Characterization of [³H]CFT binding to the norepinephrine transporter suggests that binding of CFT and nisoxetine is not mutually exclusive.
Characterization of [³H]CFT binding to the norepinephrine transporter suggests that binding of CFT and nisoxetine is not mutually exclusive.
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[‘H] CFT与去甲肾上腺素转运蛋白结合的表征表明，CFT和Nisoxetine的结合不是互斥的。
DOI：
10.1016/j.jneumeth.2011.08.044
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发表时间：
2012-01-15
影响因子：
3
通讯作者：
Reith ME
中科院分区：
文献类型：
--
作者：
Zhen J;Ali S;Dutta AK;Reith ME
去甲肾上腺素转运体（NET）是多种抗抑郁药和精神兴奋剂的重要靶点。尽管其作为药物靶点的突出性，但只有一种放射性配体用于NET竞争性结合测定，[3H]尼索西汀。然而，传统的[3H]尼索西汀结合方案往往低估了某些类别的NET配体，特别是可卡因和其他托烷的亲和力。在这里，我们探讨了使用苯托烷[3H]CFT标记人NET（hNET）在异源细胞为基础的结合研究的可行性。对时间和蛋白质含量进行了优化，并观察到特异性单位点结合。测试的NET配体抑制[3H]CFT与全细胞结合的效力（在生理[Na+]和25°C下）与在[3H]NE摄取测定中观察到的效力相似。结合测定的抑制常数（Ki）与所有测试化合物的摄取抑制常数高度相关（R2 = 0.99，p < 0.0001）。无细胞膜制剂没有显示出相同的药理学特征。在常规用于测量[3H]尼索西汀与膜制剂结合的条件下（4 ℃ 3小时，[Na+]为295 mM），尼索西汀和地昔帕明抑制[3H]CFT结合的效力变得大于在生理[Na+]下[3H]NE摄取的功能测定中测量的效力。然而，CFT和可卡因的情况正好相反。有趣的是，在研究[3H]CFT作为潜在的NET放射性配体时，我们发现了表明CFT和尼索西汀在与NET结合方面不相互排斥的证据。尼索西汀和CFT在抑制NET摄取[3H]多巴胺方面的相互作用的狄克逊图表明，这两种化合物可以同时与转运蛋白结合。
The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [3H]nisoxetine. However, traditional [3H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [3H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [3H]CFT binding to whole cells (at physiological [Na+] and 25°C) were similar to potencies observed in the [3H]NE uptake assay. Inhibition constants (Ki) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R2 = 0.99, p < 0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [3H]nisoxetine binding to membrane preparations (4°C for 3 h, [Na+] at 295 mM), the potency of nisoxetine and desipramine in inhibiting [3H]CFT binding became greater than that measured in a functional assay of [3H]NE uptake at physiological [Na+]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [3H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [3H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter.
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影响因子：
2.9
作者：
BAUER, ME;TEJANIBUTT, SM
通讯作者：
TEJANIBUTT, SM
影响因子：
3.6
作者：
Sarker S;Weissensteiner R;Steiner I;Sitte HH;Ecker GF;Freissmuth M;Sucic S
通讯作者：
Sucic S
影响因子：
4.8
作者：
Bryan-Lluka, LJ;Bönisch, H;Lewis, RJ
通讯作者：
Lewis, RJ
DOI：
10.1016/0364-7722(81)90045-x
发表时间：
1981-01-01
期刊：
PROGRESS IN NEURO-PSYCHOPHARMACOLOGY
影响因子：
--
作者：
HRDINA, PD
通讯作者：
HRDINA, PD
影响因子：
5
作者：
Dutta, Aloke K.;Ghosh, Balaram;Reith, Maarten E. A.
通讯作者：
Reith, Maarten E. A.