Investigation of rat striatal dopamine D-1 receptors solubilized by digitonin with a precipitation method.
[3H]SCH 23390 binding sites solubilized from rat striatal membranes by the detergent digitonin were investigated by using a polyethylene glycol precipitation method to separate the bound [3H]SCH 23390 from the free [3H]SCH 23390. The binding of [3H]SCH 23390 to the solubilized preparations was specific and saturable with a KD of 4.99 +/- 0.03 nM and a Bmax of 619 +/- 13 fmol/mg protein. The rank order of potency of dopamine agonists and antagonists for competing with [3H]SCH 23390 binding for the solubilized preparations was appropriate for dopamine D-1 receptors. The competition of SCH 23390 and S(-)-SCH 23388 with [3H]SCH 23390 binding for the solubilized preparations was stereoselective. However, the sensitivity of the dopamine agonist high-affinity binding to guanine nucleotide GTP was almost lost upon digitonin solubilization. Preincubating the membranes with dopamine preserved the guanine nucleotide sensitivity of agonist binding for membranes in solubilized preparations. These results proved that the polyethylene glycol precipitation method can be used for assay of digitonin-solubilized dopamine D-1 receptors in rat striatum.
Zhang
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X
Department of Pharmacology, Hiroshima University School of Medicine, Japan.
Segawa
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T
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000494:pharmacology; D000818:Animals; D001552:Benzazepines / Q000494:pharmacology; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology*; D004298:Dopamine / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000187:drug effects / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology
D001552:Benzazepines; D011954:Receptors, Dopamine; D012965:Sodium Chloride; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D006160:Guanosine Triphosphate; D004072:Digitonin; D004298:Dopamine