SELECTIVE LABELING AND LOCALIZATION OF THE M4 (M4) MUSCARINIC RECEPTOR SUBTYPE

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 mi, 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 (tn values of 65 and 75 min, respectively) favored the labeling of the mi, m2, and m4 receptor subtypes with short incubations with [H-3]NMS. Inclusion of 200 nM guanylpirenzepine and 250 nM AF-DX 116 prevented the binding of [H-3]NMS to the majority of the mi and m2 receptor subtypes, respectively, resulting in primary labeling of the m4 receptor subtype. Brief dissociation of the radioligand in the presence of 1 mu M atropine improved the ratio of m4 to m2 labeling by selectively removing [H-3]NMS from the m2 subtype. Under these conditions, the ratio of [H-3]NMS binding to the m4 versus mi, 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 (mi), 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.