3H-(-)DO 710 discriminates guanine nucleotide sensitive and insensitive dopamine binding sites

Abstract

(-)DO 710, a substituted benzamide derivative which discriminates dopamine D-2 and D-4 binding sites (Sokoloff et al. 1984), and antagonises in a differential manner several apomorphine-induced behavioral responses (Schwartz et al. 1984) was tritiated and used namely to differentially label the D-4 site. In striatum the ³H-(-)DO 710 saturation curve was best explained by the presence of two classes of sites with a 7-fold difference in affinity (K_d values of 3.0 nM and 0.42 nM) and B_max values of 316 and 106 fmol · mg protein⁻¹, respectively which correspond to the D-2 and D-4 sites (Sokoloff et al. 1984). In spite of its limited selectivity, ³H-(-)DO 710 in low concentration (0.2 nM) could be used to preferentially label striatal D-4 site as shown by the inhibition potencies of discriminant benzamide derivatives (DBD), significantly higher than at pituitary D-2 site (receptor) whereas classical neuroleptics including metoclopramide were equally potent at both sites. The affinity of a variety of agonists for striatal sites labeled with 0.2 nM ³H-(-)DO 710 generally differed from their affinity for the two states of the pituitary D-2 receptor (with high and low affinity for agonists, respectively); compounds like lisuride, N-propylnorapomorphine or pergolide had very high affinity for the striatal ³H-(-)DO 710 site. In pituitary from oestradiol-treated rats, where only D-2 site occurs, only the low-affinity site for ³H-(-)DO 710 (K_d=2.8 nM) was found. In contrast, in the olfactory bulb there was a large proportion of the high-affinity site for ³H-(-)DO 710 since binding occurred with a mean K_d value of 0.72 nM. The effects of a guanylnucleotide [0.1 mM Gpp(NH)p] differed in the three tissues. In pituitary Gpp(NH)p elicited marked effects: i) a 63% increase of specific binding of 0.2 nM ³H-(-)DO 710; ii) a 9-fold increase in the IC₅₀ value of dopamine; iii) a large increase in the pseudo-Hill coefficient of dopamine, so that the latter did not anymore differ from unity. In constrast, Gpp(NH)p produced only marginal changes in olfactory bulb in both the binding of 0.2 nM ³H-(-)DO 710 and its inhibition by dopamine. In striatum, an intermediate and more complex situation was found: Gpp(NH)p i) slightly decreased the mean K_d value of ³-H-(-)DO 710 from 1.8 to 1.2 nM; ii) increased by 30% the binding of 0.2 nM ³H-(-)DO 710; iii) decreased by 6-fold the mean affinity of dopamine. In striatum and olfactory bulb, the pseudo-Hill coefficient of dopamine was not modified by Gpp(NH)p. In addition, at a high ³H-(-)DO 710 concentration (5 nM), where part of the binding presumably occurred at ³H-(-)DO 710 low-affinity site, partial effects of Gpp(NH)p were observed. It is concluded that sites labeled with high affinity by the DBD ³H-(-)DO 710 in striatum and olfactory bulb are little or not affected by guanylnucleotides and presumably differ from either of the two agonist affinity states of the pituitary D-2 receptor.