CHARACTERIZATION AND SUBCELLULAR LOCALIZATION OF BRAIN MUSCARINIC RECEPTORS LABELLED IN VIVO BY [3H]DEXETIMIDE

Abstract— ³H]Dexetimide specifically labels brain muscarinic receptors in vivo . After i.v. injection of labelled drug into rats, radioactivity specifically accumulates in brain regions containing muscarinic receptors but not in cerebellum. This accumulation is stereospecific, saturable and displaceable by unhbelled dexetimide. In contrast, ³H]levetimide, the inactive enantiomer, does not show such preferential uptake or stereospecific displacement.
An analytical approach was used to study the subcellular distribution of ³H]dexetimide binding sites. After differential centrifugation the binding sites are mainly recovered in the microsomal fraction from different brain regions but not from the cerebellum. After displacement the radioactivity is found in the supernatant. After equilibration in a density gradient the distribution pattern of ³H]dexetimide is bimodal, like that of 5'-nucleotidase, with a major peak in a region of low density.
When the microsomal fraction was treated with digitcnin, three groups of membrane were characterized by isopycnic centrifugation on the basis of their differential shift to higher densities. Evidence is provided that the postsynaptic membranes bearing muscarinic receptors belong to the class of plasma membranes. Finally, digitonin treatment may represent a useful tool to produce subfractions enriched in postsynaptic membranes which can now be identified biochemically in binding experiments.