[³H]AF-DX 116 labels subsets of muscarinic cholinergic receptors in rat brain and heart

The in vitro binding properties of the novel muscarinic antagonist [³H]AF-DX 116 were studied using a rapid filtration technique. Association and dissociation rates of [³H]AF-DX 116 binding were rapid at 25°C (2.74 and 2.70 × 10⁷ min^-1 M^-1 for k₊₁; 0.87 and 0.93 min^-1 for k_-1) but 20-40 times slower at 0-4°C (0.13 and 0.096 × 10⁷ min^-1 M^-1 for k₊₁; 0.031 and 0.022 min^-1 for k_-1 in cerebral cortical and cardiac membranes, respectively). Kinetic dissociation constants (K_ds) were estimated to be 31.8 nM and 30.9 nM at 25°C ; 23.1 nM and 22.9 nM at 0-4°C for the cerebral cortex and heart, respectively. In saturation studies, [³H]AF-DX 116 labeled 29 percent of the total [³H](-)QNB binding sites in the cerebral cortical membranes and 87 percent in the cardiac membranes, with K_d values of 28.9 nM and 17.9 nM, respectively. Muscarinic antagonists inhibited [³H]AF-DX 116 binding in a rank order of potency of atropine > dexetimide > AF-DX 116 > PZ > levetimide in both tissues. Except for PZ/[³H]AF-DX 116 and AF-DX 116/[³H]AF-DX 116 in the cerebral cortex, all the antagonist competition curves had Hill coefficients close to one. Carbachol and oxotremorine produced shallow inhibition curves against [³H]AF-DX 116 binding in both tissues. Regional distribution studies with [³H](-)QNB, [³H]PZ and [³H]AF-DX 116 showed that most of the muscarinic receptors in the cerebral cortex, hippocampus, nucleus accumbens and corpus striattum are of the M₁ subtype while those in the brainstem, cerebellum and other lower brain regions are of the M₂ subtype. These results indicate that [³H]AF-DX 116 is a useful probe for the study of heterogeneity of muscarinic cholinergic receptors.