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CELLULAR AND MOLECULAR

Persistent Binding and Functional Antagonism by Xanomeline at the Muscarinic M₅ Receptor

Departments of Psychiatry (M.K.O.G., E.E.E.-F.), Pharmacology (E.E.E.-F.), and Neuroscience (E.E.E.-F.), University of Minnesota Medical School, Minneapolis, Minnesota

Received for publication May 27, 2005
Accepted July 6, 2005.

	Abstract

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Xanomeline is a functionally selective M₁/M₄ muscarinic acetylcholine receptor agonist. We have previously identified a novel mode of interaction of this ligand with the muscarinic M₁ receptor that involves persistent binding and activation of the receptor after extensive washout. In the present study, we tested the hypothesis that xanomeline also binds in a wash-resistant manner to muscarinic receptor subtypes where it exhibits low or no efficacy, such as the M₅ receptor subtype. A secondary hypothesis is that persistent binding of xanomeline to the M₅ receptor results in wash-resistant antagonism to the effects of full agonists. These hypotheses were tested in Chinese hamster ovary cells stably expressing the M₅ receptor. In these cells, xanomeline is a weak partial agonist and is able to inhibit carbachol-induced phosphoinositide hydrolysis to the maximal response of xanomeline in a concentration-dependent manner. Pretreatment with xanomeline followed by extensive washing resulted in a significant wash-resistant reduction in receptor affinity with no significant change in maximal cell surface receptor density. This was associated with wash-resistant antagonism of carbachol-induced activation of phosphoinositide hydrolysis at the M₅ receptor, reflected as decreased carbachol potency without a change in the maximal response. Similar experiments using the partial agonist pilocarpine demonstrated a reduction of pilocarpine potency as well as maximal response. Our results clearly indicate that wash-resistant binding of xanomeline to the muscarinic M₅ receptor is accompanied by persistent antagonism of receptor function. They also suggest a relationship between the efficacy of xanomeline and the functional consequences of its wash-resistant binding at different muscarinic receptor subtypes.

Xanomeline (3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine) has been identified as a potent muscarinic receptor agonist displaying functional selectivity for M₁ and M₄ receptors (Shannon et al., 1994; Ward et al., 1995; Bymaster et al., 1997, 1998). Because of the functional selectivity of xanomeline, interest has been generated in its potential therapeutic use in Alzheimer's disease, which is characterized by a progressive decline in cognitive function and memory deficits. Although the disease is accompanied by a marked loss of presynaptic cholinergic neurons, postsynaptic M₁ muscarinic receptors, which are involved in learning and memory, remain largely intact (Ladner and Lee, 1998). Hence, drug developmental efforts have focused on designing selective M₁ muscarinic receptor agonists in an attempt to restore cholinergic function. Clinical studies have shown that xanomeline significantly improves the cognitive function of patients with Alzheimer's disease as well as decreasing behavioral symptoms such as hallucinations and delusions (Bodick et al., 1997), which also has led to interest in xanomeline as a potential therapy for schizophrenia.

Xanomeline exhibits a novel mode of interaction with the M₁ receptor different from that used by conventional agonists. It persistently binds to and continues to activate the receptor even after extensive washing (Christopoulos et al., 1998, 1999; Jakubik et al., 2002). There is evidence that the persistent attachment of xanomeline takes place away from the classical binding site, whereas the active group of xanomeline interacts reversibly with the primary receptor activation site (Christopoulos et al., 1999; Jakubik et al., 2002).

In spite of its functional selectivity for the M₁ and M₄ muscarinic receptors (Shannon et al., 1994; Ward et al., 1995; Bymaster et al., 1997, 1998), xanomeline does not discriminate in its binding affinity among all five subtypes of muscarinic receptors (Bymaster et al., 1997; Watson et al., 1998; Wood et al., 1999). However, there is no information about whether xanomeline persistently binds to non-M₁ receptor subtypes, particularly those at which xanomeline exhibits low or no efficacy; e.g., the M₅ receptor. Such effects may have important clinical significance. In addition to supplementing the knowledge base of how ligands bind to muscarinic receptors, these studies also may help shape future drug design. We undertook the current study to determine whether the persistent binding of xanomeline seen at the M₁ receptor subtype is also evident at the M₅ receptor where it exhibits low efficacy and to assess its functional consequence.

The M₅ muscarinic acetylcholine receptor subtype was the last to be cloned. Because selective M₅ agonists or antagonists are not available, little is known about the physiological role of this receptor. However, studies using mutant mice lacking M₅ receptors have been useful in providing some insight (Wess, 2004). There is evidence that M₅ receptors are important in regulating cerebral blood flow (Yamada et al., 2003), facilitating dopamine release in the striatum (Yamada et al., 2003) and nucleus accumbens (Forster et al., 2002) and in modulating morphine- and cocaine-associated reward and withdrawal processes (Basile et al., 2002; Fink-Jensen et al., 2003).

	Materials and Methods

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Materials. [³H]N-Methylscopolamine (81 Ci/mmol) was purchased from DuPont (Wilmington, DE); [myo-³H]inositol (71 Ci/mmol) was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK); [¹⁴C]inositol-1-phosphate (300 mCi/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO); Dulbecco's modified Eagle's medium was purchased from Invitrogen (Carlsbad, CA); geneticin was obtained from Calbiochem (San Diego, CA); and bovine calf serum was supplied by Hyclone Laboratories (Logan, UT). Xanomeline tartrate was a generous gift from Eli Lilly & Co. (Indianapolis, IN); all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell Culture. CHO cells, stably expressing the human M₅ muscarinic acetylcholine receptor (CHO hM₅) (provided by Dr. M. Brann, University of Vermont Medical School, Burlington, VT) were grown for 4 days at 37°C in Dulbecco's modified Eagle's medium, supplemented with 10% bovine calf serum and 50 µg/ml geneticin, in a humidified atmosphere consisting of 5% CO₂ and 95% air. Cells were harvested 4 days after subculture by trypsinization followed by centrifugation (300g; 3 min) and resuspension of the pellet in HEPES buffer (110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1 mM MgSO₄, 25 mM glucose, 20 mM HEPES, 58 mM sucrose, pH 7.4, and 340 mOsM), repeated twice.

Pretreatment with Xanomeline. To determine the functional and binding properties of wash-resistant xanomeline, CHO hM₅ cells were incubated with increasing concentrations of xanomeline at 37°C. After 1 h, cells were diluted with HEPES buffer, centrifuged (300g; 3 min), and resuspended in buffer. This was repeated three times to remove unbound xanomeline.

Saturation Binding Assays. CHO hM₅ cells were incubated for 1 h at 37°C in the absence or presence of xanomeline (1, 10, or 30 µM) followed by centrifugation and resuspension in HEPES buffer (three times) as described above. Cells were then incubated with increasing concentrations of [³H]N-methylscopolamine (NMS) (0.02-3.5 nM) for 1 h at 37°C using 100,000 cells/assay tube in a final incubation volume of 1 ml. Nonspecific binding was determined using 10 µM atropine. The reaction was terminated by filtration on Whatman GF/C filters (Whatman Schleicher and Schuell, Keene, NH) using a Brandel cell harvester (Brandel Inc., Gaithersburg, MD). Filters were washed three times with 4-ml aliquots of ice-cold saline and dried before radioactivity (disintegrations per minute) was measured using liquid scintillation counting. Protein determinations were performed according to the method of Bradford (Bradford, 1976).

Competition Binding Assays. Three different protocols were used for competition binding assays. All used 100,000 cells/assay tube at 37°C in a final incubation volume of 1 ml. In the first protocol, CHO hM₅ cells were incubated for 1 h simultaneously with a fixed concentration of [³H]NMS (0.5 nM) and increasing concentrations of xanomeline (0.1 nM-100 µM). To assess the persistent binding of xanomeline, a second set of experiments was performed in which cells were first pretreated for 1 h with increasing concentrations of xanomeline (10 nM-1 mM) followed by extensive washing as described previously. Cells were then incubated with the radioligand (0.5 nM) for 1 h. Further experiments were designed to determine the effects of persistent binding of xanomeline on agonist binding. After pretreating cells for 1 h with vehicle or various concentrations of xanomeline (1, 10, or 30 µM) and washing extensively, cells were incubated with a fixed concentration of [³H]NMS (0.5 nM) and increasing concentrations of carbachol (0.1 µM-10 mM). In all instances, nonspecific binding was determined using 10 µM atropine. The reactions were terminated by filtration as described above.

Assay of Phosphoinositide (PI) Hydrolysis. CHO hM₅ cells were suspended in HEPES buffer and loaded with [myo-³H]inositol (8 µCi/ml) for 1 h at 37°C. Labeled cells were washed with HEPES buffer. The cells were either pretreated with xanomeline (1, 10, or 30 µM) and washed as described previously or immediately resuspended in HEPES buffer containing 10 mM LiCl. Labeled cells were distributed to assay tubes (500,000 cells/tube) and allowed to incubate for 15 min at 37°C. Concentration-response curves for the stimulation of PI hydrolysis by carbachol or pilocarpine were constructed in the absence or in the presence of xanomeline pretreatment. To assess the antagonistic effects of xanomeline at the M₅ receptor, concentration-response curves for the stimulation of PI hydrolysis by xanomeline were constructed in the absence or presence of 3 µM carbachol. In all cases, the reaction was allowed to proceed for 1 h after the addition of agonist or vehicle control before being stopped with chloroform/methanol (2:1). Samples were centrifuged (450g; 15 min), and total inositol phosphates were separated on DOWEX AG1-X8 resin using [¹⁴C]inositol-1-phosphate as a recovery standard. The amount of radioactivity (disintegrations per minute) in each sample was determined by liquid scintillation counting and adjusted for recovery.

Data Analysis. After subtraction of nonspecific binding, data from each complete saturation binding assay were analyzed via nonlinear regression with Prism 4.0 (GraphPad Software Inc., San Diego, CA) to derive individual estimates of B_max (total receptor density) and K_D (radioligand receptor equilibrium dissociation constant). Competition binding isotherms were analyzed via nonlinear regression using Prism to derive estimates of the Hill slope factor and the IC₅₀ (midpoint location or potency parameter). Data were refitted according to both one- and two-site mass action binding models, and the better model was determined by an extra sum-of-squares test using Prism. IC₅₀ values were converted to K_I values (competitor-receptor dissociation equilibrium constant) according to eq. 1 (Cheng and Prusoff, 1973),

(1)

where [D] and K_D denote the concentration and dissociation constant of the radioligand, respectively.

In functional assays of PI hydrolysis, individual agonist concentration-response curve data were fitted to the four-parameter logistic function (using Prism) as shown in eq. 2,

(2)

where E is effect, [A] is the concentration of agonist, n_H is the midpoint slope, EC₅₀ is the midpoint location parameter, and E_max and basal are the upper and lower asymptotes, respectively. Values of K_D, IC₅₀, and EC₅₀ were estimated as negative logarithms.

To determine the partial agonist properties of xanomeline, the comparative method (Barlow et al., 1967) was used whereby xanomeline and pilocarpine concentration-response curves were compared with that of a reference full agonist (carbachol) curve. The analysis was performed using global curve-fitting with Prism 4.0. Xanomeline and pilocarpine concentration-response data were fitted to the following form of the operational model of agonism (Black and Leff, 1983) (eq. 3),

(3)

where E_m is the maximum possible response of the cell above Basal, K_A is the agonist-receptor equilibrium dissociation constant, n is the slope of the transducer function linking occupancy to response, and is the operational definition of efficacy. The parameters Basal, E_m and n in eq. 3 were fixed to the values of basal, E_max, and n_H, respectively, obtained from carbachol data fitted to eq. 2, thus allowing the estimation of K_A and for xanomeline and pilocarpine.

To determine an empirical estimate of the potency of xanomeline as an antagonist (pA₂), carbachol dose-response curves were constructed in the absence or presence of different concentrations of xanomeline and globally fitted to eq. 4 using Prism 4 (Motulsky and Christopoulos, 2003),

(4)

where [A] represents the concentration of the agonist, [B] represents the concentration of the antagonist, and s represents the Schild slope factor for the antagonist. E_max, basal, n_H, and EC₅₀ were obtained from concentration response curves of the agonist in the absence of antagonist. pA₂ represents the negative logarithm of the concentration of antagonist that shifts the agonist EC₅₀ by a factor of 2.

Results are expressed as means ± S.E.M. Statistical significance was determined by paired or unpaired Student's t tests, as appropriate. A probability (p) value <0.05 was preset to indicate statistical significance.

	Results

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Inositol Phosphate Production by Various Agonists at the M₅ Muscarinic Receptor. To ascertain the functional properties of xanomeline at the M₅ muscarinic receptor, assays of muscarinic acetylcholine receptor-mediated PI hydrolysis were undertaken. The accumulation of inositol phosphates was measured in the presence of increasing concentrations of carbachol, xanomeline, or pilocarpine in CHO hM₅ cells (Fig. 1). The carbachol concentration-response curve obtained in each experiment was incorporated as a reference for a curve-fitting procedure according to the operational model of agonism (eq. 3) to obtain functional estimates of the affinity and operational efficacy of xanomeline at the M₅ receptor. As seen in Table 1, xanomeline and pilocarpine are partial agonists (log <1) in comparison with carbachol at the M₅ receptor. Furthermore, xanomeline is more potent than carbachol or pilocarpine.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Agonist-mediated PI hydrolysis in CHO cells stably expressing human M5 muscarinic acetylcholine receptors. Cells were incubated for 1 h at 37°C with increasing concentrations of carbachol (), xanomeline (), or pilocarpine (). Values represent the means ± S.E. of 4 to 10 experiments conducted in triplicate.

Antagonistic Effects of Xanomeline on the Production of Inositol Phosphates by Carbachol. Further experiments were designed to assess whether xanomeline, a partial agonist, can act as an antagonist to a full agonist at the M₅ receptor. Cells were incubated for 1 h with 3 µM carbachol in the absence or in the presence of increasing concentrations of xanomeline. As shown in Fig. 2, xanomeline was able to antagonize the ability of carbachol to stimulate PI production in a concentration-dependent manner down to the level of maximal receptor activation by xanomeline alone.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Antagonism of carbachol-induced stimulation of PI hydrolysis by xanomeline in CHO cells stably expressing human M5 muscarinic acetylcholine receptors. Cells were incubated for 1 h at 37°C with increasing concentrations of carbachol () or xanomeline in the absence () or in the presence () of 3 µM carbachol. Values represent the means ± S.E. of three experiments conducted in triplicate.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Inhibition of [3H]NMS binding by xanomeline in CHO cells stably expressing human M5 muscarinic acetylcholine receptors. The binding of 0.5 nM [3H]NMS was measured in the presence of increasing concentrations of xanomeline () or after pretreatment with increasing concentrations of xanomeline followed by washing (). Values represent the means ± S.E. of five experiments conducted in triplicate.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Effects of xanomeline pretreatment, followed by washout, on [3H]NMS binding in CHO cells stably expressing human M5 muscarinic acetylcholine receptors. Cells were pretreated with the indicated concentrations of xanomeline for 1 h at 37°C and washed extensively. Cells were subsequently incubated for 1 h with increasing concentrations of [3H]NMS. Values represent the means ± S.E. of five experiments conducted in triplicate.

Effects of Xanomeline Pretreatment on Carbachol Competition Binding. The binding of 0.5 nM [³H]NMS at the M₅ muscarinic receptor in intact CHO cells was measured in the presence of increasing concentrations of carbachol after pretreating the cells with increasing concentrations of xanomeline or vehicle for 1 h and washing extensively. Although pretreatment with 10 and 30 µM xanomeline resulted in a decrease of total counts (disintegrations per minute) (Fig. 5A), the apparent affinity of carbachol for the M₅ receptor to residual receptors did not change significantly under any of the pretreatment conditions (Fig. 5B; Table 3), and nonlinear regression analysis of the data revealed a one-site binding model in all of the groups. The results are summarized in Table 3.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Inhibition of [3H]NMS binding by carbachol in CHO cells stably expressing human M5 muscarinic acetylcholine receptors after 1 h of pretreatment with xanomeline followed by washing. Cells were pretreated with the indicated concentrations of xanomeline for 1 h at 37°C and washed extensively. The binding of 0.5 nM [3H]NMS was measured in the presence of increasing concentrations of carbachol. A, results expressed as disintegrations per minute. B, competition isotherms normalized according to corresponding controls in the absence of carbachol. Values represent the means ± S.E. of six experiments conducted in triplicate.

Effects of Xanomeline Pretreatment on Carbachol or Pilocarpine-Stimulated Production of Inositol Phosphates. Cells were incubated with either vehicle or various concentrations of xanomeline for 1 h followed by extensive washing. Full concentration-response curves to carbachol were then established (Fig. 6). Results are summarized in Table 4. A significant reduction in potency of carbachol was seen after pretreating cells with increasing concentrations of xanomeline, as evidenced by a shift in midpoint location. Basal inositol phosphate production between groups was not significantly different, and the maximal inositol phosphate production by carbachol did not differ between groups. Analysis of the data using eq. 4 determined that residual xanomeline behaved in a way corresponding to a functional antagonist pA₂ value of 6.23 ± 0.24 (n = 9). Although maximal cell surface receptor density, as determined by saturation binding assays, was not significantly altered after pretreatment with xanomeline and washing, there seemed to be a trend showing a decrease in receptor number as concentrations of xanomeline increased. It is to be noted that the concentration of carbachol required to elicit a half-maximal response is approximately 10-fold lower than that necessary for half-receptor occupancy (Tables 3 and 4). This difference implicates involvement of receptor spareness in the response. To test whether the lack of effects of persistent xanomeline binding on the maximal response to carbachol was due to spare receptors, the same experiments were repeated using the partial agonist pilocarpine instead of carbachol (Fig. 7; Table 4). Pretreatment with xanomeline resulted in decreased potency as well as a significant decrease in the maximal effect of pilocarpine. The latter effect seemed to be limited in magnitude.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Effects of xanomeline pretreatment, followed by washout, on carbachol-stimulated PI hydrolysis in CHO cells stably expressing human M5 muscarinic acetylcholine receptors. Cells were pretreated with the indicated concentrations of xanomeline for 1 h at 37°C and washed extensively. Cells were subsequently incubated for 1 h with increasing concentrations of carbachol. Values represent the means ± S.E. of nine experiments conducted in triplicate.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Effects of xanomeline pretreatment, followed by washout, on pilocarpine-stimulated PI hydrolysis in CHO cells stably expressing human M5 muscarinic acetylcholine receptors. Cells were pretreated with the indicated concentrations of xanomeline for 1 h at 37°C and washed extensively. Cells were subsequently incubated for 1 h with increasing concentrations of pilocarpine. Values represent the means ± S.E. of four experiments conducted in triplicate.

	Discussion

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In the present study, we have investigated the effects of xanomeline at the M₅ muscarinic receptor subtype. We have shown that xanomeline acts as a potent partial agonist at the M₅ receptor. Thus, xanomeline elicits only a fraction of maximal response of carbachol and also antagonizes the effects of carbachol at the M₅ receptor subtype. We also have described a wash-resistant component of xanomeline whereby this agonist binds persistently to the M₅ receptor resulting in wash-resistant antagonism of receptor activation by a full agonist.

Previous studies have shown that xanomeline acts as a muscarinic agonist with functional selectivity for the M₁ (Shannon et al., 1994; Ward et al., 1995; Bymaster et al., 1998) and M₄ (Bymaster et al., 1997) muscarinic receptor subtypes. In contrast, binding studies have shown that xanomeline has a similar affinity at all five subtypes (Bymaster et al., 1997; Watson et al., 1998; Wood et al., 1999). Xanomeline also exhibits a novel mode of interaction with the M₁ receptor, with both reversible and wash-resistant components. It has been previously shown that xanomeline persistently binds to and activates the M₁ receptor, even after extensive washing (Christopoulos et al., 1998). Evidence suggests that persistent attachment of xanomeline develops at receptor regions distinct from the classic agonist binding site (Christopoulos et al., 1998, 1999; Jakubik et al., 2002).

In this work, we have shown that xanomeline functionally acts as a potent partial agonist at the M₅ receptor in comparison with carbachol as measured by inositol phosphate production (Fig. 1). More importantly, we have shown that xanomeline can act as an antagonist to full agonists at the M₅ muscarinic receptor. Thus, the ability of carbachol to produce inositol phosphates was decreased by xanomeline in a concentration-dependent manner to the level of the maximal functional response of xanomeline as a partial agonist (Fig. 2).

In agreement with previous reports (Watson et al., 1998; Wood et al., 1999), we have demonstrated that xanomeline binds with high affinity to the M₅ receptor. Furthermore, the binding of xanomeline to the M₅ receptor subtype consists of two separate components: reversible and wash-resistant binding (Fig. 3). These results are similar to previous findings at the M₁ receptor (Christopoulos et al., 1998, 1999; Jakubik et al., 2002, 2004). As determined in saturation binding experiments, residual binding of xanomeline significantly decreased the affinity of [³H]NMS for the receptor without changing maximal receptor density (Fig. 4). Further binding experiments were conducted to determine the effects of residual xanomeline binding on agonist competition, which showed no apparent effect on carbachol potency for the receptor. Together, these data support the theory that xanomeline persistent binding affects the primary binding domain on the M₅ receptor in a competitive manner. Thus, although a portion of the hydrophobic tail of xanomeline may interact with a site different from the classical binding site in a persistent (noncompetitive) manner (Jakubik et al., 2002), the actual interaction of the active group of xanomeline at the classical binding site is reversible. We also have presented evidence that xanomeline can act as a persistent antagonist at the M₅ receptor after washout of free drug.

As shown in Fig. 3, the inhibition of [³H]NMS binding by residual xanomeline was incomplete. This could be because of negative allosteric interaction between xanomeline and NMS binding to distinct, albeit interactive, domains on the receptor (Ehlert, 1985). Jakubik et al. (2002) have previously presented evidence that persistent binding of xanomeline allosterically modulates binding at the classical binding domain on the M₁ receptor. The saturable effect of persistently bound xanomeline on the maximal response to pilocarpine is consistent with an allosteric mode of interaction (Ehlert, 1985) (Fig. 7). Alternatively, a fraction of the receptor population may not be susceptible to the wash-resistant component of xanomeline binding. More detailed experiments are necessary to determine the molecular mechanisms of interaction of xanomeline with the M₅ receptor.

Regardless of the mechanisms involved, our data present evidence that xanomeline binds in a wash-resistant manner and acts as a persistent antagonist at the M₅ muscarinic receptor. Although xanomeline binds persistently at both the M₁ and M₅ receptors, the functional consequences of this mode of binding are different, resulting in persistent agonism at the M₁ and persistent antagonism at the M₅ receptor. The ability of xanomeline to activate some subtypes of muscarinic receptors while antagonizing others in a wash-resistant manner represents a unique and complex pharmacological profile. Persistent activation of M₁ muscarinic receptors might be beneficial in the treatment of Alzheimer's dementia, unless this results in marked desensitization and down-regulation of the receptor. Prolonged antagonism of the M₅ receptor, on the other hand, may lead to decreased cerebral blood flow, which may help explain the syncopal episodes associated with xanomeline in clinical trials (Bodick et al., 1997). A small study has shown that syncope induced by xanomeline is associated with a decrease in blood pressure (Medina et al., 1997), although the mechanism remains unknown. Further experiments should be carried out to determine whether xanomeline is able to persistently antagonize other muscarinic receptor subtypes. For example, persistent blockade of presynaptic M₂ receptors would be beneficial because of potentiation of acetylcholine release. In contrast, prolonged antagonism of the function of the M₃ subtype of muscarinic receptors would result in serious side effects; e.g., dry mouth, urinary retention, and constipation. These side effects might result in decreased patient compliance.

	Footnotes

 
This work was supported by National Institutes of Health Grant NS25743.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.090134.

ABBREVIATIONS: M, muscarinic acetylcholine receptor; CHO, Chinese hamster ovary; hM₅, human M₅ muscarinic acetylcholine receptor; NMS, N-methylscopolamine; PI, phosphoinositide.

Address correspondence to: Dr. Esam E. El-Fakahany, Department of Psychiatry, MMC 392, University of Minnesota Medical School, 420 Delaware St. S.E., Minneapolis, MN 55455. E-mail: elfak001{at}umn.edu

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