Specific [3H]SCH23390 binding to dopamine D1 receptors in cerebral cortex and neostriatum: role of disulfide and sulfhydryl groups.
Receptor binding studies were performed in cerebral cortex (CTX) and neostriatum (CPU; caudate-putamen) using the dopamine D1 antagonist [3H]SCH23390. Because receptors are of protein nature, we examined the role of disulfide bonds (--SS--) and sulfhydryl groups (--SH) in the specific binding of [3H]SCH23390. Furthermore, membrane preparations contain a certain amount of lipid, so that treatments with --SH and --SS-- reagents could determine whether the fixation of the radioligand was to protein or to the lipid moiety. Pretreatment of CTX and CPU membranes with dithioerythritol, L-dithiothreitol, or 5,5'-dithiobis(2-nitrobenzoic acid), as well as with the alkylating agent N-ethylmaleimide, produced dose-dependent decreases of specific [3H]SCH23390 binding in membrane preparations from both tissues. These changes were not reversible after up to two washes, but could be prevented in part if the treatments were performed in the presence of dopamine. Additional protection experiments were conducted with (+)- and (-)-butaclamol, as well as with (+)- and (-)-SKF38393. A series of saturation experiments (with pretreated membranes in the absence of reactives) demonstrated that the alkylation of --SS-- groups reduced specific [3H]SCH23390 binding mainly through an affinity change, but L-dithiothreitol and 5,5-dithiobis(2-nitrobenzoic acid) decreased the number of binding sites. The affinity of the receptor to agonists was examined with the two enantiomers of SKF38393; the inhibition curves showed that residual binding was not affected and stereospecificity was conserved. The present results provide evidence for the participation of both --SS-- and --SH groups in the recognition site of the dopamine D1 receptor in both the CTX and the CPU.
Dewar
K M
KM
Département de Physiologie, Faculté de Médecine, Université de Montréal, Québec, Canada.
Reader
T A
TA
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D000818:Animals; D001552:Benzazepines / Q000378:metabolism* / Q000494:pharmacology; D002069:Butaclamol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D003342:Corpus Striatum / Q000378:metabolism*; D004220:Disulfides; D004226:Dithioerythritol / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008297:Male; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000187:drug effects / Q000378:metabolism*; D017447:Receptors, Dopamine D1; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds
D001552:Benzazepines; D004220:Disulfides; D011954:Receptors, Dopamine; D017447:Receptors, Dopamine D1; D013438:Sulfhydryl Compounds; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D004226:Dithioerythritol; D004228:Dithionitrobenzoic Acid; D002069:Butaclamol; D005033:Ethylmaleimide; D004229:Dithiothreitol