The kinetics of [3H]SCH 23390 dissociation from rat striatal dopamine D1 receptors: effect of dopamine.
The present study investigated possible allosteric interactions between dopamine and [3H]SCH 23390 ((R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepi n-7-ol)- labelled dopamine D1 receptors in rat striatum. As previously described, dopamine prevented [3H]SCH 23390 binding in a mixed competitive/non-competitive manner, causing both a loss of ligand affinity and a decrease in Bmax. The effect of dopamine was largely reversed following pretreatment of the membranes with 100 microM Gpp(NH)p (5'-guanylylimidodiphosphate) and was significantly enhanced by omission of Na+ from the incubation buffer. In dissociation kinetic studies, two methods of initiating ligand dissociation were used: dilution into 100-fold volume excess of buffer or addition of a molar excess of drug. Both methods yielded similar rates of [3H]SCH 23390 dissociation. Inclusion of dopamine in the volume excess of buffer did not alter the k-1 for [3H]SCH 23390 dissociation. However, when 100 microM dopamine was used instead of 1 microM piflutixol to initiate dissociation, a significant slowing of the rate of dissociation of [3H]SCH 23390 occurred. This effect of dopamine on k-1 was Na(+)-dependent since in the absence of Na+ the dopamine-induced rate of dissociation was only slightly slower than control values. Under neither condition did dopamine accelerate the rate of ligand dissociation, indicating that dopamine does not interact allosterically with [3H]SCH 23390 binding sites. These data, therefore, preclude an allosteric mechanism to explain the dopamine-induced decrease in dopamine D1 receptor density and provide direct evidence that dopamine masks ligand binding by binding to a high affinity site which can be modulated by Gpp(NH)p and Na+.
Farrell
C B
CB
Department of Pharmacology, University College Dublin, Belfield, Ireland.
O'Boyle
K M
KM
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000031:analogs & derivatives / Q000494:pharmacology; D000495:Allosteric Site / Q000187:drug effects; D000818:Animals; D001552:Benzazepines / Q000378:metabolism*; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology*; D018492:Dopamine Antagonists / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D017447:Receptors, Dopamine D1 / Q000187:drug effects / Q000378:metabolism*
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D001552:Benzazepines; D004298:Dopamine; D018492:Dopamine Antagonists; D006165:Guanylyl Imidodiphosphate; D017447:Receptors, Dopamine D1; C044846:SKF83566