The kinetics of [ 3H]SCH 23390 dissociation from rat striatal dopamine D 1 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-1 H-3-benzazepin-7-ol)-labelled dopamine D 1 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 B max. The effect of dopamine was largely reversed following pretreatment of the membranes with 100 μM 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 μM dopamine was used instead of 1 μM 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 D 1 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 +.