The characterization of [ 3H]sulpiride binding sites in rat striatal membranes

Abstract

The characteristics of [ 3H]sulpiride binding to the D 2 dopamine receptor in rat striatal membranes were examined under several conditions. In the absence of sodium ions, the specific binding of [ 3H]sulpiride could not be detected. In the direct binding experiment, at 25 °C, the affinity of [ 3H]sulpiride for D 2 receptors was increased in a dose-dependent manner of sodium ions whereas magnesium ions have opposite effects on an affinity of [ 3H]sulpiride binding. The lowering incubation temperature (4 °C) also produced a further increase in affinity of the ligand. Under all conditions, [ 3H]sulpiride labeled a single homogenous site of the receptor. On the other hand, the result from quantitative analysis of agonist/[ 3H]sulpiride competition curves indicated an existence of high (RH) and low (RL) affinity states for agonists and the proportion of two-affinity states was modulated by guanosine triphosphate (GTP), magnesium ions and lowering temperatures. GTP, together with sodium ions, caused a full conversion of RH to RL, while an increase in the affinity for agonists with a partial conversion of RL to RH could be induced by magnesium ions at 25 °C. At a lower (4 °C) temperature, the agonist competition curve indicated an existence of a single agonist low-affinity state (RL) and then, the effects of GTP and magnesium ions in the agonist affinity observed at 25 °C were abolished. These results can be incorporated into a two-step, ternary complex model involving an inhibitory guanine nucleotide binding protein for the agonist and antagonist interaction with D 2 receptors. The regulation of [ 3H]sulpiride binding by sodium ions and guanine nucleotides is analogous to that of several receptors which are inversely coupled to adenylate cyclase systems. Accordingly, the specific binding of [ 3H]sulpiride appears to represent the agonist low-affinity state of D 2 receptors.