Andersen P H
J Neurochem. 1987 Jun;48(6):1887-96. doi: 10.1111/j.1471-4159.1987.tb05752.x.
Binding of the selective dopamine (DA) uptake inhibitor [3H]GBR 12935 to rat striatal membranes was characterized biochemically and pharmacologically. [3H]GBR 12935 binding at 0 degree C was reversible and saturable and Scatchard analysis indicated a single binding site with a KD of 5.5 nM and a Bmax of 760 pmol/mg tissue. [3H]GBR 12935 labeled two binding sites. One binding site was identified as the classic DA uptake site, since methylphenidate, cocaine, diclofensine, and Lu 19-005 potently inhibited [3H]GBR 12935 binding to it. Binding to the second site was inhibited by high concentrations of the above compounds. IC50 values for inhibition of [3H]GBR 12935 binding to the DA uptake site were proportional to IC50 values for inhibition of DA uptake. However, substrates of DA uptake, e.g., DA and 1-methyl-4-phenylpyridine, and DA releasers, e.g., the amphetamines, inhibited [3H]GBR 12935 binding less than DA uptake. Rate experiments excluded the possibility that these "weak" inhibitors affected the binding by allosteric coupled binding sites. The second binding site was not a noradrenergic, serotonergic, or GABAergic uptake site. Neither was it a dopaminergic, acetylcholinergic, histaminic, serotonergic, or adrenergic receptor. However, [3H]GBR 12935 was potently displaced from it by disubstituted piperazine derivatives, i.e., flupentixol and piflutixol. DA uptake and the DA uptake binding site of [3H]GBR 12935 were located primarily in the striatum, but the piperazine acceptor site was distributed uniformly throughout the brain. Also only the DA uptake binding site was destroyed by 6-OH-DA. Thus, [3H]GBR 12935 labels the classic DA uptake site in rat striatum and also a piperazine acceptor site. Substrates for DA uptake and releasers of DA inhibited [3H]GBR 12935 binding with low potency, but did not alter the rate constants for [3H]GBR 12935 binding. Therefore inhibitors of DA uptake label the carrier site and prevent the carrier process.
对选择性多巴胺（DA）摄取抑制剂[3H]GBR 12935与大鼠纹状体膜的结合进行了生化和药理学特征分析。0℃下[3H]GBR 12935的结合是可逆且可饱和的，Scatchard分析表明存在一个单一结合位点，解离常数（KD）为5.5 nM，最大结合容量（Bmax）为760 pmol/mg组织。[3H]GBR 12935标记了两个结合位点。其中一个结合位点被鉴定为经典的DA摄取位点，因为哌醋甲酯、可卡因、双氯芬辛和Lu 19 - 005能有效抑制[3H]GBR 12935与其结合。高浓度的上述化合物可抑制与第二个位点的结合。抑制[3H]GBR 12935与DA摄取位点结合的半数抑制浓度（IC50）值与抑制DA摄取的IC50值成比例。然而，DA摄取的底物，如DA和1 - 甲基 - 4 - 苯基吡啶，以及DA释放剂，如苯丙胺类，对[3H]GBR 12935结合的抑制作用小于对DA摄取的抑制作用。速率实验排除了这些“弱”抑制剂通过变构偶联结合位点影响结合的可能性。第二个结合位点不是去甲肾上腺素能、5 - 羟色胺能或γ - 氨基丁酸能摄取位点。它也不是多巴胺能、乙酰胆碱能、组胺能、5 - 羟色胺能或肾上腺素能受体。然而，二取代哌嗪衍生物，即氟哌噻吨和匹氟噻吨能有效将[3H]GBR 12935从该位点置换出来。DA摄取以及[3H]GBR 12935的DA摄取结合位点主要位于纹状体，但哌嗪受体位点在全脑均匀分布。而且只有DA摄取结合位点会被6 - 羟基多巴胺破坏。因此，[3H]GBR 12935标记了大鼠纹状体中的经典DA摄取位点以及一个哌嗪受体位点。DA摄取底物和DA释放剂对[3H]GBR 12935结合的抑制作用较弱，但不会改变[3H]GBR 12935结合的速率常数。所以DA摄取抑制剂标记了载体位点并阻止了载体过程。
