Selective labeling and localization of the M4 (m4) muscarinic receptor subtype

G. Ferrari-Dileo, M. Waelbroeck, D. C. Mash, D. D. Flynn

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28 Scopus citations

Abstract

We report here a novel strategy for the selective labeling and localization of the M4 (m4) muscarinic receptor subtype, based on the distinct kinetics of the muscarinic antagonists dexetimide and N- methylscopolamine (NMS) and on the selectivity profile of guanylpirenzepine and AF-DX 116 for the m1-m5 muscarinic receptor subtypes expressed in CHO-K1 cells. Incubation with 10 nM dexetimide, a nonselective antagonist, resulted in >90% occupancy of each of the m1-m5 receptor subtypes. The relatively rapid rates of dexetimide dissociation from the m1, m2, and m4 receptor subtypes (t( 1/2 ) values of <12.5 min) and the slower rates of dexetimide dissociation from the m3 and m5 receptor subtypes (t( 1/2 ) values of 65 and 75 min, respectively) favored the labeling of the m1, m2, and m4 receptor subtypes with short incubations with [3H]NMS. Inclusion of 200 nM guanylpirenzepine and 250 nM AF-DX 116 prevented the binding of [3H]NMS to the majority of the m1 and m2 receptor subtypes, respectively, resulting in primary labeling of the m4 receptor subtype. Brief dissociation of the radioligand in the presence of 1 μM atropine improved the ratio of m4 to m2 labeling by selectively removing [3H]NMS from the m2 subtype. Under these conditions, the ratio of [3H]NMS binding to the m4 versus m1, m2, m3, and m5 receptor subtypes was 4:1. In vitro autoradiography combined with these m4- selective labeling conditions demonstrated that the M4 (m4) receptor subtype was localized to the primary visual area (V1, area 17, occipital cortex) and the basal ganglia, a distribution distinct from that demonstrated for the M1 (m1), M2 (m2), and M3 (m3) receptor subtypes. These results demonstrate that a combination of the distinct kinetics of dexetimide and NMS and the receptor subtype selectivity of guanylpirenzepine and AF-DX 116 provides a valuable labeling strategy to examine the distribution and localization of the M4 (m4) muscarinic receptor subtype in brain, peripheral tissues, and cell lines.

Original languageEnglish (US)
Pages (from-to)1028-1035
Number of pages8
JournalMolecular Pharmacology
Volume46
Issue number6
StatePublished - Dec 1 1994

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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    Ferrari-Dileo, G., Waelbroeck, M., Mash, D. C., & Flynn, D. D. (1994). Selective labeling and localization of the M4 (m4) muscarinic receptor subtype. Molecular Pharmacology, 46(6), 1028-1035.