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 [S-35] -guanosine 5'-O-(gamma-thiotriphosphate) ([S-35]GTP gamma S) binding assay. Binding of [S-35]GTP gamma S 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 [S-35]GTP gamma S binding by GDP could be characterized with an inhibition constant (K-i) = 18 =/- 15 mu M in the presence of 10 mu M of butaclamol, while the presence of 100 mu M of dopamine decreased it to a K-i = 0.13 +/- 0.02 mM. Dopamine increased the association rate of [S-35]GTP gamma S binding in the presence of GDP in a dose dependent manner with an EC50 = 1.45 +/- 0.48 mu M. Other dopamine receptor agonists studied displayed a potency to stimulate the [S-35]GTP gamma S binding in the order R(-)-10,11-dihydroxy-N-n-propylnorapomorphine (NPA) > pergolide greater than or equal to apomorphine > dopamine approximate to 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 [S-35]GTP gamma S binding was inhibited by different dopamine receptor antagonists in the potency order: (+)butaclamol > haloperidol approximate to clorpromazine greater than or equal to raclopride > (-)-sulpride > remoxipride > 5,6-dimethoxy-2-(dipropylamine)indan (U 991944A) > R(+)-1-chloro-8-hydroxy 3-methyl-1-phenyl-2,3,4,5-tetrahdro-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 [S-35]GTP gamma S binding in rat striatal membranes. BIOCHEM PHARMACOL 57;2: 155-162, 1999. (C) 1998 Elsevier Science Inc.