Abstract
Previous findings in our laboratory suggested that the M1muscarinic acetylcholine receptor (mAChR) agonist xanomeline exhibits a novel mode of interaction that involves persistent binding to and activation of the M1 mAChR, subsequent to extensive washout, as well as a possible insurmountable element. In the present study, we examined this interaction in greater detail, using Chinese hamster ovary cells transfected with the genes for the M1mAChR and neuronal nitric oxide synthase. Pretreatment of cells with xanomeline, followed by extensive washout, resulted in elevated basal levels of neuronal nitric oxide synthase activity that were suppressed by the antagonists atropine or pirenzepine in a concentration-dependent manner. Analysis of the data yielded estimates of Schild slope factors and pKB values for the antagonists that were consistent with a model of simple competition between these latter agents and the persistently bound form of xanomeline. The ability of the antagonists to produce parallel dextral shifts of the concentration-response curves to carbachol and xanomeline was also investigated. The interaction between xanomeline and pirenzepine appeared to be insurmountable, but this may have been due to an equilibrium artifact. In contrast, the interaction between xanomeline and atropine conformed to a model of competition, indicating that the mode of interaction of free xanomeline at the M1 mAChR is pharmacologically identical with that of the persistently bound form. Radioligand binding studies also showed that the presence of various concentrations of xanomeline had no significant effect on the calculated affinity of atropine or pirenzepine in inhibiting the binding of [3H]N-methylscopolamine. Overall, these findings suggest that the persistent attachment of xanomeline to the M1 mAChR does not prevent this agonist from interacting with the classic binding site in a competitive fashion.
Despite the multifactorial nature of both the suspected pathogenesis and proved neurochemical deficits associated with Alzheimer’s disease, one of the earliest and most consistent features of the disorder is the degeneration of basal forebrain cholinergic nuclei (Whitehouse et al., 1982), with a concurrent loss in cognitive function. To date, the only clinically approved pharmacotherapeutic agents for the treatment of Alzheimer’s disease are inhibitors of the enzymatic hydrolysis of the neurotransmitter acetylcholine (Peskind, 1998). An alternative approach that has been pursued by others, however, involves the design of directly acting M1 muscarinic acetylcholine receptor (mAChR) agonists, as the M1 mAChR has been shown to be involved in learning and memory and its numbers are relatively preserved during the progression of Alzheimer’s disease (Ladner and Lee, 1998). Xanomeline (Fig.1) is one such agent, and it has already been demonstrated to be one of the most potent M1mAChR agonists known (Shannon et al., 1994). In contrast, assays of M2 and M3 mAChR-mediated functional responses found xanomeline to display relatively weak agonistic properties (Shannon et al., 1994). Because xanomeline has similar affinity for the five mAChR subtypes (Bymaster et al., 1997), its apparent “functional” selectivity identifies this compound as a partial agonist, expressing a significant degree of efficacy only in cells or tissues with particularly efficient stimulus-response pathways and not in poorly coupled systems (Kenakin, 1997).
Functionally selective partial agonists offer a number of therapeutic advantages over full agonists (Kenakin, 1985), but a thorough pharmacological characterization of such agents is first required to allow for the extrapolation of in vitro data to the potential clinical usefulness of these agents in humans. Accordingly, recent studies in our laboratory were undertaken to further probe the molecular nature of the interaction between xanomeline and the human M1 mAChR expressed in Chinese hamster ovary (CHO) cells (Christopoulos and El-Fakahany, 1997; Christopoulos et al., 1998b). Interestingly, our findings indicated a novel mode of interaction between this compound and the M1mAChR that did not appear to be shared by conventional mAChR agonists, such as carbachol (CCh). Specifically, xanomeline displayed persistent binding to the M1 mAChR that allowed a fraction of the agent to remain in the receptor compartment and continue to activate the receptor even after extensive washout. Furthermore, a preliminary pharmacological analysis of the inhibition by the antagonist atropine of xanomeline-mediated functional responses indicated a possible element of insurmountable antagonism, but this was not evident in a series of radioligand binding paradigms. Thus, the aim of the current study was to further probe the unique binding and activating properties of xanomeline by applying a more rigorous pharmacological analysis to the interaction between this agent and mAChR antagonists at the M1 mAChR.
Experimental Procedures
Materials.
N-[3H]Methylscopolamine (NMS; 84.5 Ci/mmol) and [14C]l-citrulline (50 mCi/mmol) were purchased from NEN Dupont (Wilmington, DE). [3H]l-Arginine (64 Ci/mmol) was purchased from Amersham Life Science (Arlington Heights, IL). Dulbecco’s modified Eagle’s medium was purchased from GIBCO (Gaithersburg, MD). Geneticin and hygromycin were purchased from Calbiochem (La Jolla, CA). Bovine calf serum was purchased from Hyclone (Logan, UT). Xanomeline tartrate was a generous gift from Lilly Research Laboratories (Indianapolis, IN). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
Cell Culture.
CHO cells stably expressing the human M1 mAChR (provided by Dr. M. Brann, University of Vermont Medical School, Burlington, VT) and the neuronal isoform of nitric oxide synthase (nNOS; cDNA provided by Drs. S. H. Snyder and D. R. Bredt, Johns Hopkins University School of Medicine, Baltimore, MD) were grown for 4 days at 37°C in Dulbecco’s modified Eagle’s medium supplemented with 10% bovine calf serum, 50 μg/ml geneticin, and 50 μg/ml hygromycin in a humidified atmosphere consisting of 5% CO2 and 95% air. Cells were used 4 days after subculture and were harvested 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, 50 mM HEPES, 58 mM sucrose; pH 7.4 ± 0.02; 340 ± 5 mOsm), repeated twice.
Assay of nNOS Activity.
The activity of nNOS in intact cells was assayed by quantifying the conversion ofl-[3H]arginine tol-[3H]citrulline according to the method of Bredt and Snyder (1989) with modifications, as described previously (Christopoulos et al., 1998b). Two types of experiments were undertaken. In the first series, the residual effects of xanomeline pretreatment on basal nNOS activity in CHO cells were examined, as well as the effects of varying concentrations of the antagonists atropine or pirenzepine on this persistent xanomeline effect. Cells were initially exposed to xanomeline for 1 h at 37°C and were harvested and extensively washed to remove free agonist as follows: after centrifugation at 300g for 3 min (room temperature), the cell pellet was resuspended in 40 ml of HEPES buffer and centrifuged again. This procedure was repeated three times before a final resuspension of the resulting pellet for use in subsequent experiments. Cells (∼5 × 105/tube) were then incubated in 0.3 ml of HEPES buffer for 15 min at 37°C, after which [3H]l-arginine (0.6 μCi/tube) was added to initiate the reaction. At the same time, increasing concentrations of either atropine or pirenzepine were added, and the reaction was allowed to proceed for 1 h at 37°C before termination. In the second series of experiments, the effects of the antagonists on the concentration-response relationship to the agonists CCh or xanomeline were investigated under “standard” assay conditions (i.e., nonpretreated cells were harvested and washed, as above, and then exposed to increasing concentrations of either agonist, in the absence or presence of varying concentrations of atropine or pirenzepine). Antagonists were incubated with the cells for 20 min before the addition of agonist, after which the reaction was allowed to proceed for 1 h at 37°C. For all experiments, the reaction was stopped by adding 0.75 ml of stop solution containingl-arginine (5 mM) and EDTA (4 mM).l-[3H]Citrulline was separated froml-[3H]arginine using ion-exchange chromatography (DOWEX AG50W-X8 resin), and the amount of radioactivity, expressed as disintegrations per minute (dpm), was determined via liquid scintillation counting.
Competition Binding Experiments.
CHO cells (∼105 cells/assay tube in a total volume of 1 ml) were incubated with a fixed concentration of [3H]NMS (as described in Results) in the absence or presence of xanomeline (0.1 nM to 20 μM), atropine (0.1 nM to 2 μM), or pirenzepine (0.2 nM to 5 μM) for 1 h at 37°C. Additional atropine and pirenzepine competition experiments were conducted in the presence of both [3H]NMS and a fixed concentration of xanomeline (as indicated inResults). Nonspecific binding was defined using 10 μM atropine. Incubation was terminated by filtration through Whatman GF/C filters positioned on a Brandell cell harvester. Filters were washed three times with 4-ml aliquots of ice-cold saline and dried before radioactivity (dpm) was measured using liquid scintillation counting. Saturation binding parameters for [3H]NMS under these conditions have been determined previously (Christopoulos et al., 1998b).
Data Analysis.
In the functional assays, individual agonist concentration-response curves, in the absence and presence of antagonist, were fitted via nonlinear regression to the following four-parameter logistic function, using Prism 2.01 (GraphPAD Software, San Diego, CA):
Functional antagonist inhibition data were analyzed using a two-step procedure (Lazareno, 1997); initially, eq. 1 was applied to data describing the apparent concentration-response relationship of persistently bound xanomeline, as described in Results, to determine the best estimates of Emax, basal, nH, and EC50. Subsequently, these latter parameters were fixed in the analysis of the antagonist inhibition curves, according to the following equation (Waud, 1975; Lazareno and Birdsall, 1993a, b):
Competition binding isotherms were analyzed via nonlinear regression using Prism to derive estimates of nH(slope factor) and IC50 (midpoint location/potency parameter). Assuming simple competition, the 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 toKI values (competitor-receptor dissociation equilibrium constant) according to the following equation (Christopoulos et al., 1998b):
Data shown are the mean ± S.E.M. Comparisons between mean values were performed by paired or unpaired t tests or one-way ANOVA, as appropriate. Unless otherwise stated, values ofp < .05 were taken as statistically significant.
Results
Persistent Activation of M1 mAChR by Xanomeline.
CHO cells, expressing the human M1 mAChR and nNOS, were pretreated with either vehicle, 1 mM CCh, or 1 μM xanomeline for 1 h at 37°C before harvesting and extensive washout (see Experimental Procedures). Subsequently, basal nNOS activity (i.e., that observed in the absence of subsequent addition of agonist) was assessed by quantification of the conversion of l-[3H]arginine tol-[3H]citrulline. In agreement with our previous findings (Christopoulos and El-Fakahany, 1997;Christopoulos et al., 1998b), xanomeline displayed a remarkable ability to persistently activate the M1 mAChR after washout, in contrast to CCh (Fig. 2A). Furthermore, a comparison of the concentration dependence of the persistent effect to that of the concentration-response relationship of xanomeline under standard assay conditions (i.e., responses in the continued presence of xanomeline), as shown in Fig. 2B, indicated that the persistent effect approaches the maximal possible effect that xanomeline is capable of producing.
Quantification of Inhibition of Persistent Xanomeline Effect by mAChR Antagonists.
Previous experiments in our laboratory indicated that the persistent effects of xanomeline could be abolished by the non-subtype-selective mAChR antagonist atropine (Christopoulos and El-Fakahany, 1997; Christopoulos et al., 1998b). In the present study, the ability of increasing concentrations of atropine and the M1-selective antagonist pirenzepine to inhibit the persistent effects of xanomeline was studied in greater detail.
In CHO cells that had been pretreated with 1 μM xanomeline for 1 h at 37°C, before harvesting and extensive washout, both antagonists were able to inhibit the persistent activating effect of the agonist in a concentration-dependent manner (Fig.3). The antagonist inhibition data were subsequently analyzed according to eq. 3 in Experimental Procedures to derive quantitative estimates of the antagonist dissociation constants under these conditions. The appropriate application of this equation requires that an agonist concentration-response curve also be constructed under the same conditions for inclusion in the analysis (Lazareno and Birdsall, 1993a,b). Because the current experiments were designed to examine the effects of atropine and pirenzepine on residual, persistently bound, xanomeline, the concentration-response curve for xanomeline under standard conditions (Fig. 2B, open circles) was used as a calibration curve to obtain the apparent residual concentration of xanomeline within the receptor compartment that was yielding the persistent activating effect (Fig. 2B, solid circles). The resulting, “apparent” concentration-response curve for persistently bound xanomeline is also shown in Fig. 3 and was used in the analysis of the antagonist inhibition curves. The resulting Schild slope factors were 1.13 ± 0.19 and 0.94 ± 0.10 for atropine and pirenzepine, respectively (n = 4). Neither slope factor was significantly different from unity (p > .05), a finding consistent with simple competition, and thus was constrained to 1 for estimation of the pKB values, which were 8.49 ± 0.10 and 7.76 ± 0.07 for atropine and pirenzepine, respectively.
Analysis of Antagonism by Atropine and Pirenzepine of CCh- and Xanomeline-Mediated Responses under Standard (Continued Presence of Agonist) Assay Conditions.
A preliminary pharmacological analysis of the antagonism by a single concentration of atropine of xanomeline-mediated M1 mAChR activation indicated a significantly reduced antagonist potency, relative to the effects of the same concentration of antagonist on CCh-mediated responses (Christopoulos et al., 1998b). This finding may be suggestive of a potentially different mode of interaction between xanomeline and atropine compared with that between CCh and atropine. In the present study, this phenomenon was further investigated by undertaking a more rigorous pharmacological characterization of the effects of atropine and pirenzepine on the concentration-response relationship to CCh and xanomeline.
The presence of varying concentrations of either atropine or pirenzepine resulted in a parallel, dextral shift of the CCh concentration-response curve for M1mAChR-mediated nNOS activation (Fig. 4). The presence of either antagonist did not result in a significant (p > .05) alteration of agonist curve slope or basal or maximal response parameters, allowing the data to be assessed in terms of a competitive model of pharmacological interaction. Nonlinear regression analysis of the EC50 data for CCh, in the absence or presence of atropine, according to eq. 2 of theExperimental Procedures, allowed for the estimation of antagonist pKB and Schild slope parameters (Table 1). Figure5A illustrates a Clark plot of the data, constructed using the estimated antagonist potency value from the nonlinear regression. A similar analysis of the CCh/pirenzepine data yielded the values shown in Table 1 and Fig. 5B. In each case, the data were compatible with a model of simple competition.
When xanomeline was used as the agonist and atropine was used as the antagonist, the data were also compatible with simple competition (Figs. 5C and 6A; Table 1). In contrast, the combination of xanomeline with pirenzepine resulted in a progressive reduction in the xanomeline concentration-response curve maximum with increasing concentrations of antagonist (Fig. 6B). This latter finding is incompatible with simple, surmountable competition. Nevertheless, an empirical estimate of antagonist potency, expressed as pA2, was estimated and is shown in Table 1 and was also used to construct the Clark plot in Fig. 5D. This parameter was derived as outlined in Experimental Procedures after a determination of equieffective agonist concentrations (Fig. 6B, dashed line).
A one-way ANOVA found no significant difference (p > .05) between each antagonist’s potency estimates determined using either agonist. Additionally, no significant difference (p > .05) was found between these latter potency estimates and those determined in the antagonist inhibition experiments (Fig. 2), where the persistent xanomeline effect was being examined.
Effects of Xanomeline, Atropine, and Pirenzepine on Binding of [3H]NMS.
Xanomeline, atropine, and pirenzepine were all potent inhibitors of the binding of 0.2 nM [3H]NMS at the M1receptor in intact CHO cells (Fig. 7). Nonlinear regression analysis of the data revealed that in each instance, a one-site binding model provided an adequate fit (Table2). Additional experiments were also undertaken in which the effects were assessed of graded concentrations of xanomeline on the inhibitory potency of atropine (Fig. 7A) or pirenzepine (Fig. 7B). In this manner, any possible noncompetitive effects of xanomeline on the binding of either antagonist would have been revealed by discrepancies between estimated pKI values for each antagonist determined in the absence or presence of xanomeline. However, as shown in Table 2, the application of eq. 4 to each of the competition curves yielded affinity estimates for atropine or pirenzepine that did not vary significantly (p > .05) from those determined in the absence of xanomeline, thus providing further support for a competitive binding mode of interaction between the agonist and mAChR antagonists.
Discussion
Xanomeline represents one of the most potent agonists available for selective activation of the M1 mAChR (Shannon et al., 1994). A comparison of the xanomeline dissociation equilibrium constant (Table 2) with its EC50 value for stimulation of nNOS activity (Fig. 2B), with both determined under the same assay conditions in the present study, indicates a greater than 20-fold difference between the two parameters. Thus, the high potency of xanomeline for activating the M1 mAChR is not simply due to its relatively high affinity but also contains a significant efficacy component. Indeed, we previously demonstrated that in functional assays of both M1 mAChR-mediated phosphoinositide hydrolysis or nNOS activation, xanomeline possesses an intrinsic activity that is almost equal to that of the high-efficacy agonist CCh (Christopoulos and El-Fakahany, 1997; Christopoulos et al., 1998b). Unlike CCh, however, the potency and intrinsic activity of xanomeline show a much greater dependence on both the receptor subtype involved and the system in which the receptor is expressed (Shannon et al., 1994; Bymaster et al., 1997). On an operational level, therefore, xanomeline may be classified as a partial agonist.
The study of partial agonists has contributed greatly to the development of the concepts of affinity, efficacy, and “agonism” (Ariëns, 1954; Stephenson, 1956; Black, 1996). Although the expression of the latter property by a drug will always depend on the physiological system in which it is tested (Christopoulos and El-Fakahany, 1999), the ability of different agonists to yield different degrees of maximal receptor activation in the same system is most likely an indicator of important differences in the mode or modes of drug action at the molecular level. Indeed, some studies have suggested specific modes of interaction for mAChR partial agonists that differ from those possessed by full agonists (Gurwitz et al., 1994;Heldman et al., 1996). In the case of xanomeline, we recently proposed a model that incorporated two possible modes of interaction for this ligand with the M1 mAChR (Christopoulos et al., 1998b). The first mode involves a readily reversible, syntopic interaction with the classic binding site on the receptor, shared by other agonists, such as CCh, or antagonists, such as atropine. The second mode involves the subsequent development of a persistent attachment with specific receptor regions that allowed xanomeline to continue to activate the M1 mAChR via the classic binding site but did not allow it to be readily removed from the receptor compartment. In this manner, xanomeline may be said to behave as a “captive agonist,” a phenomenon that has already been demonstrated with the β2 adrenoceptor partial agonist salmeterol (Coleman et al., 1996). It should be noted, however, that the potential mechanism of interaction between xanomeline and the M1 mAChR is not completely analogous to that of salmeterol. It is possible that some sort of obligatory orientation must first be achieved by the xanomeline, most likely via binding to the classic attachment site on the receptor that is used by conventional agonists and antagonists before a persistent attachment to a secondary site (Christopoulos et al., 1998b). The difference between the location of the concentration-response curve to xanomeline determined under standard conditions and that determined by pretreatment followed by washout (Fig. 2B), therefore, suggests that not all of the xanomeline that interacts with the classic binding site on the receptor can form a persistent attachment with a secondary site. Alternatively, the actual mechanism of activation of the receptor by xanomeline binding at a secondary site may be different from that resulting from agonist binding to the classic site because this may also account for the observed potency differences in Fig. 2B.
Although we had previously demonstrated the specificity of the persistent effects of xanomeline with the use of the mAChR antagonist atropine (Christopoulos and El-Fakahany, 1997; Christopoulos et al., 1998b), it remained unclear whether the antagonists interacted with the persistently activated xanomeline-receptor complex in a syntopic manner. Furthermore, the apparent reduction in the ability of atropine to shift the concentration-response curve of xanomeline, relative to that of CCh, was also suggestive of a noncompetitive mode of interaction (Christopoulos et al., 1998b). This latter finding, however, remained inconclusive because the appropriate testing of the mode of agonist-antagonist interaction requires multiple concentrations of each ligand to be used (Kenakin, 1997). In the present study, both modes of activation of the M1 mAChR by xanomeline were investigated pharmacologically.
The ability of both atropine and pirenzepine to reduce the persistent activating effect of xanomeline in a concentration-dependent manner (Fig. 2A) further confirmed the specificity of the latter phenomenon. More importantly, the application of an inhibition curve design and accompanying analysis (Lazareno, 1997; Lazareno and Birdsall, 1993a,b) allowed for the interaction between the antagonists and the persistently bound xanomeline to be quantified and assessed in terms of a competitive model. It should be noted that the “control” activation curve included in this analysis was that of the effects of persistently bound xanomeline, indirectly determined via the use of a “calibration” curve (see Results). The actual pretreatment concentrations (Fig. 2B, solid circles) that were used to establish the persistent effect before washout could not be used in the analysis of the data because they represent a different experimental condition (i.e., continued presence of free agonist). The inclusion of this latter dataset in the analysis of the antagonist inhibition curves would have resulted in a significant underestimation of antagonist potency (not shown). Indeed, without the incorporation of a corrected control curve, the shape and location of the antagonist inhibition curves could not be used in any tests of conformity of the data to competition (Lazareno and Birdsall, 1993a; Lazareno, 1997). The finding of Schild slope factors not significantly different from unity for both antagonists may be taken as presumptive evidence that the interaction between these compounds and the persistently bound xanomeline is consistent with simple competition (Kenakin, 1997). Furthermore, the calculated pKB values were not different from such values determined for each antagonist using other functional (Table 1) or binding (Table 2) assays. To our knowledge, this is the first application of an antagonist inhibition curve design to the functional effects of a persistently bound agonist.
The second functional experimental paradigm used in the current study was designed to further examine the effects of antagonist inhibition of the responses to xanomeline concentrations that are introduced to the receptor compartment after preequilibration of the preparation with antagonist. This paradigm, therefore, represents the classic approach to the determination and quantification of competitive antagonism in functional assays (Kenakin, 1997). The use of multiple antagonist concentrations against the actions of CCh or xanomeline in mediating M1 mAChR-mediated nNOS activation allows for the nature of interaction between each agonist and antagonist to be tested for deviations from competitivity by means of the estimation of a Schild slope parameter. This was not possible in previous preliminary, in vitro functional studies of xanomeline because only single antagonist concentrations were tested (Shannon et al., 1994;Christopoulos et al., 1998b). The application of a full Schild design in our current functional experiments revealed two important findings. First, analysis of the inhibition of the CCh-mediated responses by both antagonists and of the xanomeline-mediated responses by atropine shows the data to be adequately described by a competitive model of interaction (Figs. 4-6A; Table 1); the Schild slope parameters were not significantly different from unity, and the pKB values were in good agreement with those obtained in the radioligand binding assays (Table 2). In contrast to our previous suggestion, therefore, it appears that the interaction between xanomeline and atropine in functional assays is compatible with simple competition. Second, the interaction between xanomeline and pirenzepine did not appear to satisfy the minimum criterion of a competitive interaction because a progressive reduction in xanomeline concentration-response curve maxima was observed in the presence of increasing concentrations of antagonist (Fig. 6B). This may be a manifestation of a true, noncompetitive interaction between these latter two agents, but additional data and alternative explanations must first be considered. For example, in the radioligand binding assays (Fig. 7; Table 2), data were obtained that argue against a noncompetitive mode of interaction between xanomeline and pirenzepine. Specifically, the affinity of pirenzepine for the M1 mAChR, assessed via its ability to inhibit the binding of [3H]NMS, was unaltered in the presence of increasing concentrations of xanomeline (Table 2). A similar finding was demonstrated for the interaction between xanomeline and atropine, both in the present study and in our previous work (Christopoulos et al., 1998b). Thus, at the binding level, the interaction between xanomeline and pirenzepine is consistent with simple competition.
Even though both an agonist and an antagonist can interact with a receptor in a readily reversible, syntopic fashion, apparent insurmountability of the antagonism by an agonist may still occur in functional assays due to equilibrium artifacts (Kenakin, 1997). In particular, Paton and Waud (1967) have described a condition known as the “quasiequilibrium” or “hemiequilibrium,” whereby agonists that require a significant proportion of receptor occupancy to achieve their maximal effect (i.e., partial agonists) cannot do so in the presence of slowly dissociating antagonists. Preliminary experiments in our laboratory have found pirenzepine to possesses an approximately 8-fold slower dissociation rate constant than atropine (A. Christopoulos, A. M. Parsons, M. J. Lew, and E. E. El-Fakahany, in preparation), and because xanomeline is a partial agonist relative to CCh, the combination of the xanomeline and pirenzepine may predispose them toward achieving a hemiequilibrium state, relative to the real-time scale of response. At this point, it should also be noted that the “response” in the present study was not obtained in real-time but rather was obtained using a stop-time assay that measured the cumulative formation of product over time. Although the reaction was allowed to proceed for 1 h, ensuring that the interacting agents eventually equilibrated with the receptor, any hemiequilibrium effects that may have been manifested at the early time points of receptor activation would still be expected to influence the estimates of total product formed over time. This latter point warrants further study because many such assays are commonly used in the measurement of proximal receptor-mediated events (e.g., second messenger formation). Furthermore, if a hemiequilibrium state did exist between xanomeline and pirenzepine at the M1mAChR in CHO cells, then real-time measurements of rapid, transient responses (e.g., intracellular calcium mobilization) would be expected to reveal a similar pattern of antagonism to that observed in the present study. Preliminary experiments in our laboratory have found this to be the case, whereby the antagonism by atropine or pirenzepine of either CCh- or xanomeline-mediated calcium mobilization appears to be insurmountable in all instances (A. Christopoulos, A. M. Parsons, M. J. Lew, and E. E. El-Fakahany, in preparation). Thus, it is possible that the functional insurmountability of the antagonism by pirenzepine of xanomeline responses observed in the present study is not due to a noncompetitive interaction but rather represents an equilibrium artifact. Although the kinetic argument is simply the most parsimonious explanation for our present findings, we cannot rule out the possibility of a true, noncompetitive mode of interaction for the specific combination of xanomeline and pirenzepine because the latter antagonist has been suggested to interact allosterically with the M1 mAChR under certain conditions (Christopoulos et al., 1998a). In any case, a pKB value was not estimated for this particular drug combination, but an empirical estimate of antagonist potency, expressed as a pA2, was calculated instead (Table 1).
Therapy aimed at targeting functional M1 mAChRs with xanomeline or similar agents remains a viable option in the treatment of memory deficits in Alzheimer’s disease (Peskind, 1998). Although xanomeline appears to be able to bind to and activate the M1 mAChR in a persistent manner, our present results indicate that this novel mode of interaction does not preclude xanomeline from acting at the classic binding site on the mAChR in a manner that is consistent with simple competition. This is particularly important when considering the potential clinical use of xanomeline in the treatment of Alzheimer’s disease because xanomeline has already been demonstrated in clinical trials (Bodick et al., 1997a,b) to improve the cognitive function of patients with the disorder. For example, although the persistent activating effects of xanomeline have important implications with regard to the dosage regimen (Christopoulos et al., 1998b), the findings in the present study suggest that the pharmacodynamic properties of xanomeline will most likely be more akin to those of traditional, readily reversible agonists. Furthermore, concomitant antagonist therapy would be expected to result in a reversal of xanomeline effects, whether the latter are due to the persistently bound agonist form or not. This latter point is important in terms of possible xanomeline overdose or in those instances of the coadministration of additional therapeutic agents that possess antimuscarinic properties.
Footnotes
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Send reprint requests to: Prof. Esam E. El-Fakahany, Departments of Psychiatry, Neuroscience and Pharmacology, Box 392 Mayo Memorial, University of Minnesota Medical School, Minneapolis, MN 55455. E-mail:elfak001{at}maroon.tc.umn.edu
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↵1 This work was supported by National Institutes of Health Grant NS25743.
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↵2 Present address: University of Wisconsin-Stout, Biology Department, Menomonie, WI 54751.
- Abbreviations:
- mAChR
- muscarinic acetylcholine receptor
- CHO
- Chinese hamster ovary
- CCh
- carbachol
- NMS
- N-methylscopolamine
- nNOS
- neuronal nitric oxide synthase
- dpm
- disintegrations per minute
- Received October 29, 1998.
- Accepted January 27, 1999.
- The American Society for Pharmacology and Experimental Therapeutics