B values of 8.85 and 8.60, respectively (Fig. 5). However, atropine did not similarly inhibit xanomeline-induced bradycardia.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of atropine (3.0 × 108 M) to antagonize the bradycardia produced by carbamylcholine (top), sabcomeline (middle), and xanomeline (bottom) in mouse atria. Points are mean values and vertical bars represent the standard error of the mean for the number of tissues indicated in parentheses.

	Discussion

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Both sabcomeline and xanomeline are functional M₁ receptor agonists that have shown clinical efficacy in treating the cognitive deficits associated with Alzheimer's disease (Cooper et al., 1996; Kumar and Orgogozo, 1996; Bodick et al., 1997). Both compounds possess high affinity at M₂ receptors, raising the possibility that these agents may alter cardiac function. In fact, previous in vivo studies in rodents, although not conducted under similar conditions, suggested that sabcomeline produced bradycardia, whereas xanomeline induced tachycardia (Shannon et al., 1994; Loudon et al., 1997). The present studies indicate that although xanomeline and sabcomeline can both induce a bradycardic effect in isolated mouse atria, marked qualitative and quantitative differences exist in the characteristics of the M₂ receptor interactions between sabcomeline and xanomeline.

In addition, the present studies heightened our awareness of the possible physiological relevance of the high- and low-affinity states of the M₂ receptor to atrial bradycardia. In heart (Gies and Landry, 1988; Dawson and Poretski, 1990; Fryer et al., 1990; Haddad et al., 1990; Daeffler et al., 1999) and other preparations (Peralta et al., 1987; Gies and Landry, 1988; Herawi et al., 1988; Fraeyman et al., 1991; Burford et al., 1995b; Rinken, 1996; Vogel et al., 1997), carbamylcholine can interact with at least two states of the M₂ receptor characterized by high- and low-carbamylcholine affinity. In general, it is the high M₂ receptor affinity site that is most often measured and considered to be physiologically relevant. However, the present studies demonstrated that activation of the low- rather than high-affinity binding state of the M₂ receptor is likely to be responsible for the bradycardia induced by carbamylcholine. Calculation of receptor occupancy for carbamylcholine, a classical M₂ receptor full agonist known to induce marked bradycardia, revealed that the bradycardic efficacy of carbamylcholine was consistent with receptor occupancy theory only when efficacy was calculated using the affinity constant for carbamylcholine for the low-affinity rather than the high-affinity state of the M₂ receptor. These data are the first to extend to mammalian heart, the previous suggestion that "the low agonist affinity form of the cardiac muscarinic receptor is the physiologically active form" in embryonic chick heart (Halvorsen and Nathanson, 1981).

In mouse atria, sabcomeline was a weak, but potent M₂ receptor partial agonist producing a maximal of 50% reduction in heart rate. These data at M₂ receptors in mouse atria agree nicely with the partial agonist effects of sabcomeline to inhibit M₂-mediated acetylcholine release in rat cortical slices (Loudon et al., 1997). Partial agonist activity at M₂ receptors was further confirmed by the demonstration that low concentrations of sabcomeline markedly antagonized carbamylcholine-induced bradycardia in mouse atria with an antagonist dissociation constant of 3.0 × 10⁸ M, a value close to the EC₅₀ value for the bradycardic effects of sabcomeline and close to the previously reported K_A (Loudon et al., 1997). Sabcomeline-induced bradycardia was antagonized by atropine and the antagonist dissociation constant for atropine was similar when either sabcomeline or carbamylcholine served as agonist. Thus, sabcomeline was functionally a partial agonist of M₂ receptors with low concentrations capable of inhibiting carbamylcholine-induced bradycardia.

Interestingly, although xanomeline like sabcomeline also induced bradycardia in mouse atria, the in vitro bradycardic potency of xanomeline was lowest of the three agonists studied (sabcomeline > carbamylcholine > xanomeline with regard to bradycardic potency). Furthermore, although high concentrations of xanomeline induced a marked bradycardia, xanomeline (even as high as 10⁵ M) did not inhibit the bradycardic response to carbamylcholine, suggesting that xanomeline, unlike sabcomeline, was not a partial agonist at the M₂ receptors responsible for bradycardia.

Xanomeline and sabcomeline also possessed different kinetic profiles with regard to the onset of the bradycardic effect. Sabcomeline, like carbamylcholine, produced a rapid reduction in heart rate, which reached maximal effect within 5 min at all concentrations examined consistent with M₂ receptor activation. In contrast, the bradycardic effect produced by xanomeline occurred with a slower onset requiring approximately 15 min before maximal bradycardia occurred with each concentration of xanomeline. This observation suggested that xanomeline may not be activating mouse M₂ atrial receptors. In fact, an analysis of the receptor occupancy required for M₂ agonist activation with sabcomeline and xanomeline was consistent with this hypothesis. Using reported M₂ receptor affinities, sabcomeline possessed higher efficacy to activate atrial M₂ receptors than xanomeline (Fig. 3) and the receptor occupancy calculated for xanomeline using its reported high affinity for M₂ receptors was not consistent with classical theory. Xanomeline required greater than 95% of the receptors to be occupied before a bradycardic response could be observed, whereas for sabcomeline, bradycardia was observed with only 10 to 40% of receptors occupied. Last, the bradycardia produced by xanomeline was not antagonized by atropine, a potent nonselective muscarinic receptor antagonist, which blocked carbamylcholine and sabcomeline-induced bradycardia. Thus, although high concentrations of xanomeline were capable of producing bradycardia, the bradycardia was not mediated via activation of M₂ receptors as indicated by the slow development of bradycardia, weak efficacy of xanomeline, the inability of xanomeline to block carbamylcholine-induced bradycardia, and the inability of atropine to block xanomeline-induced bradycardia.

This conclusion is also consistent with the observation that the low- rather than high-affinity state of the M₂ receptor is most relevant to functional bradycardia. The reported high affinity of xanomeline for the M₂ receptor (Shannon et al., 1994) was likely a reflection of its affinity for the high-affinity state of the M₂ receptor, and thus was not relevant to the bradycardia observed with high xanomeline concentrations.

The inability of xanomeline to activate atrial M₂ receptors is consistent with the lack of effect of xanomeline on heart rate in humans as measured with continuous ambulatory monitoring (Bodick et al., 1997), and with the inability of xanomeline to produce a distinct bradycardic effect in vivo in rats (Shannon et al., 1994). In contrast, sabcomeline did produce a small but significant reduction in heart rate after intravenous administration to anesthetized rats (Loudon et al., 1997). To date, no clinical data on heart rate in humans with sabcomeline are available.

In summary, although sabcomeline and xanomeline can produce atrial bradycardia in vitro, marked differences were apparent in the characteristics of the bradycardic response to these agonists. Sabcomeline was a potent M₂ receptor partial agonist in mouse atria, producing a rapid bradycardic response that resulted in a maximal 50% decrease in heart rate. Sabcomeline was also capable of inhibiting the bradycardic effect of carbamylcholine. In contrast, xanomeline was considerably less potent than sabcomeline as a bradycardic agonist in mouse atria, produced a slower bradycardic effect, which was not blocked by atropine, and was incapable of inhibiting carbamylcholine-induced bradycardia. These data along with analysis of receptor occupancy curves suggest that the reported M₂ receptor affinity for xanomeline is not relevant in assessing its bradycardic potential. The fact that atropine did not block xanomeline-induced bradycardia suggested that the bradycardia was not a result of muscarinic receptor interactions. Furthermore, and most importantly, analysis of receptor occupancy curves revealed that muscarinic-induced bradycardia most likely resulted from activation of the low- rather than high-affinity state of the M₂ receptor.