- Email: [email protected]
The M5 (m5) receptor subtype: Fact or fiction?
Life Scicnccs, Vol. 60, Nos. 13/14, pp. llM-1112,1!297 Copyright 0 1997 Ekvier Science Inc. Printed in the USA. All rights resewed txm-3205/w $17.00 t .oa
PI1 800243205(97)00054-4
ELSEVIER
TEJE M5 (m5) RECEPTOR
SUBTYPE:
Carolyn M. Reever, Gaby Ferrari-DiLeo
FACT OR FICTION? and Donna D. Flynn
Department
of Molecular & Cellular Pharmacology P.O. Box 016189 University of Miami School of Medicine Miami, FL 33101
By comparison to the other subtypes of muscarinic receptors, very little is known about the binding properties, locations, mechanisms and physiological finctions of the M5 (m5)* receptor subtype. Studies of the m5 receptor have been hampered by the lack of mS-selective ligands or antibodies and a source that endogenously expresses predominantly the m5 receptor subtype. We have developed a pharmacological labeling strategy using the non-selective muscarinic antagonist [3HJNMS, in the presence of muscarinic antagonists and toxins in green mamba venom to occlude the ml-m4 receptor subtypes, to selectively label the m5 receptor subtype. This m5-selective labeling approach, along with those developed for the other four receptor subtypes, has permitted for the first time a comparison of the relative expression levels and anatomical localizations of the five muscarinic receptor subtypes in the brain. The distribution profile of the m5 receptor is distinct from the other four subtypes and is enriched in the outer layers of the cortex, specific subfields of the hippocampus, caudate putamen, olfactory tubercle and nucleus accumbens. These studies have also demonstrated that the levels of m5 receptor protein expression are apparently higher and more widespread than anticipated from previous in situ hybridization and immunoprecipitation studies. Taken together, the results suggest a unique and potentially physiologically important role for the m5 receptor subtype in modulating the actions of acetylcholine in the brain. Key Work
autoradiography,
muscarinic
receptors,
m5 receptor
Not long after the original cloning of four muscarinic receptor genes from mammalian brain, a fifth muscarinic receptor gene was identified (1,2). Nonetheless, subsequent Northern blot *The nomenclature for muscarinic receptor subtypes has designated the big “M” terminology for the pharmacologicallydefincd receptors and the small “m” terminology for the molecularly-defined receptors. It is now clear that the ml sequence corresponds to the Ml receptor, the m2 to the M2, etc. Since the receptor subtype-selectivity of the pharmacological labeling strategies described here have been validated using the individual molecular receptor expressed in transfected cells, we have utilized the small “m” terminology to refer to the respective receptors labeled by each of the subtype-selective conditions.
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analyses failed to demonstrate the expression of rnRNA for the mS muscarinic receptor subtype in brain or in selected peripheral tissues under conditions that revealed the presence of the ml-m4 transcripts (1). These results suggested that the m5 receptor was expressed at very low levels, if at all, compared to the other four muscarinic receptors. In situ hybridization histochemical studies confirmed the relative low abundance of m5 receptor mRNA in the brain, but demonstrated a distinct localization pattern for this receptor subtype in restricted subfields of the hippocampus and basal ganglia (3,4). Within the substantia nigra pars compacta @NC) and ventral tegmental area (VTA), m5 receptor mRNA was the only muscarinic receptor transcript co-expressed with D2 dopamine receptor mRNA (4). This discrete and highly concentrated expression of the m5 subtype suggested a unique and physiologically important role for this receptor.
The MS Receptor: What Do We Know? Very little is known about the pharmacology, mechanisms, locations and physiological fimctions of the m5 receptor compared to the other four receptor subtypes. Table 1 summarizes what we currently know about the m5 receptor. Structurally, the m5 receptor is the second largest muscarinic receptor and is most like the m3 receptor with a large i3 loop. The third cytoplasmic loop accounts for the greatest sequence diversity between receptor subtypes and also between receptors from different species. Of the five muscarinic receptors, the m5 subtype demonstrates the least homology in the i3 region between the rat and human sequences (1). Pharmacologically, no ligands have been identified with high affinity or selectivity for the m5 subtype. However, the m5 receptor demonstrates significantly lower affinities for AQ-R4 741 and AF-DX 384 compared to the ml-m4 receptors (5). Solubiliition of the m5, and also the m3 receptor, is least efficient and results in large decreases in affinity of the m5 receptor for most muscarinic receptor ligands (6). Functional studies of the m5 receptor have been limited to studies in clonal cell lines over-expressing the m5 receptor protein, since no tissues or cell lines that express a predominance of the native m5 receptor have been identified. These studies have
TABLE I. THE M5 RECEPTOR STRUCTURE
SUBTYPE:
WHAT DO WE KNOW?
) 532 amino acids (compare to 460-480 of ml, m2 and m4) ) like m3 with large i3 loop ) least homology (80%) in i3 loop between rat and human
PHARMACOLOGY
’ no selective high affinity ligand ) 1 lo-fold lower affinities than ml-m4 for AQ-RA 741 and AF-DX 384 ) E-fold decrease in affinity for NMS with solubilization
) least efficient solubilization MECHANISMS
) follows subtype specific pattern for odd-numbered receptors: PLQ3 via Gq, PL&, PLD, NOS, Ca” influx, CAMP, reverse transformation
LOCATION
) most restricted mRNA distribution of the five subtypes ) transcript expressed only in brain (also melanoma cells and microglia /macrophzges) ’ highest densities of mRNA in SNc, VTA, hippocampus, cortex and striatum ) co-localizes with D2 receotor in SNc
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elucidated some of the biochemical signaling pathways modulated by the m5 receptor and have demonstrated that mS-mediated signal transduction follows the subtype-specific pattern for the odd-numbered muscarinic receptor subtypes. Recent studies have identified a human melanoma cell line that endogenously expresses the m5 receptor and may facilitate studies on the timctional characteristics of this subtype (7). Tools to Study Muscarinic Receptor Subtypes Studies of the m5 receptor protein have been hampered by the lack of appropriate tools to identity, label and localize it, Pharmacological characterization and anatomical localization studies have been impossible since no ligands, toxins or antibodies are available with sufficient selectivity to specifically label the m5 receptor. Molecular probes have identified m5 receptor transcripts but not functional receptor proteins. Antibodies have been the most usetil tools to Receptor subtype-specific antisera have been useful for study individual receptors. immunoprecipitation studies demonstrating the relative abundance and distribution of the five receptor proteins in brain and peripheral tissues. While these antisera have provided immunocytochemical maps of the ml-m4 receptors in the brain, similar studies of the m5 subtype have not been done due to the low sensitivity of the polyclonal antisera to this receptor and to the lack of cross-reactivity of antibodies made to portions of the human m5 sequence with the rat receptor. Novel Pharmacological Strategies to Selectively Label Muscarinic Receptor Subtypes Pharmacological classification of the Ml-M4 receptors has for the most part relied on equilibrium binding assays with partially subtype-selective antagonists. These studies have been complicated somewhat by the overlapping atlinities of most available muscarinic ligands. Thus, while these assays were the first to establish the existence of muscarinic receptor subclasses, they have not been optimal for the direct labeling and localization of the individual receptor proteins in the brain. At present, pirenzepine, a putative Ml (ml) receptor subtype-selective antagonist, remains the only radio-labeled ligand with sufficient selectivity that permits labeling of a single receptor subtype. We have recently achieved selective labeling of the ml-m4 receptor subtypes by combining the use of the distinct kinetic and equilibrium binding properties of muscarinic receptor antagonists (8,9). Selective ml receptor labeling was achieved with low concentrations of [3H]pirenzepine as accomplished by other laboratories (8,lO). Selective m2 labeling was achieved with [3H]NMS by taking advantage of 1) the subtype-selective toxins in green mamba venom to occlude the ml and m4 subtypes (11, 12), 2) the relatively lower afhnity of the m2 receptor compared to the m3 receptor for 4-DAMP (5), and 3) the rapid kinetics of antagonist binding to the m2 compared to the m5 receptor (9). Selective m3 receptor labeling with [3H]NMS was also achieved in the presence of venom to occupy the ml and m4 receptors, AQRA 741 to occupy most of the m2 receptor (5, Reever and Flynn unpublished), and by short incubations to minimize labeling of the m5 subtype (9). Selective m4 labeling (9) was achieved with C3H]NMS in the presence of 1) guanylpirenzepine to occupy the ml receptor, 2) AF-DX 116 to occupy the m2 receptor (13) with a subsequent dissociation to minimize [3H]NMS labeling of the m2 subtype, and 3) dexetimide to occupy the m3 and m5 receptors. Each of these four labeling strategies resulted primarily in the labeling of the respective molecular receptor subtype (g,9). Selective Labeling of the MS Receptor Subtype Currently, there are no subtype-selective ligands or labeling conditions available for the m5 receptor. However, the m5 receptor demonstrates at least lo-fold lower afhnities for AQ-RA
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741 and AP-DX 384 compared to the affinities of the ml-m4 receptor subtypes for these antagonists (5). This suggested that concentrations of these compounds that occupy most of the ml, m2, m3 and m4 receptors may leave a large fraction of the m5 receptor unoccupied and available for labeling with a non-selective tritiated ligand like NMS. In the presence of 1 p.M AQRA 741,20% or less of the ml-m4 receptors were labeled with [3H’JNMS while nearly 60% of the m5 receptors were labeled. Although a significant proportion of m5 receptors were labeled compared to the other four subtypes, the higher levels of expression of the ml, m2 and m4 receptors relative to the m5 receptor in the brain, suggested that little if any m5-selective labeling would be achieved in this tissue. Thus, in order to minimize the contribution of these more abundant subtypes to the total labeling, we explored the use of venom from the green mamba, which has been shown by a number laboratories to contain muscarinic receptor subtype-selective toxins (11,12). In the presence of 30 ug/ml crude venom, virtually none of the ml, less than 10% of the m4 receptors, and nearly all of the m5 receptors were labeled with [‘H]NMS. In the presence of both AQ-KA 741 and venom, 55% of the mS receptors, 12% of the m3 and none of the ml, m2 and m4 receptors were labeled with [311JNMS (Pig. 1). This 4:l ratio of mS:m3 labeling represented labeling primarily of the m5 subtype in the brain, since the m5 and m3 receptors are expressed at approximately equivalent levels in this tissue (14,15)
60
0 ml
m2
m3
m4
m5
Figure 1. Proportions of ml-m5 receptors, individually expressed in CHO-Kl cells, labeled with 0.5 nM [31-IjNlvIS in the presence of 30 &ml green mamba venom and 1 pM AQ-RA 741. These labeling conditions resulted in nearly exclusive labeling of the m5 receptor, with a minor contribution of m3 labeling. Bars, means + standard deviation of triplicate determinations from 3 separate experiments.
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Anatomical Distribution of the m5 Receptor in the Brain Development of an mS-selective labeling strategy permitted for the first time visualization of the anatomical distribution of the mS receptor protein. The autoradiographic [%jNMS labeling patterns for the total, m3 and m5 muscarinic receptor populations are shown in Figure 2. Total muscarinic receptors were visualized in the presence of 0.5 nM [3H]NMS and m3 and m5 receptors were labeled as described above. The m5 labeling pattern was partially overlapping but distinct from the m3 pattern, as well as from the patterns previously noted for the ml-m4 receptors (8,9). The highest densities of m5 labeling were observed in the outermost layer of the cortex and the caudate putamen. Within the basal forebrain, the olfactory tubercle was the most intensely labeled region. The hippocampus showed distinct labeling of portions of the dentate gyms and CA1 and CA2 subfields, with no labeling of the CA3 subfield. In more caudal regions, the superficial gray layer of the superior colliculus was apparent while there was an absence of labeling in the inferior colliculus. There was no distinctive m5 labeling within more posterior regions of the brain or brainstem areas. Although not visible in sagittal sections, distinct m5 labeling of the SNNTA was apparent in coronal sections (data not shown). The SNc demonstrated the most intense labeling consistent with the noted high level of expression of m5 mRNA in this region (3,4). While there was some overlap of m3 with m5 labeling across the entire brain, there were some striking differences. Labeling of the m3 receptor was lower in the outer layer of the cortex, caudate putamen, hippocampus and olfactory tubercle compared to m5 labeling in these regions. Distinct m3 labeling was apparent in the more ventral layers of the superior colliculus, the inferior colliculus, cerebellum and specific brainstem nuclei (pontine, motor trigeminal and facial) while m5 labeling in these regions was very low or absent. These differences suggest that two distinct populations of receptors were labeled by the m3 and m5 conditions. Physiological Role of the m5 Receptor Since this is the first opportunity we have had to visualize the distribution of the m5 receptor protein in the brain, some speculation is warranted. The distinct m5 labeling of the outermost cortical layer is intriguing since this layer consists primarily of glial cells, apical dendrites and axons that run laterally, parallel to the pial surface. While we have previously suggested that the m5 receptor is the predominant muscarinic subtype expressed on microglia (16), it is uncertain whether this unique m5 labeling pattern results from labeling of glial cells in the superficial layers of the cortex. The m5 subtype also has been shown to be the muscarinic receptor subtype most efficiently coupled to nitric oxide synthase (NOS; 17). Taken together with the high levels of expression of the m5 receptor in the basal ganglia, a region also high in NOS expression, these findings suggest the m5 receptor may play a role in mediating NOS activation. The rather extensive distribution of the m5 subtype is surprising baaed on the early demonstrations by in situ hybridization of the relatively restricted m5 mRNA localization within the SNMA and its notable absence in the striatum and cortex (3,4). However, more recent studies using RT-PCR (18) have shown that the m5 subtype was uniformly expressed throughout the brain. Consistent with these studies, our autoradiograms demonstrated high levels of m5 labeling in the cortex, hippocampus, and particularly the striatum. The high level of expression of m5 receptor protein along with the co-localization of m5 receptor mRNA with dopamine D2 receptor mRNA in the SNc suggested to us that m5 receptors may be present on nigro-striatal dopaminergic terminals. We tested this hypothesis by performing 6-hydroxy-dopamine lesions of the SNNTA in rats and examining the m5 labeling pattern in the striatum 21 days post lesion. There was nearly a complete loss of tyrosine hydroxylase staining, a marker for dopaminergic cell bodies, in the SN as well as of [“‘I-RTI-121 binding, a high afiinity probe for the dopamine transporter, in the striatum (data not shown). These results are consistent with the degeneration
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Figure 2. Grayscale images of autoradiograms of total (T), m3 and mS muscarinic receptors in sag&al sections (2.4 mm lateral) of rat brain (20). Computergenerated grayscale coding was generated from scanned autoradiograms (while, The range highest densities; gruy, intermediate densities; black, lowest densities). of optical densities was the same for the m3 and m5 autoradiograms (O-3.9) but expanded (O-20) to compare the distribution of the more abundant total muscarinic receptor population. IC, inferior colliculus; Pn, pontine nuclei; 7, nucleus of the facial nerve; CA, CA subfield of Ammon’s horn; SuG, superficial gray layer of the superior colliculus; C’, caudate-putamen.
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of both dopaminergic cell bodies in the SNNTA and terminals in the striatum, respectively. While m5 labeling was lost in the SN/VTA region, there was little or no decrease in m5 labeling in the striatum. This result suggested that only a minor fraction of the m5 receptors in the striatum are on terminals of dopaminergic neurons originating in the SN/VTA, while the majority are on intrinsic or afferent striatal neurons. Due to the limits of resolution of light microscopic autoradiography, the identification of the specific population of neurons that express the m5 receptors in the striatum awaits the development of an m5-selective antibody. Conclusions: What More Do We Know? Selective labeling conditions for the M5 (m5) receptor subtype have been developed that have permitted for the first time autoradiographic localization studies of the m5 receptor in the brain. These m5-selective labeling conditions have assisted in studies of m5 receptor distribution in brain regions and peripheral tissues using in vitro test tube binding assays (19). The m5 receptor was enriched in the outer layers of the cortex, certain subfields of the hippocampus, caudate putamen, and olfactory tubercle. The availability of m5 receptor labeling conditions also has provided the first opportunity to directly compare the regional distribution patterns of all five muscarinic receptor subtypes. The distribution of the m5 receptor was distinct from the m3 and other muscarinic receptor subtypes. The relative levels of expression of the m5 receptor appeared to be higher and more widespread than previously anticipated from in situ hybridization studies. Results from lesioning experiments have provided evidence for the expression of the m5 receptor on both cell bodies and terminals of dopaminergic neurons in the SN/VTA, as well as on other intrinsic striatal neurons. While the studies described here represent a small step forward in our understanding of the M5 (m5) receptor subtype, it is anticipated that they will enhance interest and further study of this forgotten muscarinic receptor subtype. The development of m5-selective ligands and antibodies and identification of tissues and/or cell lines that express the native m5 receptor protein are necessary in order to determine the tinctions and physiological significance of the m5 muscarinic receptor subtype. Acknowledgments We gratefully acknowledge the technical assistance of Dr. David Liskowsky with the SN/VTA lesioning experiments and Dr. Weizhao Zhou with the computer-based image analysis of autoradiograms. AQ-R4 741 was generously provided by Boehringer Ingelheim, Ridgefield, CT. This work was supported by PHS grants NS19065 and AG12738. References 1. 2. 3. 4. 5. 6. 7.
BONNER, A.C. YOUNG, M.R. BRANN, and N.J. BUCKLEY, Neuron 1403-410(1988). LIAO, A.P.N. THEMMBN, R. JOHO, C. BABERIS, M. BIRNBAUMER, and L. BIRNBAUMBR, J. Biol. Chem. 264 7328-7337 (1989). VILARO, J.M. PALACIOS, and G. MENGOD, Neurosci Lett. 114 154-159 (1990). WEINER, A.I. LEVEY, and M.R. BRANN, Proc. Natl. Acad. Sci. USA 87 7050-7054 (1990). DOIUB, J. WESS, G. LAMBRECHT, R. TACKE, E. MUTSCHLER, and M.R. BRANN, J. Pharmacol. Exp. Ther. 256 727-733 (1991). RINKEN, K. KAMEYAMA, T. HAGA, and L. ENGSTROM, Biochem. Pharmacol. 48 1245-1251 (1994). A. KOHN, R. ALESSANDRO, J. PROBST, W. JACOBS, E. BRILLEY, and CC. PELDER, J. Biol. Chem. 271 17476-17484 (1996).
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12. 13. 14. 15. 16. 17. 18. 19. 20.
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FLYNN and D.C. MASH, Synapse 14 283-296 (1993). FERRARI-DlLEO, M. WAELBROECK, D.C. MASH and D.D. FLYNN, Mol. Pharmacol. 46 1028-1035 (1994). SPENCER, E. HORVATH, and J. TRABER, Brain Res. 380 59-68 (1986). ADEM, A. ASBLOM, G. JOHANSSON, P.M. MBUGUA and E. KARLSON, Biochim. Biophys. Acta 968 340-345 (1988). S.I. MAX, J.S. LIANG, and L.T. POTTER J. Neurosci. 13 4293-4300 (1993). M. WAELBROECK, M. TASTENOY, J. CAMUS, and J. CHRISTOPHE, Mol. Pharmacol. 38 267-273 (1990). A.I. LEVEY, S.M. EDMUND& C.J. HEILMAN, T.J. DESMOND, and K.A. FREY Neurosci. 63 207-221 (1994). R.P. YASUDA, W. CIESLA, L.R. FLORES, S.J. WALL, M. LI, S.A. SATKUS, J.S. WEISSTEIN, B.V. SPAGNOLA, and B.B. WOLFE, Mol. Pharmacol. 43 149-157 (1993). G. FERRARI-DILEO and D.D. FLYNN, Life Sci 56 1037 (1995). S.Z. WANG, S.Z. ZHU, and E.E. EL-FAKAHANY, J. Pharmacol. and Exp. Ther. 268 552-557 (1994). J. WEI, E.A. WALTON, A. MlLICI, and J.J. BUCCAFUSCO, J. Neurochem. 63 8 15-821 (1994). D.D. FLYNN, C.M. REEVER and G. FERRARI-DlLEO, Drug Dev. Res., in press. G. PAXINOS AND C. WATSON, The Rat Brain in Stereotaxic Coordinates, Academic Press, Australia (1986).
PI1 800243205(97)00054-4
ELSEVIER
TEJE M5 (m5) RECEPTOR
SUBTYPE:
Carolyn M. Reever, Gaby Ferrari-DiLeo
FACT OR FICTION? and Donna D. Flynn
Department
of Molecular & Cellular Pharmacology P.O. Box 016189 University of Miami School of Medicine Miami, FL 33101
By comparison to the other subtypes of muscarinic receptors, very little is known about the binding properties, locations, mechanisms and physiological finctions of the M5 (m5)* receptor subtype. Studies of the m5 receptor have been hampered by the lack of mS-selective ligands or antibodies and a source that endogenously expresses predominantly the m5 receptor subtype. We have developed a pharmacological labeling strategy using the non-selective muscarinic antagonist [3HJNMS, in the presence of muscarinic antagonists and toxins in green mamba venom to occlude the ml-m4 receptor subtypes, to selectively label the m5 receptor subtype. This m5-selective labeling approach, along with those developed for the other four receptor subtypes, has permitted for the first time a comparison of the relative expression levels and anatomical localizations of the five muscarinic receptor subtypes in the brain. The distribution profile of the m5 receptor is distinct from the other four subtypes and is enriched in the outer layers of the cortex, specific subfields of the hippocampus, caudate putamen, olfactory tubercle and nucleus accumbens. These studies have also demonstrated that the levels of m5 receptor protein expression are apparently higher and more widespread than anticipated from previous in situ hybridization and immunoprecipitation studies. Taken together, the results suggest a unique and potentially physiologically important role for the m5 receptor subtype in modulating the actions of acetylcholine in the brain. Key Work
autoradiography,
muscarinic
receptors,
m5 receptor
Not long after the original cloning of four muscarinic receptor genes from mammalian brain, a fifth muscarinic receptor gene was identified (1,2). Nonetheless, subsequent Northern blot *The nomenclature for muscarinic receptor subtypes has designated the big “M” terminology for the pharmacologicallydefincd receptors and the small “m” terminology for the molecularly-defined receptors. It is now clear that the ml sequence corresponds to the Ml receptor, the m2 to the M2, etc. Since the receptor subtype-selectivity of the pharmacological labeling strategies described here have been validated using the individual molecular receptor expressed in transfected cells, we have utilized the small “m” terminology to refer to the respective receptors labeled by each of the subtype-selective conditions.
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analyses failed to demonstrate the expression of rnRNA for the mS muscarinic receptor subtype in brain or in selected peripheral tissues under conditions that revealed the presence of the ml-m4 transcripts (1). These results suggested that the m5 receptor was expressed at very low levels, if at all, compared to the other four muscarinic receptors. In situ hybridization histochemical studies confirmed the relative low abundance of m5 receptor mRNA in the brain, but demonstrated a distinct localization pattern for this receptor subtype in restricted subfields of the hippocampus and basal ganglia (3,4). Within the substantia nigra pars compacta @NC) and ventral tegmental area (VTA), m5 receptor mRNA was the only muscarinic receptor transcript co-expressed with D2 dopamine receptor mRNA (4). This discrete and highly concentrated expression of the m5 subtype suggested a unique and physiologically important role for this receptor.
The MS Receptor: What Do We Know? Very little is known about the pharmacology, mechanisms, locations and physiological fimctions of the m5 receptor compared to the other four receptor subtypes. Table 1 summarizes what we currently know about the m5 receptor. Structurally, the m5 receptor is the second largest muscarinic receptor and is most like the m3 receptor with a large i3 loop. The third cytoplasmic loop accounts for the greatest sequence diversity between receptor subtypes and also between receptors from different species. Of the five muscarinic receptors, the m5 subtype demonstrates the least homology in the i3 region between the rat and human sequences (1). Pharmacologically, no ligands have been identified with high affinity or selectivity for the m5 subtype. However, the m5 receptor demonstrates significantly lower affinities for AQ-R4 741 and AF-DX 384 compared to the ml-m4 receptors (5). Solubiliition of the m5, and also the m3 receptor, is least efficient and results in large decreases in affinity of the m5 receptor for most muscarinic receptor ligands (6). Functional studies of the m5 receptor have been limited to studies in clonal cell lines over-expressing the m5 receptor protein, since no tissues or cell lines that express a predominance of the native m5 receptor have been identified. These studies have
TABLE I. THE M5 RECEPTOR STRUCTURE
SUBTYPE:
WHAT DO WE KNOW?
) 532 amino acids (compare to 460-480 of ml, m2 and m4) ) like m3 with large i3 loop ) least homology (80%) in i3 loop between rat and human
PHARMACOLOGY
’ no selective high affinity ligand ) 1 lo-fold lower affinities than ml-m4 for AQ-RA 741 and AF-DX 384 ) E-fold decrease in affinity for NMS with solubilization
) least efficient solubilization MECHANISMS
) follows subtype specific pattern for odd-numbered receptors: PLQ3 via Gq, PL&, PLD, NOS, Ca” influx, CAMP, reverse transformation
LOCATION
) most restricted mRNA distribution of the five subtypes ) transcript expressed only in brain (also melanoma cells and microglia /macrophzges) ’ highest densities of mRNA in SNc, VTA, hippocampus, cortex and striatum ) co-localizes with D2 receotor in SNc
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elucidated some of the biochemical signaling pathways modulated by the m5 receptor and have demonstrated that mS-mediated signal transduction follows the subtype-specific pattern for the odd-numbered muscarinic receptor subtypes. Recent studies have identified a human melanoma cell line that endogenously expresses the m5 receptor and may facilitate studies on the timctional characteristics of this subtype (7). Tools to Study Muscarinic Receptor Subtypes Studies of the m5 receptor protein have been hampered by the lack of appropriate tools to identity, label and localize it, Pharmacological characterization and anatomical localization studies have been impossible since no ligands, toxins or antibodies are available with sufficient selectivity to specifically label the m5 receptor. Molecular probes have identified m5 receptor transcripts but not functional receptor proteins. Antibodies have been the most usetil tools to Receptor subtype-specific antisera have been useful for study individual receptors. immunoprecipitation studies demonstrating the relative abundance and distribution of the five receptor proteins in brain and peripheral tissues. While these antisera have provided immunocytochemical maps of the ml-m4 receptors in the brain, similar studies of the m5 subtype have not been done due to the low sensitivity of the polyclonal antisera to this receptor and to the lack of cross-reactivity of antibodies made to portions of the human m5 sequence with the rat receptor. Novel Pharmacological Strategies to Selectively Label Muscarinic Receptor Subtypes Pharmacological classification of the Ml-M4 receptors has for the most part relied on equilibrium binding assays with partially subtype-selective antagonists. These studies have been complicated somewhat by the overlapping atlinities of most available muscarinic ligands. Thus, while these assays were the first to establish the existence of muscarinic receptor subclasses, they have not been optimal for the direct labeling and localization of the individual receptor proteins in the brain. At present, pirenzepine, a putative Ml (ml) receptor subtype-selective antagonist, remains the only radio-labeled ligand with sufficient selectivity that permits labeling of a single receptor subtype. We have recently achieved selective labeling of the ml-m4 receptor subtypes by combining the use of the distinct kinetic and equilibrium binding properties of muscarinic receptor antagonists (8,9). Selective ml receptor labeling was achieved with low concentrations of [3H]pirenzepine as accomplished by other laboratories (8,lO). Selective m2 labeling was achieved with [3H]NMS by taking advantage of 1) the subtype-selective toxins in green mamba venom to occlude the ml and m4 subtypes (11, 12), 2) the relatively lower afhnity of the m2 receptor compared to the m3 receptor for 4-DAMP (5), and 3) the rapid kinetics of antagonist binding to the m2 compared to the m5 receptor (9). Selective m3 receptor labeling with [3H]NMS was also achieved in the presence of venom to occupy the ml and m4 receptors, AQRA 741 to occupy most of the m2 receptor (5, Reever and Flynn unpublished), and by short incubations to minimize labeling of the m5 subtype (9). Selective m4 labeling (9) was achieved with C3H]NMS in the presence of 1) guanylpirenzepine to occupy the ml receptor, 2) AF-DX 116 to occupy the m2 receptor (13) with a subsequent dissociation to minimize [3H]NMS labeling of the m2 subtype, and 3) dexetimide to occupy the m3 and m5 receptors. Each of these four labeling strategies resulted primarily in the labeling of the respective molecular receptor subtype (g,9). Selective Labeling of the MS Receptor Subtype Currently, there are no subtype-selective ligands or labeling conditions available for the m5 receptor. However, the m5 receptor demonstrates at least lo-fold lower afhnities for AQ-RA
1108
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Subtype
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741 and AP-DX 384 compared to the affinities of the ml-m4 receptor subtypes for these antagonists (5). This suggested that concentrations of these compounds that occupy most of the ml, m2, m3 and m4 receptors may leave a large fraction of the m5 receptor unoccupied and available for labeling with a non-selective tritiated ligand like NMS. In the presence of 1 p.M AQRA 741,20% or less of the ml-m4 receptors were labeled with [3H’JNMS while nearly 60% of the m5 receptors were labeled. Although a significant proportion of m5 receptors were labeled compared to the other four subtypes, the higher levels of expression of the ml, m2 and m4 receptors relative to the m5 receptor in the brain, suggested that little if any m5-selective labeling would be achieved in this tissue. Thus, in order to minimize the contribution of these more abundant subtypes to the total labeling, we explored the use of venom from the green mamba, which has been shown by a number laboratories to contain muscarinic receptor subtype-selective toxins (11,12). In the presence of 30 ug/ml crude venom, virtually none of the ml, less than 10% of the m4 receptors, and nearly all of the m5 receptors were labeled with [‘H]NMS. In the presence of both AQ-KA 741 and venom, 55% of the mS receptors, 12% of the m3 and none of the ml, m2 and m4 receptors were labeled with [311JNMS (Pig. 1). This 4:l ratio of mS:m3 labeling represented labeling primarily of the m5 subtype in the brain, since the m5 and m3 receptors are expressed at approximately equivalent levels in this tissue (14,15)
60
0 ml
m2
m3
m4
m5
Figure 1. Proportions of ml-m5 receptors, individually expressed in CHO-Kl cells, labeled with 0.5 nM [31-IjNlvIS in the presence of 30 &ml green mamba venom and 1 pM AQ-RA 741. These labeling conditions resulted in nearly exclusive labeling of the m5 receptor, with a minor contribution of m3 labeling. Bars, means + standard deviation of triplicate determinations from 3 separate experiments.
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Anatomical Distribution of the m5 Receptor in the Brain Development of an mS-selective labeling strategy permitted for the first time visualization of the anatomical distribution of the mS receptor protein. The autoradiographic [%jNMS labeling patterns for the total, m3 and m5 muscarinic receptor populations are shown in Figure 2. Total muscarinic receptors were visualized in the presence of 0.5 nM [3H]NMS and m3 and m5 receptors were labeled as described above. The m5 labeling pattern was partially overlapping but distinct from the m3 pattern, as well as from the patterns previously noted for the ml-m4 receptors (8,9). The highest densities of m5 labeling were observed in the outermost layer of the cortex and the caudate putamen. Within the basal forebrain, the olfactory tubercle was the most intensely labeled region. The hippocampus showed distinct labeling of portions of the dentate gyms and CA1 and CA2 subfields, with no labeling of the CA3 subfield. In more caudal regions, the superficial gray layer of the superior colliculus was apparent while there was an absence of labeling in the inferior colliculus. There was no distinctive m5 labeling within more posterior regions of the brain or brainstem areas. Although not visible in sagittal sections, distinct m5 labeling of the SNNTA was apparent in coronal sections (data not shown). The SNc demonstrated the most intense labeling consistent with the noted high level of expression of m5 mRNA in this region (3,4). While there was some overlap of m3 with m5 labeling across the entire brain, there were some striking differences. Labeling of the m3 receptor was lower in the outer layer of the cortex, caudate putamen, hippocampus and olfactory tubercle compared to m5 labeling in these regions. Distinct m3 labeling was apparent in the more ventral layers of the superior colliculus, the inferior colliculus, cerebellum and specific brainstem nuclei (pontine, motor trigeminal and facial) while m5 labeling in these regions was very low or absent. These differences suggest that two distinct populations of receptors were labeled by the m3 and m5 conditions. Physiological Role of the m5 Receptor Since this is the first opportunity we have had to visualize the distribution of the m5 receptor protein in the brain, some speculation is warranted. The distinct m5 labeling of the outermost cortical layer is intriguing since this layer consists primarily of glial cells, apical dendrites and axons that run laterally, parallel to the pial surface. While we have previously suggested that the m5 receptor is the predominant muscarinic subtype expressed on microglia (16), it is uncertain whether this unique m5 labeling pattern results from labeling of glial cells in the superficial layers of the cortex. The m5 subtype also has been shown to be the muscarinic receptor subtype most efficiently coupled to nitric oxide synthase (NOS; 17). Taken together with the high levels of expression of the m5 receptor in the basal ganglia, a region also high in NOS expression, these findings suggest the m5 receptor may play a role in mediating NOS activation. The rather extensive distribution of the m5 subtype is surprising baaed on the early demonstrations by in situ hybridization of the relatively restricted m5 mRNA localization within the SNMA and its notable absence in the striatum and cortex (3,4). However, more recent studies using RT-PCR (18) have shown that the m5 subtype was uniformly expressed throughout the brain. Consistent with these studies, our autoradiograms demonstrated high levels of m5 labeling in the cortex, hippocampus, and particularly the striatum. The high level of expression of m5 receptor protein along with the co-localization of m5 receptor mRNA with dopamine D2 receptor mRNA in the SNc suggested to us that m5 receptors may be present on nigro-striatal dopaminergic terminals. We tested this hypothesis by performing 6-hydroxy-dopamine lesions of the SNNTA in rats and examining the m5 labeling pattern in the striatum 21 days post lesion. There was nearly a complete loss of tyrosine hydroxylase staining, a marker for dopaminergic cell bodies, in the SN as well as of [“‘I-RTI-121 binding, a high afiinity probe for the dopamine transporter, in the striatum (data not shown). These results are consistent with the degeneration
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Figure 2. Grayscale images of autoradiograms of total (T), m3 and mS muscarinic receptors in sag&al sections (2.4 mm lateral) of rat brain (20). Computergenerated grayscale coding was generated from scanned autoradiograms (while, The range highest densities; gruy, intermediate densities; black, lowest densities). of optical densities was the same for the m3 and m5 autoradiograms (O-3.9) but expanded (O-20) to compare the distribution of the more abundant total muscarinic receptor population. IC, inferior colliculus; Pn, pontine nuclei; 7, nucleus of the facial nerve; CA, CA subfield of Ammon’s horn; SuG, superficial gray layer of the superior colliculus; C’, caudate-putamen.
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of both dopaminergic cell bodies in the SNNTA and terminals in the striatum, respectively. While m5 labeling was lost in the SN/VTA region, there was little or no decrease in m5 labeling in the striatum. This result suggested that only a minor fraction of the m5 receptors in the striatum are on terminals of dopaminergic neurons originating in the SN/VTA, while the majority are on intrinsic or afferent striatal neurons. Due to the limits of resolution of light microscopic autoradiography, the identification of the specific population of neurons that express the m5 receptors in the striatum awaits the development of an m5-selective antibody. Conclusions: What More Do We Know? Selective labeling conditions for the M5 (m5) receptor subtype have been developed that have permitted for the first time autoradiographic localization studies of the m5 receptor in the brain. These m5-selective labeling conditions have assisted in studies of m5 receptor distribution in brain regions and peripheral tissues using in vitro test tube binding assays (19). The m5 receptor was enriched in the outer layers of the cortex, certain subfields of the hippocampus, caudate putamen, and olfactory tubercle. The availability of m5 receptor labeling conditions also has provided the first opportunity to directly compare the regional distribution patterns of all five muscarinic receptor subtypes. The distribution of the m5 receptor was distinct from the m3 and other muscarinic receptor subtypes. The relative levels of expression of the m5 receptor appeared to be higher and more widespread than previously anticipated from in situ hybridization studies. Results from lesioning experiments have provided evidence for the expression of the m5 receptor on both cell bodies and terminals of dopaminergic neurons in the SN/VTA, as well as on other intrinsic striatal neurons. While the studies described here represent a small step forward in our understanding of the M5 (m5) receptor subtype, it is anticipated that they will enhance interest and further study of this forgotten muscarinic receptor subtype. The development of m5-selective ligands and antibodies and identification of tissues and/or cell lines that express the native m5 receptor protein are necessary in order to determine the tinctions and physiological significance of the m5 muscarinic receptor subtype. Acknowledgments We gratefully acknowledge the technical assistance of Dr. David Liskowsky with the SN/VTA lesioning experiments and Dr. Weizhao Zhou with the computer-based image analysis of autoradiograms. AQ-R4 741 was generously provided by Boehringer Ingelheim, Ridgefield, CT. This work was supported by PHS grants NS19065 and AG12738. References 1. 2. 3. 4. 5. 6. 7.
BONNER, A.C. YOUNG, M.R. BRANN, and N.J. BUCKLEY, Neuron 1403-410(1988). LIAO, A.P.N. THEMMBN, R. JOHO, C. BABERIS, M. BIRNBAUMER, and L. BIRNBAUMBR, J. Biol. Chem. 264 7328-7337 (1989). VILARO, J.M. PALACIOS, and G. MENGOD, Neurosci Lett. 114 154-159 (1990). WEINER, A.I. LEVEY, and M.R. BRANN, Proc. Natl. Acad. Sci. USA 87 7050-7054 (1990). DOIUB, J. WESS, G. LAMBRECHT, R. TACKE, E. MUTSCHLER, and M.R. BRANN, J. Pharmacol. Exp. Ther. 256 727-733 (1991). RINKEN, K. KAMEYAMA, T. HAGA, and L. ENGSTROM, Biochem. Pharmacol. 48 1245-1251 (1994). A. KOHN, R. ALESSANDRO, J. PROBST, W. JACOBS, E. BRILLEY, and CC. PELDER, J. Biol. Chem. 271 17476-17484 (1996).
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