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CELLULAR AND MOLECULAR

Long-Term Changes in the Muscarinic M₁ Receptor Induced by Instantaneous Formation of Wash-Resistant Xanomeline-Receptor Complex

Division of Neuroscience Research in Psychiatry, University of Minnesota Medical School, Minneapolis, Minnesota

Received August 10, 2007; accepted September 7, 2007.

	Abstract

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Unlike other M₁ muscarinic acetylcholine receptor agonists, xanomeline demonstrates a unique mode of binding to the receptor. It not only binds reversibly to the receptor's conventional orthosteric site but also binds persistently at a secondary binding domain(s) on the M₁ receptor. This results in persistent activation of the receptor even after extensive washout, and allosteric modulation of the orthosteric site. In the current study, we investigated how the effects of very brief exposure (1 min) of intact Chinese hamster ovary cells expressing M₁ receptors to xanomeline followed by washout change with time. Pretreatment with xanomeline for 1 min resulted in a concentration-dependent wash-resistant inhibition of [³H]N-methylscopolamine (NMS) binding, with a lower potency than that observed in the continuous presence of xanomeline in the binding assay medium. This effect was associated with wash-resistant receptor activation. Incubation of pretreated and washed cells in control medium for 24 h transformed the monophasic xanomeline wash-resistant binding curve to one that exhibits two distinct potencies. This was the result of the appearance of a new very high-potency binding component without a change in the low-potency state. The delayed effects of persistently bound xanomeline are mainly due to reduction of the maximal binding of [³H]NMS without a change in its affinity. These treatment conditions also reversed persistent receptor activation by xanomeline. Our results imply that brief exposure to xanomeline followed by washing and prolonged waiting may result in delayed receptor desensitization accompanied by internalization or down-regulation.

The M₁ mAChR is part of the muscarinic acetylcholine receptor family consisting of five receptor subtypes (M₁–M₅) and the superfamily of G protein-coupled receptors (Hulme et al., 1990). Finding a selective M₁ agonist for the treatment of Alzheimer's dementia is necessary to avoid serious side effects, such as salivation and gastrointestinal complications, due to activation of other muscarinic receptor subtypes (Bodick et al., 1997; Messer, 2002). The search for a selective M₁ agonist has been hampered by the highly conserved nature of the orthosteric site among the five receptor subtypes where acetylcholine and other conventional agonists bind (Bonner et al., 1991). A promising breakthrough was the discovery of multiple allosteric binding domains on the M₁ mAChR (Birdsall and Lazareno, 2005; Espinoza-Fonseca and Trujillo-Ferrara, 2006). These domains are distant from the orthosteric site, and they demonstrate enough variance in their sequence among receptor subtypes (Bonner et al., 1991; Espinoza-Fonseca and Trujillo-Ferrara, 2006) to possibly serve as targets for M₁-selective allosteric agonists (Spalding et al., 2002).

One such agonist, xanomeline (3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine), has been under intense study due to its unique characteristics of interaction with the M₁ mAChR. Xanomeline exhibits high potency and functional selectivity at the M₁ and M₄ mAChR (Shannon et al., 1994; Bymaster et al., 1997, 1998). More importantly, xanomeline exhibits multiple modes of binding to the M₁ mAChR. In addition to its reversible interaction with the orthosteric binding site on the receptor, xanomeline also demonstrates persistent binding to the M₁ mAChR accompanied by long-lasting receptor activation after extensive washing (Christopoulos and El-Fakahany, 1997; Christopoulos et al., 1998). There is evidence that the latter mode of xanomeline binding takes place at a secondary binding domain(s) on the receptor, resulting in allosteric modulation of the orthosteric site (Christopoulos et al., 1998; Jakubík et al., 2002, 2006).

A major component of wash-resistant xanomeline binding to the M₁ mAChR takes place almost instantaneously upon addition of xanomeline to cells that express the receptor followed by immediate washing (Jakubík et al., 2002, 2006). Interestingly, a similar rapid phase of xanomeline wash-resistant binding is absent at the M₂ mAChR (Jakubík et al., 2006), suggesting the existence of receptor subtype-specific conformations induced by xanomeline that might underlie its functional selectivity. Our laboratory recently reported that although very low concentrations of xanomeline do not exhibit immediate wash-resistant effects on radioligand binding to the M₁ mAChR, such effects are revealed upon incubating xanomeline-pretreated and washed cells in control medium for 24 h (De Lorme et al., 2006). These effects involve binding of xanomeline at a secondary allosteric domain on the receptor, and they are time-dependent. The purpose of the present study was to further explore the immediate and long-term effects of very brief exposure of the receptor to xanomeline on radioligand binding and receptor function.

	Materials and Methods

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Materials. [³H]N-Methylscopolamine (NMS) (81 Ci/mmol) was purchased from DuPont (Wilmington, DE); myo-[³H]inositol (71 Ci/mmol) was purchased from GE Healthcare (Chalfont, St. Giles, 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); G-418 (Geneticin) was obtained from Calbiochem (San Diego, CA); and bovine calf serum was supplied by Hyclone Laboratories (Logan, UT). Xanomeline tartarate was a generous gift from Eli Lilly & Co. (Indianapolis, IN). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell Culture. Chinese hamster ovary cells stably transfected with the human M₁ mAChR (M₁-CHO) (provided by Dr. M. Brann, University of Vermont Medical School, Burlington, VT) were grown for 4 days in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum and 50 µg/ml G-418. Cells were grown to confluence in culture flasks or 24-well plates at 37°C in a humidified atmosphere consisting of 5% CO₂, 95% air.

Pretreatment with Xanomeline. Confluent M₁-CHO cells were incubated in monolayer in the absence or presence of increasing concentrations of xanomeline at 37°C for 1 min, followed by three washes with media to remove unbound drug. The cells were either used in the assay immediately after washing, or they were allowed to incubate in monolayer for 24 h in the absence of free drug. A third group of cells was pretreated with xanomeline for 24 h, washed, and used immediately for assays. In all cases, the cells were washed with iso-osmotic HEPES buffer (110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1 mM MgSO₄, 25 mM glucose, 20 mM HEPES, and 58 mM sucrose; pH 7.4 ± 0.02; 340 ± 5 m Osm) three times before their use in binding or functional assays.

Displacement Binding Assays. M₁-CHO cells grown in 24-well plates were incubated for 1 h at 37°C simultaneously with a fixed concentration of the muscarinic receptor radioligand [³H]NMS (0.2 nM) and increasing concentrations of xanomeline (1 nM–100 µM). To assess the persistent binding of xanomeline, cells were first pretreated with increasing concentrations of xanomeline (1 pM–100 µM) followed by three washes with or without further incubation in the absence of free xanomeline as described previously. Cells were then incubated with 0.2 nM [³H]NMS for 1 h at 37°C. In all instances, nonspecific binding was determined using 10 µM atropine. Free radioligand was removed by surface washing, and labeled cells removed by the addition of 1 M NaOH. Radioactive content was determined by liquid scintillation spectrometry. Decreases in cell density were visually observed after long-term pretreatment conditions with concentrations of xanomeline greater than 0.1 µM. To quantitatively assess the extent of this decrease, protein content and cell counts were measured following long-term pretreatment conditions. Protein determinations were performed according to the method of Bradford (Bradford, 1976). Cell counts were performed manually using a hemocytometer. Displacement curves were normalized to correct for changes in protein content or cell number (Figs. 2 and 3).

View larger version (13K): [in this window] [in a new window]   Fig. 2. A, changes in protein content following long-term xanomeline pretreatment. Cells were pretreated with increasing concentrations of xanomeline for 1 min at 37°C, washed extensively, and then allowed to incubate in the absence of free xanomeline for 24 h (closed triangles), or continuously incubated with increasing concentrations of xanomeline for 24 h, followed by washing (open diamonds). Protein content was measured by the method of Bradford; 100% was defined as protein content measured in untreated cells. Values represent the means ± S.E.M. of four to six experiments performed in triplicate. B, data from individual displacement experiments (shown as means in Fig. 1) were adjusted for the changes in protein content. All other details are as for Fig. 1.

View larger version (13K): [in this window] [in a new window]   Fig. 3. A, changes in cell number following long-term xanomeline pretreatment. Cells were pretreated with increasing concentrations of xanomeline for 1 min at 37°C, washed extensively, and then allowed to incubate in the absence of free xanomeline for 24 h (closed triangles), or continuously incubated with increasing concentrations of xanomeline for 24 h, followed by washing (open diamonds). Cell counts were performed manually using a hemocytometer; 100% was defined as cell number measured in untreated control cells. Values represent the means ± S.E.M. of three to five experiments performed in triplicate. B, data from individual displacement experiments (shown as means in Fig. 1) were adjusted to reflect the changes in cell number. All other details are as for Fig. 1.

Dissociation Kinetics Assays. M₁-CHO cells grown in culture flasks were pretreated in the absence or presence of 10 µM xanomeline as described previously. Cells were harvested by trypsinization followed by centrifugation and resuspension three times in iso-osmotic HEPES buffer. Cells were then incubated with a fixed concentration of [³H]NMS (0.2 nM) for 1 h at 37°C using 100,000 cells/assay tube. After this period, 10 µM atropine was added to initiate ligand dissociation by inhibiting its reassociation. Nonspecific binding was defined as noted above. The dissociation reaction was terminated by filtration as described above. The amount of remaining bound radioactivity was measured at various time intervals to determine the dissociation rate of [³H]NMS.

Assay of Phosphoinositide Hydrolysis. Two different protocols were used for the PI assay. In the first set of experiments, the agonistic effects of wash-resistant xanomeline binding were compared with those induced by free xanomeline or carbachol. M₁-CHO cells grown in 24-well plates were loaded in monolayer with 1 µCi/ml myo-[³H]inositol for 24 h at 37°C. Cells were washed three times in iso-osmotic HEPES buffer to remove unincorporated myo-[³H]inositol, and then they were exposed to increasing concentrations of xanomeline or carbachol in the presence of 10 mM LiCl for 1 h at 37°C. To assess the persistent activation induced by brief xanomeline pretreatment, cells preloaded with myo-[³H]inositol were incubated in the presence of increasing concentrations of xanomeline for 1 min followed by three washes in iso-osmotic HEPES buffer. The reaction was allowed to proceed for 1 h at 37°C in the presence of 10 mM LiCl without further addition of agonist. In all cases, the reaction was stopped with 0.3 M HClO₄ and neutralized with 0.15 M K₂CO₃. Concentration-response curves for the stimulation of PI hydrolysis by carbachol, xanomeline, or wash-resistant xanomeline were constructed. In the second set of experiments, the effect of xanomeline pretreatment on PI hydrolysis elicited by carbachol was investigated. M₁-CHO cells were grown in culture flasks, loaded in monolayer with 1 µCi/ml myo-[³H]inositol for 24 h at 37°C, and pretreated in the absence or presence of 300 nM xanomeline as described previously. Cells were harvested by trypsinization followed by centrifugation and resuspension three times in isoosmotic HEPES buffer. Cells were resuspended in iso-osmotic HEPES buffer containing 10 mM LiCl, and then they were distributed to assay tubes (500,000 cells/tube). Concentration-response curves for the stimulation of PI hydrolysis by carbachol were constructed in the absence or presence of xanomeline pretreatments. The reaction was allowed to proceed for 1 h at 37°C after the addition of carbachol before being stopped with chloroform/methanol (2:1). For all experiments, samples were centrifuged (450g; 15 min), and total inositol phosphates were separated by ion-exchange chromatography (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 the value was adjusted for recovery.

Data Analysis. Displacement binding isotherms were analyzed via nonlinear regression using Prism (GraphPad Software Inc., San Diego, CA) to derive estimates of 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 4.0 (GraphPad Software Inc.). The nonreversible nature of xanomeline binding did not permit transforming IC₅₀ values to inhibition constants because such transformation assumes reversible competitive interaction. After subtraction of nonspecific binding, data from each complete saturation binding assay were analyzed via nonlinear regression with Prism to derive individual estimates of total receptor density (B_max) and radioligand-receptor equilibrium dissociation constant (K_D). Data from dissociation kinetic experiments were analyzed by Prism according to both monoexponential and biexponential dissociation models and the two fits compared by an extra-sum-of-squares test to estimate the radioligand dissociation rate constant (k_off). In functional assays of PI hydrolysis, individual concentration-response curve data were fitted to a four-parameter logistic function using Prism. Data shown are the means ± S.E.M. Comparisons between mean values were performed by unpaired t tests or one-way analysis of variance (ANOVA), as appropriate. A value of p < 0.05 was taken to indicate statistical significance.

	Results

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Effects of Prolonged Waiting following Brief Xanomeline Pretreatment on Radioligand Binding to the M₁ Muscarinic Receptor in Displacement-Type Experiments. Coincubation of M₁-CHO cells simultaneously with [³H]NMS and xanomeline resulted in concentration-dependent inhibition of radioligand-specific binding (Fig. 1). Preincubation of cells with increasing concentrations of xanomeline for 1 min followed by washing away free drug revealed progressive residual inhibition of [³H]NMS-specific binding. However, xanomeline wash-resistant inhibition of [³H]NMS binding was incomplete, with estimated maximal inhibition of 85% (Fig. 1, open triangles). Additionally, the potency of xanomeline wash-resistant binding was 1.5 orders of magnitude lower in comparison with that obtained by xanomeline continuously present during the binding assay. In both cases, nonlinear regression of the data indicated a best fit to a one-site binding model (Table 1). It is unlikely that the observed wash-resistant effects of xanomeline are due to inefficiency of washing off free xanomeline. Increasing the number of washes in cells preincubated with 10 µM xanomeline from 3 to 6 did not change the magnitude of xanomeline wash-resistant inhibition of [³H]NMS binding. Moreover, the supernatant of the third or seventh wash had a negligible effect when added in the binding assay medium.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Inhibition of [3H]NMS binding by xanomeline in M1-CHO cells. The binding of 0.2 nM [3H]NMS was measured in the presence of increasing concentrations of xanomeline (closed squares) or after the following pretreatment conditions. Cells were pretreated with increasing concentrations of xanomeline for 1 min at 37°C, washed extensively, and then used immediately in binding assays (open triangles) or allowed to incubate in the absence of free xanomeline for 24 h (closed triangles). Another group of cells was continuously incubated with increasing concentrations of xanomeline for 24 h, followed by washing and immediate determination of [3H]NMS binding (open diamonds). Receptor binding was assayed by incubation of intact cells for 1 h at 37°C with 0.2 nM [3H]NMS. Values represent the means ± S.E.M. of four to nine experiments performed in triplicate, and they are depicted as a percentage of the specific binding in control cells treated with buffer.

To examine the delayed effects of brief exposure to xanomeline, cells were preincubated in monolayer with increasing concentrations of xanomeline for 1 min, washed, and incubated for an additional 24 h in control culture medium. This delay resulted in the emergence of a high-potency component in the [³H]NMS displacement curve (Fig. 1, closed triangles), representing approximately half of the total receptor population. The potency of xanomeline at this new binding component was 2.5 orders of magnitude greater than that at the single site detected before prolonged waiting (Table 1). In contrast, the potency of xanomeline at the low-potency binding component was comparable with that seen without prolonged waiting (Table 1). Pretreatment with xanomeline for 1 min followed by washing and waiting also exhibited a ceiling effect in inhibition of [³H]NMS binding (88% maximal inhibition) similar to that seen in the latter group. Similarly, cells pretreated with increasing concentrations of xanomeline for 24 h followed by washing and immediate use for binding assays showed two equally distributed components of inhibition of [³H]NMS binding, albeit with much higher potencies at both sites than observed after 1-min pretreatment with a 24-h wait in the absence of free drug (Fig. 1; Table 1).

Changes in cell density were visually apparent following long-term pretreatment with concentrations of xanomeline higher than 100 nM, or with brief preincubation with these concentrations followed by washing and prolonged waiting. Therefore, control experiments were designed to quantitate changes in cell number and protein content in these experimental groups. These measurements indicated marked concentration-dependent decreases in cell number, with a lesser effect on protein content (Figs. 2A and 3A). Displacement binding data were reanalyzed to account for these variances. As shown in Fig. 2B and Table 1, correction for protein content has virtually no effect on the observed potency of xanomeline at the high- and low-potency binding components or on their relative distribution in the 1-min pretreatment/24-h waiting or the 24-h pretreatment groups. Reanalyzing the 24-h pretreatment data to correct for decreased cell number reduced the proportion of its high-potency component from 44 to 38% (Fig. 3B; Table 1) in the absence of marked alteration in xanomeline potency at the two binding components. Correction for changes in cell number in cells that were exposed briefly to xanomeline followed by 24 h in the absence of free drug still revealed xanomeline interaction with two distinct potency components. The potency of xanomeline at its high-potency component was 4 orders of magnitude higher than that of the single-site detected before prolonged waiting (Table 1). This high-potency component represented 20% of total receptors. Taken together, the two potency components resulting from long-term treatment with xanomeline or prolonged waiting after brief exposure are still evident after correction for xanomeline effects on cell number and protein content. Under similar conditions, dimethyl sulfoxide (xanomeline vehicle) did not cause significant effects on radioligand binding or protein content. At 1% (the highest concentration used), dimethyl sulfoxide reduced cell number by 40% following continuous treatment for 24 h (data not shown).

Effects of Prolonged Waiting following Brief Xanomeline Pretreatment on Radioligand Affinity and Maximal Binding to the M₁ Muscarinic Receptor. The ability of increasing concentrations of [³H]NMS to bind to the M₁ receptor in control cells was compared with that following different protocols of xanomeline pretreatment (Fig. 4). Although brief pretreatment for 1 min with 300 nM xanomeline followed by washing did not result in changes in the maximal binding of [³H]NMS, a profound decrease was observed 24 h after washing away free drug (Fig. 4A; Table 2). The magnitude of this decrease was similar to that detected in cells incubated continuously with xanomeline for 24 h. No changes in radioligand affinity were observed in any of the treatment groups. Similar changes in maximal binding were observed when the concentration of xanomeline used for pretreatments was increased to 10 µM (Fig. 4B). In contrast, a decrease in [³H]NMS affinity was observed in this case following pretreatment with xanomeline for 1 min and 24 h. However, the effects of brief pretreatment with xanomeline on radioligand affinity were fully reversed when washed cells were incubated for 24 h in the absence of free xanomeline (Table 2).

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effects of xanomeline pretreatment, followed by washout, on [3H]NMS saturation binding in M1-CHO cells. Cells were pretreated with 300 nM xanomeline (A) or 10 µM xanomeline (B) for 1 min at 37°C, washed extensively, and then used immediately in binding assays (open triangles), or allowed to incubate in the absence of free xanomeline for 24 h (closed triangles). Another group of cells was continuously incubated with increasing concentrations of xanomeline for 24 h, followed by washing and immediate determination of [3H]NMS binding (open diamonds). Untreated (closed squares) and pretreated cells were subsequently incubated for 1 h with increasing concentrations of [3H]NMS. Values represent the means ± S.E.M. of four to nine experiments conducted in triplicate.

Effects of Xanomeline Pretreatment on the Rate of [³H]NMS Dissociation. Wash-resistant binding of xanomeline to the M₁ muscarinic receptor at a site different from that of conventional muscarinic ligands, e.g., [³H]NMS, results in allosteric modulation of the conformation of the receptor primary binding domain (Christopoulos et al., 1998; Jakubík et al., 2002, 2006). This is reflected in an altered rate of [³H]NMS dissociation maximally induced by a receptor-saturating concentration of atropine. We used this property to determine whether the observed long-term effects of xanomeline pretreatment are associated with its persistent binding to the receptor, even after incubating xanomeline-pretreated cells for 24 h in the absence of free drug. M₁-CHO cells were subjected to the various xanomeline pretreatment protocols described above, and then they were incubated with 0.5 nM [³H]NMS for 1 h. Dissociation of the radioligand was initiated by the addition of 10 µM atropine, and the dissociation reaction was allowed to proceed for various time intervals. In all instances, radioligand dissociation was best described by a monoexponential model. All xanomeline pretreatment conditions resulted in a significant decrease in the rate of [³H]NMS dissociation (Table 3).

Effects of Prolonged Waiting after Brief Pretreatment with Xanomeline on M₁ Receptor-Mediated Accumulation of Inositol Phosphates. Receptor-mediated stimulation of PI hydrolysis was used to ascertain the short- and long-term functional consequences of xanomeline binding to the M₁ receptor. Accumulation of inositol phosphates was measured in naive M₁-CHO cells in the presence of increasing concentrations of xanomeline or carbachol, or in cells that had been pretreated for 1 min with xanomeline and then extensively washed, without further addition of agonists (Fig. 5). The maximal accumulation of inositol phosphates induced by xanomeline in naive M₁-CHO cells was similar in magnitude to that of the full agonist carbachol, although xanomeline was 2 orders of magnitude more potent than carbachol. In agreement with previous findings (De Lorme et al., 2006), preincubation of cells with xanomeline for 1 min followed by washing resulted in persistent activation of the M₁ receptor, albeit with a lower apparent potency than observed in its continuous presence. Furthermore, the maximal stimulation elicited in cells that had been pretreated for 1 min and washed was approximately 25% lower than that obtained in the continuous presence of xanomeline (Fig. 5; Table 4).

View larger version (10K): [in this window] [in a new window]   Fig. 5. Wash-resistant xanomeline-induced PI hydrolysis in M1-CHO cells. Cells were labeled with myo-[3H]inositol and then pretreated with increasing concentrations of xanomeline for 1 min followed by extensive washing. Pretreated cells were subsequently incubated for 1 h in the absence of agonist to allow accumulation of inositol phosphates (open triangles). Comparisons were made with naive cells that were treated continuously with either xanomeline (closed circles) or carbachol (closed squares) for 1 h at 37°C. [3H]Inositol phosphates were separated by ion-exchange chromatography, and radioactivity was corrected for recovery of [14C]inositol phosphate. Values represent the means ± S.E.M. of three to four experiments conducted in triplicate.

Further experiments were designed to ascertain the acute and delayed effects of persistent xanomeline binding on M₁ muscarinic receptor function. Stimulation of inositol phosphate hydrolysis was measured in the presence of increasing concentrations of the full agonist carbachol in naive cells or following various pretreatment conditions with xanomeline. Parallel experiments using carbachol for pretreatments were undertaken to ensure the effectiveness of the washing procedure in removal of free drug from the culture medium (data not shown). Pretreatment with 300 nM xanomeline for 1 min resulted in an increase in basal receptor activity following washout (Fig. 6, open triangles). Addition of carbachol to xanomeline-pretreated cells produced a concentration-dependent increase in inositol phosphate hydrolysis, with a maximal response equal to that of carbachol alone. Interestingly, the effects of wash-resistant xanomeline on basal levels were reversed when xanomeline-pretreated cells were allowed to incubate in the absence of free xanomeline for 24 h, with no alteration in the potency or maximal response of carbachol (Fig. 6, closed triangles). Results are summarized in Table 5.

View larger version (10K): [in this window] [in a new window]   Fig. 6. Effects of xanomeline pretreatment, followed by washout, on carbachol-stimulated PI hydrolysis in M1-CHO cells. Cells were pretreated with 300 nM xanomeline for 1 min at 37°C, washed extensively, and then incubated for 1 h with increasing concentrations of carbachol (open triangles). Another group of cells was similarly pretreated with xanomeline and washed, but these cells were incubated for 24 h in control medium before the addition of carbachol (closed triangles). Carbachol-stimulated PI hydrolysis in untreated cells is represented by closed squares. Values represent the means ± S.E.M. of three experiments conducted in triplicate.

	Discussion

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Xanomeline is a unique muscarinic receptor agonist in that it interacts with the receptor both in a reversible and wash-resistant manner (Christopoulos et al., 1998; Jakubík et al., 2002, 2004, 2006). There is strong evidence that the latter mode of xanomeline binding is due to its interaction at a secondary binding domain that allosterically modulates binding of ligands to the receptor orthosteric site (Jakubík et al., 2002, 2006). In a recent preliminary report from our laboratory, we have demonstrated potentiation of the wash-resistant effects of a single low xanomeline concentration in inhibiting [³H]NMS binding to the M₁ muscarinic receptor in CHO cells by incubating washed cells for a day in control medium (De Lorme et al., 2006). In our current study, we undertook an in-depth investigation of the long-term effects of very brief exposure to different concentrations of xanomeline on M₁ receptor binding and function.

In agreement with previous reports from our laboratory (De Lorme et al., 2006; Jakubík et al., 2006), we show that wash-resistant xanomeline binding to the M₁ muscarinic receptor takes place immediately in a concentration-dependent manner, with a potency that is 1.5 orders of magnitude less than that observed in the continuous presence of xanomeline in the binding assay medium. In both cases, xanomeline demonstrates binding to the receptor with a single potency. Although the formation of the stable receptor-xanomeline complex is immediate, slow changes occur over time to further alter the ability of [³H]NMS to bind to the receptor. Most markedly, the monophasic wash-resistant binding curve shown in cells pretreated with increasing xanomeline concentrations for 1 min becomes biphasic 24 h later (Fig. 1). This biphasic behavior in inhibition of [³H]NMS binding remains evident after correction for the effects of xanomeline on cell counts or protein content, and it suggests different mechanisms of receptor regulation at low and high xanomeline concentrations. Interestingly, the potency of xanomeline at the high-potency component is even markedly higher than that seen when xanomeline is continuously present in the binding assay. In contrast, the low-potency component closely corresponds to effects of xanomeline wash-resistant binding in the absence of waiting. Thus, only the effects of low concentrations of xanomeline are potentiated with time due to the appearance of a new high-potency component in inhibiting [³H]NMS binding. Noteworthy, effects of various xanomeline treatments on radioligand binding were not mimicked by dimethyl sulfoxide, the solvent used to solubilize xanomeline.

It is well known that prolonged activation of receptors by agonists results in receptor internalization and down-regulation (Bunemann and Hosey, 1999; Ferguson, 2001). Our current results indicate that wash-resistant binding of xanomeline may result in latent alteration, blockade, or down-regulation of M₁ receptors, making them less available for [³H]NMS binding. We therefore included an additional experimental group where cells were treated with xanomeline continuously for 24 h before being washed and used in the binding assay. One would expect to see a high degree of receptor internalization or down-regulation under the latter condition. Continuous treatment for 24 h with increasing xanomeline concentrations results in a biphasic inhibition curve of [³H]NMS binding (Fig. 1). The low-potency component is indistinguishable from that of xanomeline when continuously present in the binding assay (Table 1). However, the potency of xanomeline at both the high- and low-potency components is significantly higher than that observed in cells pretreated with xanomeline for 1 min, washed, and then incubated for 24 h in the absence of xanomeline. As shown in Fig. 4 and Table 2, maximal binding of [³H]NMS obtained in saturation binding experiments is greatly reduced in both the 24-h pretreatment group and after–1-min pretreatment with low or high xanomeline concentrations followed by washout and 24-h wait. The affinity of [³H]NMS for the receptor in the latter treatment group remains virtually unchanged. These results support the possibility that receptor internalization or down-regulation may be occurring during the 24 h following removal of free xanomeline, where one would expect to see a decrease in maximal binding, while the affinity for the receptor remains unchanged. We performed new preliminary experiments comparing the effects of 1-min xanomeline pretreatment and waiting on the specific binding of receptor-saturating concentrations of [³H]NMS (a hydrophilic ligand that labels only cell surface receptors) and [³H]quinuclidinyl benzilate (a lipophilic cell-permeable ligand that labels both cell surface and internalized receptors) (Feigenbaum and El-Fakahany, 1985). The profile of effects of xanomeline on the binding of the two ligands was similar (data not shown), suggesting that receptor down-regulation rather than internalization takes place. An incremental decrease in the number of receptors caused by increasing concentrations of xanomeline during the pretreatment phase would result in a progressive decrease in [³H]NMS binding and, therefore, the late emergence of a new "apparent" xanomeline high-potency site. However, this interpretation does not explain why the effects of pretreatment with higher concentrations of xanomeline, which are expected to result in greater receptor down-regulation, did not demonstrate potentiation of inhibition of [³H]NMS binding with time.

We have also shown that pretreatment with xanomeline for 1 min followed by washing results in persistent activation of the M₁ receptor in a concentration-dependent manner, displaying a lower potency and slightly lower maximal response compared with the effects of continuous presence of xanomeline (Fig. 5). The decrease in potency parallels the corresponding decrease in binding potency following washing of cells briefly preincubated with xanomeline. One potential interpretation of the observed lower maximal receptor activation in xanomeline-pretreated and washed cells is receptor desensitization. However, the ability of carbachol to produce further receptor activation argues against this possibility. Another plausible interpretation is the induction of a different conformation of an activated receptor state, evidenced by the observed change in the rate of dissociation of [³H]NMS binding in xanomeline pretreated cells (Table 3). Interestingly, the increase in basal levels of receptor activation seen after 1 min of pretreatment with xanomeline is reversed after 24 h (Fig. 6). This reversal cannot be due to dissociation of the xanomeline-receptor complex, because the radioligand binding assay data indicate that the effects of xanomeline on [³H]NMS binding are actually more evident following waiting (Fig. 1). The latter conditions were also associated with changes in the rate of radioligand dissociation, providing additional proof that xanomeline is still bound to the allosteric site following washing and prolonged waiting. Surprisingly, there were no significant changes in either the potency or the maximal effects of carbachol in stimulating PI hydrolysis following xanomeline pretreatment, washing, and prolonged waiting. This is in spite of the observed marked decrease in maximal [³H]NMS binding that suggests a decrease in the number of available receptors. One possible explanation of this paradox is the involvement of a large receptor reserve, where only a small fraction of the total receptor population is required for eliciting a maximal functional response to carbachol. In fact, preliminary experiments showed that the concentration of carbachol required to elicit half-maximal activation of PI hydrolysis (0.2 µM) is markedly lower than the concentration needed to occupy 50% of the receptors (235 µM). Furthermore, 90% of the maximal PI response to carbachol requires occupancy of only 0.8% of the receptors. Thus, there is significant receptor reserve in the system. However, in a system with receptor reserve, progressive reduction of the number of available receptors should elicit an incremental decrease in agonist potency, with reduction in maximal response ensuing once receptor reserve is exhausted (Furchgott, 1966; Van Rossum, 1968).

Our current results indicate that there is profound time-dependent enhancement of the effects of xanomeline bound to the M₁ receptor in a wash-resistant manner on [³H]NMS binding. This is reflected in the appearance of a xanomeline high-potency site, which represents the most novel aspect of our findings. The most likely underlying mechanism of these delayed changes in the effects of xanomeline is receptor down-regulation. However, the possible contribution of other mechanisms could not be discounted at present. These potential mechanisms include changes in the conformation of the receptor with time to one that is less capable of binding [³H]NMS. This new conformation might be the result of time-dependent interaction of xanomeline with additional residues on the receptor protein, binding of more than one molecule of xanomeline to multiple sites on the receptor, or the formation of receptor dimers. The molecular mechanisms underlying the observed latent effects of wash-resistant xanomeline will be under intensive investigation in our laboratory.

	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.107.129940.

ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor(s); NMS, N-methylscopolamine; CHO, Chinese hamster ovary; PI, phosphoinositide; ANOVA, analysis of variance.

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

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