Rinken A, Finnman U B, Fuxe K
Institute of Chemical Physics, University of Tartu, Estonia.
Biochem Pharmacol. 1999 Jan 15;57(2):155-62. doi: 10.1016/s0006-2952(98)00287-1.
Functional activation of dopamine receptors in the crude membranes from rat striatum was studied by a [35S]-guanosine 5'-O-(gamma-thiotriphosphate) ([35S]GTPgammaS) binding assay. Binding of [35S]GTPgammaS could be characterized with a dissociation constant (Kd) = 14.6+/-0.8 nM and this did not depend on the presence of dopamine. The displacement of [35S]GTPgammaS binding by GDP could be characterized with an inhibition constant (K(i)) = 78+/-15 microM in the presence of 10 microM of butaclamol, while the presence of 100 microM of dopamine decreased it to a K(i) = 0.13+/-0.02 mM. Dopamine increased the association rate of [35S]GTPgammaS binding in the presence of GDP in a dose-dependent manner with an EC50 = 1.45+/-0.48 microM. Other dopamine receptor agonists studied displayed a potency to stimulate the [35S]GTPgammaS binding in the order R(-)-10,11dihydroxy-N-n-propylnorapomorphine (NPA) > pergolide > or = apomorphine > dopamine approximately 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]be nzopyrano[4,3-b]-1,4-oxazin-9-ol hydrocholoride (PD 128,907). The dopamine-induced stimulation of [35S]GTPgammaS binding was inhibited by different dopamine receptor antagonists in the potency order: (+)butaclamol > haloperidol approximately clorpromazine > or = 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-benzaz epine (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 D2 subtype, supporting the idea that this subtype is most likely responsible for the dopaminergic activation of [35S]GTPgammaS binding in rat striatal membranes.
通过[35S]-鸟苷5'-O-(γ-硫代三磷酸)([35S]GTPγS)结合试验研究了大鼠纹状体粗制膜中多巴胺受体的功能激活。[35S]GTPγS的结合可用解离常数(Kd)=14.6±0.8 nM来表征，且这并不依赖于多巴胺的存在。在10 μM布他拉莫存在的情况下，GDP对[35S]GTPγS结合的置换可用抑制常数(Ki)=78±15 μM来表征，而100 μM多巴胺的存在使其降至Ki=0.13±0.02 mM。多巴胺在GDP存在的情况下以剂量依赖方式增加[35S]GTPγS结合的缔合速率，EC50=1.45±0.48 μM。所研究的其他多巴胺受体激动剂刺激[35S]GTPγS结合的效力顺序为：R(-)-10,11-二羟基-N-正丙基去甲阿扑吗啡(NPA)>培高利特>或=阿扑吗啡>多巴胺≈喹吡罗>R(+)-1-苯基-2,3,4,5-四氢-(1H)-3-苯并氮杂卓-7,8-二醇盐酸盐(SKF-38393)>S(+)(4aR,10bR)-3,4,4a,10b-四氢-4-丙基-2H,5H-[1]苯并吡喃并[4,3-b]-1,4-恶嗪-9-醇盐酸盐(PD 128,907)。多巴胺诱导的[35S]GTPγS结合刺激被不同的多巴胺受体拮抗剂以以下效力顺序抑制：(+)布他拉莫>氟哌啶醇≈氯丙嗪>或=雷氯必利>(-)-舒必利>瑞莫必利>5,6-二甲氧基-2-(二丙胺)茚满(U 991944A)>R(+)-7-氯-8-羟基-3-甲基-1-苯基-2,3,4,5-四氢-1H-3-苯并氮杂卓(SCH-23390)。将所得数据与这些配体对不同亚型多巴胺受体的解离常数进行比较，仅与D2亚型的常数有良好的相关性，支持了该亚型最有可能负责大鼠纹状体膜中[35S]GTPγS结合的多巴胺能激活这一观点。
