The kinetics of [3H]SCH 23390 dissociation from rat striatal dopamine D1 receptors: effect of dopamine

Abstract

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+.