title: Evaluation of muscarinic agonist-induced analgesia in muscarinic acetylcholine receptor knockout mice.
abstract: Centrally active muscarinic agonists display pronounced analgesic effects. Identification of the specific muscarinic acetylcholine receptor (mAChR) subtype(s) mediating this activity is of considerable therapeutic interest. To examine the roles of the M(2) and M(4) receptor subtypes, the two G(i)/G(o)-coupled mAChRs, in mediating agonist-dependent antinociception, we generated a mutant mouse line deficient in both M(2) and M(4) mAChRs [M(2)/M(4) double-knockout (KO) mice]. In wild-type mice, systemic, intrathecal, or intracerebroventricular administration of centrally active muscarinic agonists resulted in robust analgesic effects, indicating that muscarinic analgesia can be mediated by both spinal and supraspinal mechanisms. Strikingly, muscarinic agonist-induced antinociception was totally abolished in M(2)/M(4) double-KO mice, independent of the route of application. The nonselective muscarinic agonist oxotremorine showed reduced analgesic potency in M(2) receptor single-KO mice, but retained full analgesic activity in M(4) receptor single-KO mice. In contrast, two novel muscarinic agonists chemically derived from epibatidine, CMI-936 and CMI-1145, displayed reduced analgesic activity in both M(2) and M(4) receptor single-KO mice, independent of the route of application. Radioligand binding studies indicated that the two CMI compounds, in contrast to oxotremorine, showed >6-fold higher affinity for M(4) than for M(2) receptors, providing a molecular basis for the observed differences in agonist activity profiles. These data provide unambiguous evidence that muscarinic analgesia is exclusively mediated by a combination of M(2) and M(4) mAChRs at both spinal and supraspinal sites. These findings should be of considerable relevance for the development of receptor subtype-selective muscarinic agonists as novel analgesic drugs.
authors.affiliation: Laboratory of Bioorganic Chemistry, National Institute of Diabetes Digestive and Kidney Diseases, Bethesda, Maryland 20892-0810, USA.
authors.forename: Alokesh
authors.initials: A
authors.lastname: Duttaroy
authors.forename: Jesus
authors.initials: J
authors.lastname: Gomeza
authors.forename: Jai-Wei
authors.initials: JW
authors.lastname: Gan
authors.forename: Nasir
authors.initials: N
authors.lastname: Siddiqui
authors.forename: Anthony S
authors.initials: AS
authors.lastname: Basile
authors.forename: W Dean
authors.initials: WD
authors.lastname: Harman
authors.forename: Philip L
authors.initials: PL
authors.lastname: Smith
authors.forename: Christian C
authors.initials: CC
authors.lastname: Felder
authors.forename: Allan I
authors.initials: AI
authors.lastname: Levey
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wess
mesh_terms: D000698:Analgesia; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D007276:Injections, Intraventricular; D051379:Mice; D018345:Mice, Knockout; D018721:Muscarinic Agonists; D019832:N-Methylscopolamine / Q000494:pharmacology; D010276:Parasympatholytics / Q000494:pharmacology; D043585:Receptor, Muscarinic M2; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013116:Spinal Cord / Q000378:metabolism; D014316:Tritium
chemical_list: D018721:Muscarinic Agonists; D010276:Parasympatholytics; D043585:Receptor, Muscarinic M2; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic; D014316:Tritium; D019832:N-Methylscopolamine