Pharmacological characterization of dopamine-stimulated [ 35S]-Guanosine 5′-(γ-thiotriphosphate) ([ 35S]GTPγS) binding in rat striatal membranes

Functional activation of dopamine receptors in the crude membranes from rat striatum was studied by a [ 35S]-guanosine 5′- O-(γ-thiotriphosphate) ([ 35S]GTPγS) binding assay. Binding of [ 35S]GTPγS could be characterized with a dissociation constant (K d) = 14.6 ± 0.8 nM and this did not depend on the presence of dopamine. The displacement of [ 35S]GTPγS binding by GDP could be characterized with an inhibition constant (K i) = 78 ± 15 μM in the presence of 10μM of butaclamol, while the presence of 100 μM of dopamine decreased it to a K i = 0.13 ± 0.02 mM. Dopamine increased the association rate of [ 35S]GTPγS binding in the presence of GDP in a dose-dependent manner with an ec 50 = 1.45 ± 0.48 μM. Other dopamine receptor agonists studied displayed a potency to stimulate the [ 35S]GTPγS binding in the order R(−)-10,11-dihydroxy- N- n-propylnorapomorphine (NPA) > pergolide ≥ apomorphine > dopamine ≈ quinpirole > R(+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF-38393) > S(+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol hydrocholoride (PD 128,907). The dopamine-induced stimulation of [ 35S]GTPγS binding was inhibited by different dopamine receptor antagonists in the potency order: (+)butaclamol > haloperidol ≈ clorpromazine ≥ raclopride > (−)-sulpride > remoxipride > 5,6-dimethoxy-2-(dipropylamine)indan (U 991944A) > R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH-23390). Comparison of the obtained data with the dissociation constants of these ligands to different subtypes of dopamine receptors gave a good correlation only with constants for the D 2 subtype, supporting the idea that this subtype is most likely responsible for the dopaminergic activation of [ 35S]GTPγS binding in rat striatal membranes.