Effects of monovalent cations on neostriatal dopamine D2 receptors labeled with [3H]raclopride.

Specific [3H]raclopride binding to dopamine D2 receptors in the rabbit neostriatum was investigated in the presence of the monovalent cations sodium, lithium and potassium. NaCl and LiCl produced concentration-dependent elevations in specific [3H]raclopride binding with sodium inducing approximately 50% more binding than lithium. Inhibition of [3H]raclopride binding by the antagonist (+)-butaclamol was unaffected by the presence of sodium or lithium in the incubation medium. In contrast, the potency of dopamine to compete with [3H]raclopride was decreased by these two ions. This effect was more pronounced in the presence of sodium than lithium and was observed for both the high- and low-affinity states of the D2 receptor. The guanine nucleotide derivative 5'-guanylylimidodiphosphate (Gpp(NH)p) reduced the potency of dopamine to compete with [3H]raclopride binding in both the presence and absence of cations; however, this effect of Gpp(NH)p was a shift of the D2 receptors from a high to a lower affinity state. Saturation binding curves in the presence of sodium or lithium were compared with experiments carried out in the absence of monovalent cations (sucrose) and demonstrated that these ions increased the affinity (judged by the equilibrium dissociation constant Kd) of the neostriatal [3H]raclopride binding sites. While NaCl produced a significantly greater change in the Kd of [3H]raclopride binding as compared to LiCl, no differences were apparent in the maximum binding capacity (Bmax) values determined in the presence of these two cations. In conclusion, the results indicate that [3H]raclopride binding to rabbit neostriatal membranes exhibits a sensitivity to monovalent cations that is consistent with the ionic regulatory properties of the D2 receptor. Moreover, although lithium and sodium influence specific [3H]raclopride binding in a similar manner, there appear to be quantitative differences between these two ions.

Specific [3H]raclopride binding to neostriatal dopamine D2 receptors: role of disulfide and sulfhydryl groups.

Receptor binding studies were performed in rabbit neostriatum (caudate-putamen) using the dopamine D2 antagonist [3H]raclopride. Treatment of the membrane preparations with the reducing agent L-dithiothreitol (L-DTT) as well as with the alkylating compound N-ethylmaleimide (NEM), produced dose-dependent decreases of specific [3H]raclopride binding; the IC50 values were of 3.1 and 1.2 mM, respectively. Saturation experiments showed that the reduction of disulfide (-S-S-) bonds by L-DTT (1 mM) decreased the number of binding sites, with only a slight increase in the affinity. On the other hand, alkylation of sulfhydryl (-SH) groups by NEM (1 mM) decreased both receptor number and affinity. The properties of the remaining binding sites were examined in competition curves with the physiological substrate dopamine and the dopaminergic antagonist (+)butaclamol. The IC50 values for (+)butaclamol in control and in L-DTT and NEM treated membranes were between 3.4 and 4.8 nM, with Hill coefficients (nH) of 1, indicating that the remaining binding sites conserved a high affinity for antagonist binding. In the case of dopamine, the curves were shallow (nH 0.45-0.64) and both compounds increased the IC50 from 0.7 microM (control) to 8 microM and 11 microM, for L-DTT and NEM respectively. Iterative analysis revealed that L-DTT produced a very important (greater than 60%) decrease in the number of high-affinity (RH) binding. After NEM, there was a decrease in both the number of (RH) and the affinity (KH) of the high-affinity binding sites, and in the affinity (KL) of the low-affinity sites. These results demonstrate the participation of -S-S- and -SH groups in the agonist conformation of the primary ligand recognition site of the dopamine D2 receptor. Alternatively, -S-S- and -SH groups could be related to the coupling of the primary ligand recognition protein with adenylate cyclase by means of an inhibitory type of G protein.