QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.122093v1 322/1/316    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Machová, E. Articles by Dolezal, V. Search for Related Content PubMed PubMed Citation Articles by Machová, E. Articles by Dolezal, V.

NEUROPHARMACOLOGY

Wash-Resistantly Bound Xanomeline Inhibits Acetylcholine Release by Persistent Activation of Presynaptic M₂ and M₄ Muscarinic Receptors in Rat Brain

E. Machová, J. Jakubík, E. E. El-Fakahany, and V. Doleal

Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic (E.M., J.J., V.D.); and Department of Psychiatry, University of Minnesota Medical School, Minneapolis, Minnesota (E.E.E.)

Received March 1, 2007; accepted April 18, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
We studied the effects of 3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) wash-resistant binding on presynaptic muscarinic regulation of electrically evoked [³H]acetylcholine (ACh) release from rat brain slices. In both cortical and striatal tissues that possess M₂ and M₄ autoreceptors, respectively, immediate application of 10 µM xanomeline had no effect on evoked [³H]ACh release or its inhibition by 10 µM carbachol. In contrast, preincubation with 1, 10, or 100 µM xanomeline for 15 min decreased evoked release of ACh measured after 53 min of washing in xanomeline-free medium in a concentration-dependent manner. The maximal inhibitory effect equaled the immediate effect of the muscarinic full agonist carbachol, and it was completely (at 1 and 10 µM xanomeline) or partially (at 100 µM xanomeline) blocked by 1 µM N-methylscopolamine. Neither presence of N-methylscopolamine during 100 µM xanomeline treatment nor previous irreversible inactivation of the classical receptor binding site using propylbenzylcholine mustard in cortical slices prevented the inhibitory effect of wash-resistantly bound xanomeline. Treatment of cortical slices with xanomeline slightly decreased the number of muscarinic binding sites, and it markedly decreased affinity for N-methylscopolamine. When applied as in acetylcholine release experiments, xanomeline did not impair presynaptic ₂-adrenoceptor-mediated regulation of noradrenaline release. The functional studies in brain tissue reported in this work demonstrate that xanomeline can function as a wash-resistant agonist of native presynaptic muscarinic M₂ and M₄ receptors with both competitive and allosteric components of action.

We observed previously that xanomeline interacts with two binding sites on the muscarinic receptor, i.e., the orthosteric binding site and another distinct site where it binds in a wash-resistant manner (Christopoulos et al., 1998; Jakubík et al., 2002, 2004). Wash-resistantly bound xanomeline activates guanosine 5'-O-(3-thio)triphosphate binding at the M₁ receptor, and, although with lower efficacy but similar affinity, also at the M₂ receptor (Jakubík et al., 2006). Likewise, wash-resistantly bound xanomeline induces durable antagonism of M₅ receptor activation (Grant and El-Fakahany, 2005). Together, these findings strongly indicate that the complicated pharmacological profile of xanomeline action may be due not only to the selective stimulation of M₁/M₄ receptors but also to not yet well characterized effects of wash-resistant xanomeline at all subtypes of muscarinic receptors thus far tested.

All these observations on the effects of wash-resistant xanomeline on receptor function were derived in experiments on human muscarinic receptors heterologously expressed in fibroblasts (Chinese hamster ovary cells). It is therefore very important to elucidate whether such unusual effects of xanomeline are also evident in case of receptors expressed in their natural environment. To approach this question, we performed ex vivo experiments with rat brain cortical and striatal tissue. We investigated delayed wash-resistant effects of xanomeline on the regulation of stimulation-evoked release of acetylcholine (ACh) that is mediated by M₂ receptors in brain cortex and M₄ receptors in striatum (Doleal and Tuek, 1998; Zhang et al., 2002; Bymaster et al., 2003). We show that wash-resistant xanomeline binding decreases evoked ACh release by stimulating both M₂ and M₄ receptors and that both the allosteric and orthosteric binding sites participate in this effect.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Immediate application of xanomeline does not influence electrically evoked [3H]ACh release and its presynaptic M2 (cortex) and M4 (striatum) receptor-mediated inhibition. Brain cortical (left column) and striatal (right column) slices were stimulated (2-ms monopolar pulses at 1 Hz for 1 min at 25 mA) at the beginning of the 3rd, 9th, and 15th collection fraction (abscissa). A and D, no drug (closed circles) or 10 µM xanomeline (open circles) was present 4 min before and during the second stimulation and 10 µM NMS 4 min before and during the third stimulation as indicated. [3H]ACh release is expressed as fractional release in percentage of tissue content of radioactivity (ordinate). B and E, slices were stimulated as in described in previous experiments but in the presence of 10 µM carbachol (closed squares) or 10 µM carbachol and 10 µM xanomeline together during the second stimulation. Points are mean ± S.E.M. of five to nine samples from two (cortex) or three (striatum) animals in independent experiments. Ordinate, fractional release of radioactivity. Abscissa, time from the end of loading. C and F, influence of tested drugs is expressed (ordinate) as Sn/S1 ratios (relative to the first, always control, stimulation; letters Ct, x, c, and cx denote superfusion with control medium, 10 µM xanomeline, 10 µM carbachol, or 10 µM xanomeline and 10 µM carbachol together before and during the second stimulation, respectively). **, p < 0.01, significantly different from corresponding preceding control stimulation and following stimulation in the presence of 1 µM NMS by ANOVA followed by Tukey's test.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

View larger version (23K): [in this window] [in a new window]   Fig. 5. A, inhibition of electrically evoked [3H]ACh release by wash-resistantly bound xanomeline after irreversible blockade of the muscarinic receptor orthosteric binding site. Cortical slices were treated during the last 15 min of loading with [3H]Ch with 100 nM PRBCM to irreversibly inactivate the muscarinic orthosteric binding site and afterward without (closed squares) or with (opened squares) 100 µM xanomeline for another 15 min. Slices were then superfused in the absence of free xanomeline and stimulated in control medium, then in the presence of 10 µM carbachol, and finally in the presence of 10 µM carbachol and 1 µM NMS together as described in Fig. 2A. Each point is mean ± S.E.M. of samples derived from at least two independent experiments. Ordinate, fractional release of radioactivity. Abscissa, time from the end of loading. Values of evoked [3H]ACh release and statistical evaluation are given in Table 4. B, effect of treatment with increasing concentrations of PRBCM for 15 min on [3H]NMS binding to brain cortex membranes prepared after 53 min of washing. Abscissa: concentration of PRBCM during treatment. Ordinate: [3H]NMS-specific binding is expressed in dpm per aliquot of 500 µg of protein. Each point is mean ± range of two measurements in triplicates. Control binding was 52,139 ± 23 dpm (mean ± range). C, effects of increasing concentraions of xanomeline applied for 15 min on [3H]NMS binding to brain cortex membranes prepared after 53 min of washing. [3H]NMS-specific binding (ordinate) to brain cortex membranes is expressed in dpm per aliquot of 50 to 60 µgof protein. Points are mean ± S.E.M. of representative measurement in triplicates. Abscissa, [3H]NMS in nanomolar. Parameters of [2H]NMS binding are summarized in Table 2.

Binding Experiments. [³H]N-methylscopolamine binding and wash-resistant xanomeline binding were determined as described by Jakubík et al. (2006). Curve fitting and statistical evaluation of data were done using Prism 4 (GraphPad Software Inc., San Diego, CA).

View larger version (30K): [in this window] [in a new window]   Fig. 2. Concentration-response relationship of wash-resistant xanomeline effects on electrically evoked [3H]ACh and [3H]NA release. Cortical (A) and striatal (B) slices were loaded with tritiated choline and then preincubated for 15 min without or with 1 to 100 µM xanomeline as indicated and then superfused with xanomeline-free medium. Three consecutive electrical stimulation were applied as in experiments shown in Fig. 1. Electrically evoked [3H]ACh release was measured during superfusion in control medium, in the presence of 10 µM carbachol, and in the presence of 10 µM carbachol and 1 µM NMS together, respectively. C, cortical slices were loaded with [3H]NA, treated for 15 min without or with 1 to 100 µM xanomeline as indicated, and then superfused in medium containing 1 µM desipramine. Electrically evoked [3H]NA release was measured with time as in A and B. The 2-adrenoceptor agonist UK-14,304 (1 µM) was present during the second stimulation and UK-14,304 together with 1 µM 2-adrenoceptor antagonist yohimbine during the third stimulation. Ordinate, fractional release of transmitter. Abscissa, time from the end of loading. Each point is mean ± S.E.M. of samples derived from at least two animals. Values of evoked [3H]ACh and [3H]NA release, number of observations, and statistical evaluation are given in Table 1.

All experiments were performed on male albino Wistar rats bred in the animal house of the Institute of Physiology (Czech Academy of Sciences, Prague, Czech Republic). The experiments were approved by the Animal Care and Use Committee of the Institute of Physiology to be in agreement with the Animal Protection Law of the Czech Republic that is fully compatible with European Community Council directives 86/609/EEC.

	Results

Top Abstract Materials and Methods Results Discussion References

 
In control slices, sequential stimulations applied at the 9th and 33rd min of superfusion (third and ninth collected fractions) evoked the release of [³H]ACh that slightly declined from 0.92 ± 0.08 to 0.78 ± 0.06% of tissue content of radioactivity (mean ± S.E.M.; n = 6) in cortex and from 2.99 ± 0.44 to 2.53 ± 0.41 (n = 9) in striatum (Fig. 1, A and D). Addition of 10 µM xanomeline to the superfusion medium had no effect on either basal outflow of radioactivity (0.85 ± 0.04 versus 0.80 ± 0.02% of tissue content of radioactivity in cortex and 1.13 ± 0.07 versus 1.11 ± 0.18 in striatum for control and xanomeline treatments, respectively) or electrically evoked release of [³H]ACh evaluated as S₂/S₁ (Fig. 1C for cortex and F for striatum). As expected, 10 µM carbachol significantly inhibited the evoked release of [³H]ACh and 1 µM NMS abolished its inhibitory influence in both tissues. Xanomeline at a concentration of 10 µM present together with carbachol did not influence its inhibitory effect (Fig. 1, B and C, for cortex and E and F, for striatum).

Pretreatment of choline-loaded cortical and striatal slices with 1, 10, or 100 µM xanomeline for 15 min followed by extensive washing had no effect on basal outflow of radioactivity, but it resulted in a concentration-dependent inhibition of evoked [³H]ACh release from cortical slices by 49, 77, and 86% and striatal slices by 23, 48, and 67%, respectively (Fig. 2, A and B; Table 1). Carbachol at 10 µM further caused a significant decrease in cortical slices pretreated with 1 µM xanomeline and in striatal slices pretreated with 1 and 10 µM xanomeline. In both tissues, 1 µM NMS fully prevented the inhibition of ACh release induced by pretreatment with 1 and 10 µM xanomeline, but it only partially reversed the inhibition induced by pretreatment with 100 µM xanomeline. Unlike [³H]ACh release, pretreatment with up to 10 µM xanomeline had no influence on either basal outflow of radioactivity or evoked release of [³H]NA and its regulation by presynaptic ₂-adrenoceptors (Fig. 2C; Table 1). Compared with all other groups, pretreatment with 100 µM xanomeline significantly increased basal outflow of ³H radioactivity to 4.18 ± 0.17% of tissue content of radioactivity from 2.84 ± 0.20, 2.64 ± 0.09, and 2.46 ± 0.18 in controls and after pretreatment with 1 and 10 µM xanomeline, respectively (p < 0.001 compared with all other groups by ANOVA and Tukey's test). As in ACh release experiments, pretreatment with 100 µM xanomeline significantly reduced by approximately 35% evoked release of [³H]NA. However, regulation of evoked [³H]NA release by presynaptic ₂-adrenoceptors was preserved as demonstrated by inhibition of evoked [³H]NA release by the selective agonist of ₂-adrenoceptors, UK-14,304, and full reversal of this effect by the ₂-adrenoceptors antagonist yohimbine.

Binding experiments with membranes of CHO cells expressing human M₁–M₅ subtypes of muscarinic receptors demonstrated that wash-resistant xanomeline binding occurs with similar potency at all subtypes (Fig. 3). Likewise, pretreatment of cortical slices with 100 µM xanomeline followed by washing as in superfusion experiments slightly decreased maximal binding of [³H]NMS determined in cortical membranes by 28% (from 675 to 486 fmol/mg protein), but it induced a large (approximately 10 times) decrease of [³H]NMS affinity from 495 to 4847 pM (Fig. 5C; Table 2).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Wash-resistant xanomeline binding to the M1-M5 subtypes of muscarinic receptors. Membranes prepared from M3 (triangles), M4 (diamonds), and M5 (closed circles) receptor expressing CHO cells were preincubated 60 min with increasing concentrations of xanomeline (abscissa), extensively washed, and then incubated with [3H]NMS. Wash-resistant binding of xanomeline to muscarinic receptors was determined by its ability to decrease binding of 1 nM [3H]NMS (ordinate, specific binding in percentage of control) as described in Jakubík et al. (2006). Nonspecific binding was determined in the presence of 10 µM NMS. Incubation with [3H]NMS was terminated after 60 min by filtration through glass fiber filters. Data are means ± S.E.M. of three to four independent experiments performed in triplicates. Hill slopes are not significantly different from unity. Inset, pIC50 values. Data for M1 and M2 receptors (open circles and open squares, respectively) were taken from Jakubík et al. (2006) for comparison.

View this table: [in this window] [in a new window]   TABLE 2 Parameters of [3H]N-methylscopolamine binding to cortical membranes after treatment with xanomeline Data shown in Fig. 5C were evaluated as described by Jakubik et al. (2006). Results are mean ± S.E.M. of four independent samples run in triplicates.

In the next experiments, we further investigated features of the irreversible agonistic effects of pretreatment with 100 µM xanomeline in cortical slices, i.e., at M₂ receptors. As shown in Fig. 4A and Table 3, the release of acetylcholine after xanomeline pretreatment was stable for at least three stimulations. Neither presence of 1 µM NMS during xanomeline treatment (Fig. 4B; Table 3) nor continuous washing with 1 µM NMS for 57 min before stimulation (Fig. 4C; Table 3) prevented inhibition of ACh release. In experiments summarized in Fig. 5, A and B and Table 4, we irreversibly inactivated the orthosteric binding site using propylbenzylcholine mustard to further support the finding that activation of the M₂ receptor by prolonged pretreatment with 100 µM xanomeline does not involve the orthosteric binding site. Treatment of slices with 100 nM PRBCM for 15 min reduced specific binding of the orthosteric ligand [³H]NMS at 2 nM to 4.5 ± 0.8% of control. As expected, inactivation of the orthosteric binding site by PRBCM treatment markedly attenuated presynaptic modulation of ACh release by carbachol (Fig. 5A; Table 4). The small remaining response to carbachol was not receptor-mediated, because it was not blocked by NMS. However, in line with our observation that NMS present together with xanomeline does not prevent its delayed inhibitory effects on ACh release, preincubation of PRBCM-treated slices with 100 µM xanomeline for 15 min still strongly inhibited ACh release, and this inhibition was not reversed in the presence of NMS during stimulation, contrary to what is expected in case of drugs acting through the orthosteric binding site.

View larger version (25K): [in this window] [in a new window]   Fig. 4. N-Methylscopolamine does not prevent wash-resistant xanomeline effect. A, three consecutive control stimulations (in the absence of muscarinic ligands) evoke comparable [3H]ACh release in control slices as well as in 100 µM xanomeline-treated slices. B, 1 µM NMS present during xanomeline treatment (closed squares) does not influence the inhibitory effect of wash-resistant xanomeline on [3H]ACh release. C, extensive washing of slices in medium containing 1 µM NMS (closed triangles) prevents only partially the inhibitory effect of wash-resistant xanomeline. Ordinate, fractional release of transmitter. Abscissa, time from the end of loading. Points are mean ± S.E.M. of samples derived from two independent experiments. Values of evoked [3H]ACh release, number of observations, and statistical evaluation are given in Table 3.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Xanomeline is a muscarinic agonist that binds equally well to all subtypes of muscarinic receptors, but its immediate application in functional assays in vitro points to M₁/M₄ selectivity (Bymaster et al., 1997). Another remarkable feature of xanomeline interaction with muscarinic receptors is its wash-resistant binding demonstrated at M₁ (Christopoulos et al., 1998, 1999; Jakubík et al., 2002, 2004), M₂ (Jakubík et al., 2006), and M₅ (Grant and El-Fakahany, 2005) receptors. We also demonstrate wash-resistant binding of xanomeline to the M₃ and M₄ receptor subtypes that has similar affinity with other subtypes (Fig. 3). In addition, it has been demonstrated that in the absence of free ligand, wash-resistantly bound xanomeline exhibits different potency, time course, and efficacy in activating M₁ and M₂ receptors (Jakubík et al., 2006) and antagonizes the M₅ subtype (Grant and El-Fakahany, 2005). However, all these observations were obtained using muscarinic receptors heterogenously expressed in cell lines. The main finding of the present experiments is the confirmation of the presence of persistent wash-resistant effects of xanomeline at M₂ and M₄ receptors expressed in their natural environment.

In line with previous findings on M₂ receptors expressed in membranes of CHO cells, xanomeline at concentrations that saturate the orthosteric binding site had no immediate effect on either basal efflux of radioactivity or evoked release of labeled ACh release from cortical slices. Conversely, preincubation of cortical slices for 15 min with xanomeline induced a delayed concentration-dependent decrease of evoked ACh release estimated 53 min after xanomeline washout. The half-maximal inhibition of evoked ACh release by wash-resistant xanomeline was reached at around 1 µM. The maximal effect of xanomeline amounted to that of the full agonist carbachol and maximal effects of xanomeline and carbachol were not additive, indicating common mechanism of action, i.e., activation of M₂ receptors. Although the potency of wash-resistant xanomeline in inhibiting ACh release and in inducing coupling of M₂ receptor expressed in CHO cell membranes to G_i/o G proteins (Jakubík et al., 2006) is reasonably comparable, its efficacy in inhibiting evoked acetylcholine release is higher. This discrepancy may be due to receptor reserve of autoreceptors on cholinergic endings.

Selectivity of xanomeline pretreatment with regard to muscarinic receptor-mediated effects was tested using presynaptic ₂-adrenoceptors that mediate presynaptic inhibition of evoked noradrenaline release (Starke, 2001). Noradrenaline release from rat brain cortex in vivo is not influenced by administration of xanomeline (Perry et al., 2001). In concert, as shown in Fig. 2C and Table 1, xanomeline pretreatment at concentrations up to 10 µM had no appreciable effect on evoked noradrenaline release or on its inhibition by the ₂-adrenoceptor agonist UK-14,304. Pretreatment with 100 µM xanomeline, however, significantly increased basal outflow of radioactivity and somehow reduced evoked noradrenaline release. However, the inhibition of evoked release by an ₂-adrenergic agonist remained preserved.

An interesting feature of wash-resistant xanomeline inhibitory action was observed in experiments testing the involvement of the orthosteric site in xanomeline effects on ACh release. Presence of the orthosteric antagonist N-methylscopolamine in the medium during stimulation abolishes inhibition of ACh release observed after treatment with low concentrations of xanomeline (1 and 10 µM), whereas only partial prevention was found after treatment with 100 µM xanomeline for both cortex and striatum. The inhibition of ACh release from cortical slices induced by pretreatment with 100 µM xanomeline was not abolished by either presence of N-methylscopolamine during pretreatment or extensive washing in the presence of N-methylscopolamine. Likewise, irreversible inactivation of the orthosteric binding site using propylbenzylcholine mustard before xanomeline treatment reduced [³H]N-methylscopolamine binding by more than 95% and abolished carbachol-induced inhibition of evoked ACh release, but it did not prevent the inhibitory action of wash-resistantly bound xanomeline. These results apparently indicate that binding of xanomeline to the orthosteric site is not imperative for formation of its wash-resistant interaction with the receptor. Furthermore, they demonstrate that wash-resistant xanomeline activates the receptor even when the orthosteric site is obstructed as evidenced by abolition of N-methylscopolamine binding and inhibition of evoked ACh release by an orthosteric agonist after propylbenzylcholine treatment.

The inhibitory action of xanomeline on ACh release was similar in cortex and striatum in that it did not display immediate effects; the concentration-responses were roughly the same, in line with comparable affinity of wash-resistant binding (Fig. 3); and the delayed inhibitory effects were not prevented by pretreatment with xanomeline in the presence of antagonist (data not shown for striatum). This observation was a bit surprising in striatum because of reported M₁/M₄ agonistic profile (Bymaster et al., 2002, 2003). Because striatum contains cholinergic interneurons, we verified that the inhibitory action of xanomeline on ACh release was effected through presynaptic M₄ receptors in experiments with 50 mM potassium stimulation that precludes a possible involvement of action potential propagation from cell bodies. As with electrical stimulation, xanomeline had no immediate effect when applied 8 min before and during potassium stimulation. However, preincubation with 100 µM xanomeline for 15 min followed by 53-min washing significantly reduced evoked ACh release (data not shown). We speculate that wash-resistant xanomeline binding is necessary for agonistic effects of xanomeline. It may be a matter of kinetics of wash-resistant binding formation that is very fast in M₁ receptor; therefore, receptor activation seems immediately. This is in contrast to much slower onset in case of the M₂ subtype (Jakubík et al., 2006) and perhaps also in the M₄ subtype. Alternatively, free xanomeline acting only through the orthosteric binding site might have antagonistic effects. This possibility is unlikely because continuous presence of xanomeline does not interfere with carbachol inhibitory influence on ACh release (Fig. 1, B and E). Moreover, we did not observe a reduction of wash-resistant xanomeline-induced receptor activation by free xanomeline in M₁ and M₂ receptors (Jakubík et al., 2006).

In conclusion, results of our experiments demonstrate wash-resistant delayed agonistic effects of xanomeline at muscarinic M₂ and M₄ receptors in functional tests using natural brain tissue. They are in line with observations obtained in binding and functional experiments in CHO cells expressing individual subtypes of muscarinic receptors, and they provide evidence for a complex mode of xanomeline action that encompasses both orthosteric and allosteric components. Interaction of xanomeline at an allosteric site on the muscarinic receptor is probably involved in its wash-resistant binding and agonistic effects (Jakubík et al., 2002). Thus, our findings support the potential of allosteric agonists (Lazareno and Birdsall, 1995; Jakubík et al., 1996, 1998, 2006; Sur et al., 2003; Langmead et al., 2006), and they provide an example of an agonist drug with prolonged activity that lingers in the absence of free ligand.

	Acknowledgements

 
Xanomeline was kindly provided by Dr. C. Felder. CHO cells expressing individual subtypes of muscarinic receptors were kindly supplied by Prof. T. I. Bonner (National Institute of Mental Health, Bethesda, MD).

	Footnotes

 
This work was supported by project of Czech Academy of Sciences AV0Z50110509, Czech Science Foundation Grant GACR305/05/0452, National Institutes of Health Grant NS25743, and Ministry of Education, Youth, and Sport of Czech Republic Grant LC554.

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

doi:10.1124/jpet.107.122093.

ABBREVIATIONS: ACh, acetylcholine; PRBCM, propylbenzylcholine mustard; NMS, N-methylscopolamine; NA, noradrenaline; CHO, Chinese hamster ovary; UK-14,304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine.

Address correspondence to: Dr. Vladimír Doleal, Department of Neurochemistry, Institute of Physiology, Czech Academy of Sciences, Vídeská 1083, 14220 Prague, Czech Republic. E-mail: dolezal{at}biomed.cas.cz

	References

Top Abstract Materials and Methods Results Discussion References

 

Andersen MB, Fink-Jensen A, Peacock L, Gerlach J, Bymaster F, Lundbaek JA, and Werge T (2003) The muscarinic M1/M4 receptor agonist xanomeline exhibits antipsychotic-like activity in Cebus apella monkeys. Neuropsychopharmacology 28: 1168-1175.[Medline]

Bymaster FP, Carter PA, Peters SC, Zhang W, Ward JS, Mitch CH, Calligaro DO, Whitesitt CA, DeLapp N, Shannon HE, et al. (1998) Xanomeline compared to other muscarinic agents on stimulation of phosphoinositide hydrolysis in vivo and other cholinomimetic effects. Brain Res 795: 179-190.[CrossRef][Medline]

Bymaster FP, Felder C, Ahmed S, and McKinzie D (2002) Muscarinic receptors as a target for drugs treating schizophrenia. Curr Drug Targets CNS Neurol Disord 1: 163-181.[CrossRef][Medline]

Bymaster FP, McKinzie DL, Felder CC, and Wess J (2003) Use of M1–M5 muscarinic receptor knockout mice as novel tools to delineate the physiological roles of the muscarinic cholinergic system. Neurochem Res 28: 437-442.[CrossRef][Medline]

Bymaster FP, Whitesitt CA, Shannon HE, DeLapp N, Ward JS, Calligaro DO, Shipley LA, Buelke-Sam JL, Bodick NC, Farde L, et al. (1997) Xanomeline: a selective muscarinic agonist for the treatment of Alzheimer's disease. Drug Dev Res 40: 158-170.[CrossRef]

Christopoulos A, Parsons AM, and El-Fakahany EE (1999) Pharmacological analysis of the novel mode of interaction between xanomeline and the M1 muscarinic acetylcholine receptor. J Pharmacol Exp Ther 289: 1220-1228.[Abstract/Free Full Text]

Christopoulos A, Pierce TL, Sorman JL, and El-Fakahany EE (1998) On the unique binding and activating properties of xanomeline at the M1 muscarinic acetylcholine receptor. Mol Pharmacol 53: 1120-1130.[Abstract/Free Full Text]

Doleal V, Jackisch R, Hertting G, and Allgaier C (1992) Activation of dopamine D1 receptors does not affect D2 receptor-mediated inhibition of acetylcholine release in rabbit striatum. Naunyn Schmiedebergs Arch Pharmacol 345: 16-20.[Medline]

Doleal V and Tuek S (1998) The effects of brucine and alcuronium on the inhibition of ³H acetylcholine release from rat striatum by muscarinic receptor agonists. Br J Pharmacol 124: 1213-1218.[CrossRef][Medline]

Grant MK and El-Fakahany EE (2005) Persistent binding and functional antagonism by xanomeline at the muscarinic M5 receptor. J Pharmacol Exp Ther 315: 313-319.[Abstract/Free Full Text]

Jakubík J, Baáková L, Lisá V, El-Fakahany EE, and Tuek S (1996) Activation of muscarinic acetylcholine receptors via their allosteric binding sites. Proc Natl Acad Sci U S A 93: 8705-8709.[Abstract/Free Full Text]

Jakubík J, El-Fakahany EE, and Doleal V (2006) Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M(1) and M(2) muscarinic acetylcholine receptors. Mol Pharmacol 70: 656-666.[Abstract/Free Full Text]

Jakubík J, Haga T, and Tuek S (1998) Effects of an agonist, allosteric modulator, and antagonist on guanosine--[³⁵S]thiotriphosphate binding to liposomes with varying muscarinic receptor/G_o protein stoichiometry. Mol Pharmacol 54: 899-906.[Abstract/Free Full Text]

Jakubík J, Tuek S, and El-Fakahany EE (2002) Allosteric modulation by persistent binding of xanomeline of the interaction of competitive ligands with the M1 muscarinic acetylcholine receptor. J Pharmacol Exp Ther 301: 1033-1041.[Abstract/Free Full Text]

Jakubík J, Tuek S, and El-Fakahany EE (2004) Role of receptor protein and membrane lipids in xanomeline wash-resistant binding to muscarinic M1 receptors. J Pharmacol Exp Ther 308: 105-110.[Abstract/Free Full Text]

Langmead CJ, Fry VA, Forbes IT, Branch CL, Christopoulos A, Wood MD, and Herdon HJ (2006) Probing the molecular mechanism of interaction between 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the muscarinic M(1) receptor: direct pharmacological evidence that AC-42 is an allosteric agonist. Mol Pharmacol 69: 236-246.[Abstract/Free Full Text]

Lazareno S and Birdsall NJ (1995) Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors. Mol Pharmacol 48: 362-378.[Abstract]

Lazareno S, Doleal V, Popham A, and Birdsall NJ (2004) Thiochrome enhances acetylcholine affinity at muscarinic M4 receptors: receptor subtype selectivity via cooperativity rather than affinity. Mol Pharmacol 65: 257-266.[Abstract/Free Full Text]

Mirza NR, Peters D, and Sparks RG (2003) Xanomeline and the antipsychotic potential of muscarinic receptor subtype selective agonists. CNS Drug Rev 9: 159-186.[Medline]

Perry KW, Nisenbaum LK, George CA, Shannon HE, Felder CC, and Bymaster FP (2001) The muscarinic agonist xanomeline increases monoamine release and immediate early gene expression in the rat prefrontal cortex. Biol Psychiatry 49: 716-725.[CrossRef][Medline]

Shannon HE, Bymaster FP, Calligaro DO, Greenwood B, Mitch CH, Sawyer BD, Ward JS, Wong DT, Olesen PH, Sheardown MJ, et al. (1994) Xanomeline: a novel muscarinic receptor agonist with functional selectivity for M1 receptors. J Pharmacol Exp Ther 269: 271-281.[Abstract/Free Full Text]

Starke K (2001) Presynaptic autoreceptors in the third decade: focus on alpha2-adrenoceptors. J Neurochem 78: 685-693.[CrossRef][Medline]

Sur C, Mallorga PJ, Wittmann M, Jacobson MA, Pascarella D, Williams JB, Brandish PE, Pettibone DJ, Scolnick EM, and Conn PJ (2003) N-Desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates N-methyl-D-aspartate receptor activity. Proc Natl Acad Sci U S A 100: 13674-13679.[Abstract/Free Full Text]

Ward JS, Merritt L, Calligaro DO, Bymaster FP, Shannon HE, Sawyer BD, Mitch CH, Deeter JB, Peters SC, Sheardown MJ, et al. (1995) Functionally selective M1 muscarinic agonists. 3. Side chains and azacycles contributing to functional muscarinic selectivity among pyrazinylazacycles. J Med Chem 38: 3469-3481.[CrossRef][Medline]

Watson J, Brough S, Coldwell MC, Gager T, Ho M, Hunter AJ, Jerman J, Middlemiss DN, Riley GJ, and Brown AM (1998) Functional effects of the muscarinic receptor agonist, xanomeline, at 5-HT1 and 5-HT2 receptors. Br J Pharmacol 125: 1413-1420.[CrossRef][Medline]

Wood MD, Murkitt KL, Ho M, Watson JM, Brown F, Hunter AJ, and Middlemiss DN (1999) Functional comparison of muscarinic partial agonists at muscarinic receptor subtypes hM1, hM2, hM3, hM4 and hM5 using microphysiometry. Br J Pharmacol 126: 1620-1624.[CrossRef][Medline]

Zhang W, Basile AS, Gomeza J, Volpicelli LA, Levey AI, and Wess J (2002) Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice. J Neurosci 22: 1709-1717.[Abstract/Free Full Text]

This article has been cited by other articles:

				T. P. Kenakin Seven Transmembrane Receptors as Nature's Prototype Allosteric Protein: De-emphasizing the Geography of Binding Mol. Pharmacol., September 1, 2008; 74(3): 541 - 543. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.122093v1 322/1/316    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Machová, E. Articles by Dolezal, V. Search for Related Content PubMed PubMed Citation Articles by Machová, E. Articles by Dolezal, V.