Characterization, solubilization, and separation of two distinct dopamine receptors in canine caudate nucleus.

Two types of [3H]dopamine binding were found in the synaptic membrane fractions of canine caudate nucleus and referred to as Dl (associated with adenylate cyclase) and Dz (independent of adenylate cyclase) dopamine receptors. Both receptors were present primarily in the membrane fractions and showed a similar subcellular distribution except for the microsomal fractions. Binding of [3H]dopamine to the Dl and Dz receptors was rapid, reversible, saturable, temperature-dependent, and of high affinity with the equilibrium dissociation constants (a) of 3.6 pM and 12 I~M, respectively. Based on the selective inhibition of r3H]dopamine binding to the Dl and DZ receptors, dopamine antagonists could be tentatively classified into three classes, namely Dl-selective (YM-09151-2: cis-N-(1-benzyl-2-methyl-pyrrolidin-3-yl)-5-chloro-2-methoxy-4methylaminobenzamide), Dz-selective (sulpiride, a-ergocryptine) and nonselective (haloperidol, chlorpromazine). Ultraviolet illumination of the incubation mixture containing synaptic membranes and E3H]dopamine gave a stable association between the two due to the formation of a possibly covalent bond so that no dissociation took place even after gel filtration on a Sephadex G-200 column following solubilization with 2% Lubrol PX. Photochemically [3H]dopamine-labeled Dl and Dz receptors were clearly separable from one another by the gel filtration. Radioactivity of the [3H]dopamine-Dl receptor complex was mostly eluted at the elution volume corresponding to the Stokes radius of 4.5 nm and a major peak of the [3H]dopamine-D2 complex was found slightly behind the void volume corresponding to the Stokes radius of approximately 6.5 nm. Both elution peaks of the radioactive complexes were almost entirely eliminated by the presence of each specific antagonist at the time of incubation.