[S-35] 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

Recombinant human dopamine D-4.4 receptor-mediated G protein activatio n was characterized in membranes of transfected mammalian (Chinese ham ster ovary) cells by the use of [S-35]guanosine-5'-O-(3-thio)triphosph ate ([S-35]GTP gamma S) binding. An initial series of experiments defi ned the conditions (3 mu M GDP, 100 mM NaCl, 3 mM MgCl2) under which o ptimal stimulation (2.2-fold increase in specific [S-35]GTP gamma S bi nding) was achieved with the endogenous agonist dopamine. The number o f dopamine-activated G proteins in Chinese hamster ovary-D-4.4 membran es was determined through [S-35]GTP gamma S isotopic dilution saturati on binding, yielding a B-max value of 2.29 pmol/ mg. This compared wit h a D-4.4 receptor B-max value of 1.40 pmol/mg determined by [H-3]spip erone saturation binding, indicating that 1 or 2 G proteins were activ ated per D-4.4 receptor and that there were few or no ''spare receptor s'' in this cell line. Under these conditions, the efficacy for stimul ation of [S-35]GTP gamma S binding at D-4.4 receptors of 12 dopaminerg ic 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 EC,, values for agonist stimulation of [S-3 5]GTP gamma S binding correlated well with the inhibition constants de rived from competition binding with [H-3]spiperone (r = +.99). However , other antiparkinsonian drugs (bromocriptine, L-DOPA and terguride) s howed low affinity and/or were devoid of agonist activity at D-4.4 rec eptors. The potency at D-4.4 receptors of the novel, selective D-4.4 r eceptor antagonist L 745,870 was determined, indicating that it has hi gh affinity (K-i = 1.99 nM) without detectable agonist activity. Furth ermore, L 745,870 completely inhibited dopamine-stimulated [S-35]GTP g amma S binding with a K-b value of 1.07 nM. The action of an additiona l 20 chemically diverse dopaminergic ligands, including clozapine, zip rasidone, sertindole, olanzapine and several other ''atypical'' antips ychotics, in advanced development was investigated. Each of these liga nds shifted the dopamine stimulation curve to the right in a parallel manner consistent with competitive antagonism at this site and yieldin g 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 contr ast, 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 antagonis t fananserin (RP 62203), the sigma ligand BMY 14,802 and the D-3 recep tor antagonist GR 103,691. In conclusion, dopamine D-4.4 receptor acti vity is unlikely to be an important factor in the clinical effectivene ss of antiparkinsonian drugs, although low agonist efficacy at D-4.4 r eceptors might be associated with a lesser incidence of side effects. Furthermore, antagonist activity at D-4.4 receptors is a common proper ty of many typical and atypical antipsychotic agents.