Dopamine D 2 receptor-mediated G protein activation assessed by agonist-stimulated [ 35S]guanosine 5′- O-(γ-thiotriphosphate) binding in rat striatal membranes

Abstract

In order to investigate the functional interaction between the native dopamine receptors and their coupled guanine nucleotide-binding regulatory (G) proteins, dopamine-stimulated [ 35S]guanosine 5′- O-(γ-thiotriphosphate) ([ 35S]GTPγS) binding was pharmacologically characterized in rat striatal membranes. Following optimizing the experimental conditions as to the concentrations of GDP, MgCl 2 and NaCl in the assay medium, the agonist and antagonist properties for a series of dopamine receptor ligands were determined mainly under the standard assay condition. The pharmacological profile of this response clearly indicated the involvement of dopamine D 2-like receptors, but not of dopamine D 1-like receptors. Among the types of dopamine D 2-like receptors, dopamine D 2 receptors most likely appeared to be involved in dopamine-stimulated [ 35S]GTPγS binding in rat striatal membranes, because the affinities of agonists and antagonists determined in the present study were significantly correlated with those reported in the previous literature only for dopamine D 2 receptors, but not for dopamine D 3 or D 4 types. Though the concentration-dependent inhibition curves of dopamine-stimulated [ 35S]GTPγS binding by spiperone and S(−)-raclopride were apparently biphasic, the origin of the low-affinity minor components was not fully determined. The antiparkinsonian drugs with the properties of dopamine receptor agonism were shown to behave as stimulants with varied affinities and relative efficacies in the current assay system. On the other hand, neither phencyclidine (PCP) nor ketamine stimulated the specific [ 35S]GTPγS binding, in contrast with the previous report demonstrating that these two N-methyl- d-aspartic acid (NMDA) receptor antagonists behaved as agonists at human dopamine D 2 receptors expressed in Chinese hamster ovary (CHO) cells. These results provide important information about the functional activation of G proteins coupled with dopamine D 2 receptors as well as agonist actions of various compounds at native dopamine D 2 receptors, which are potentially involved in pathophysiology and pharmacotherapy of neuropsychiatric diseases such as Parkinson's disease, schizophrenia and depression.