Vol. 282, Issue 1, 181-191, 1997

[³⁵S]Guanosine-5-O-(3-thio)triphosphate Binding as a Measure of Efficacy at Human Recombinant Dopamine D_4.4 Receptors: Actions of Antiparkinsonian and Antipsychotic Agents

A. Newman-Tancredi, V. Audinot, C. Chaput and L.Verrièle and M. J. Millan

Department of Psychopharmacology, Institut de Recherches Servier, 78290 Croissy-sur-Seine, France

Recombinant human dopamine D_4.4 receptor-mediated G protein activation was characterized in membranes of transfected mammalian (Chinese hamster ovary) cells by the use of [³⁵S]guanosine-5-O-(3-thio)triphosphate ([³⁵S]GTPS) binding. An initial series of experiments defined the conditions (3 µM GDP, 100 mM NaCl, 3 mM MgCl₂) under which optimal stimulation (2.2-fold increase in specific [³⁵S]GTPS binding) was achieved with the endogenous agonist dopamine. The number of dopamine-activated G proteins in Chinese hamster ovary-D_4.4 membranes was determined through [³⁵S]GTPS isotopic dilution saturation binding, yielding a B_max value of 2.29 pmol/mg. This compared with a D_4.4 receptor B_max value of 1.40 pmol/mg determined by [³H]spiperone saturation binding, indicating that 1 or 2 G proteins were activated per D_4.4 receptor and that there were few or no "spare receptors" in this cell line. Under these conditions, the efficacy for stimulation of [³⁵S]GTPS binding at D_4.4 receptors of 12 dopaminergic agonists was determined. Several antiparkinsonian drugs, including ropinirole, quinerolane and lisuride, exhibited agonist activity at D_4.4 receptors (E_max = 74.3%, 72.4% and 32.2%, respectively, compared with dopamine = 100%). The EC₅₀ values for agonist stimulation of [³⁵S]GTPS binding correlated well with the inhibition constants derived from competition binding with [³H]spiperone (r = +.99). However, other antiparkinsonian drugs (bromocriptine, L-DOPA and terguride) showed low affinity and/or were devoid of agonist activity at D_4.4 receptors. The potency at D_4.4 receptors of the novel, selective D_4.4 receptor antagonist L 745,870 was determined, indicating that it has high affinity (K_i = 1.99 nM) without detectable agonist activity. Furthermore, L 745,870 completely inhibited dopamine-stimulated [³⁵S]GTPS binding with a K_b value of 1.07 nM. The action of an additional 20 chemically diverse dopaminergic ligands, including clozapine, ziprasidone, sertindole, olanzapine and several other "atypical" antipsychotics, in advanced development was investigated. Each of these ligands shifted the dopamine stimulation curve to the right in a parallel manner consistent with competitive antagonism at this site and yielding K_b values (32.6, 22.4, 17.2 and 26.5 nM, respectively) that agreed closely with their K_i values (38.0, 14.9, 18.5 and 26.1 nM). In contrast, raclopride and seroquel exhibited low affinity at D_4.4 receptors (K_i > 1000 nM). Other compounds that showed antagonist activity at D_4.4 receptors included the 5-hydroxytryptamine_2A receptor antagonist fananserin (RP 62203), the sigma ligand BMY 14,802 and the D₃ receptor antagonist GR 103,691. In conclusion, dopamine D_4.4 receptor activity is unlikely to be an important factor in the clinical effectiveness of antiparkinsonian drugs, although low agonist efficacy at D_4.4 receptors might be associated with a lesser incidence of side effects. Furthermore, antagonist activity at D_4.4 receptors is a common property of many typical and atypical antipsychotic agents.