Characterization of High‐Affinity Dopamine D₂ Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate‐Solubilized Bovine Striatal Preparations

Abstract

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.

Design, Synthesis, and Dopamine Receptor Modulating Activity of Diketopiperazine Peptidomimetics ofl-Prolyl-l-leucylglycinamide

“…[ 3 H]Spiroperidol/ N - Propylnorapomorphine Binding Competition Assay. Bovine striatal membranes were prepared essentially as described in previous reports. , The tissues were homogenized in 10 volumes of 0.25 M sucrose in a Potter-Elvehjem homogenizer and centrifuged at 1000 g for 15 min. The supernatant was then centrifuged for 1 h at 100000 g .…”

Characterisation of the Binding of [3H]WAY‐100635, a Novel 5‐Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

“…PICS n (Lohse et al ., 1987) and dopamine DZ receptors (Kazmi et al ., 1986) . It is recognised that divalent canons are required for the stabilisation of an agonist-specific high-affinity state at the expense of the low-affinity state by promoting the formation of the 5-HT, A receptor-ligand-G protein ternary complex .…”

Design, Synthesis, X-ray Analysis, and Dopamine Receptor-Modulating Activity of Mimics of the "C5" Hydrogen-Bonded Conformation in the Peptidomimetic 2-Oxo-3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-1-pyrrolidineacetamide

“…The bovine striatal membranes were prepared essentially as described in previous reports. 15 The tissues were homogenized in 10 volumes of 0.25 M sucrose in a Potter-Elvehjem homogenizer and centrifuged at lOOOg for 15 min. The supernatant was then centrifuged for 1 h at lOOOOOg.…”