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Molecular and pharmacological analysis of the noradrenaline transporter

去甲肾上腺素转运蛋白的分子和药理学分析

基本信息

批准号：
09680778
负责人：
TOYOHIRA Yumiko
金额：
$ 2.11万
依托单位：
University of Occupational and Environmental Health
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Noradrenaline transporters (NAT) located on plasma membranes of noradrenergic nerve terminals remove noradrenaline (NA) from the extracellular milieu via the Na^+/Cl^--dependent cotransport process and terminate the action of NA in the synapse. The pharmacological properties of NAT in adrenal medullary cells are similar to those of the NAT of central and peripheral noradrenergic neurons. NAT expressed in adrenal medullary cells provides a convenient model system to study the effects of drugs on this transporter.Treatment of cultured adrenal medullary cells with IFN-alpha caused a decrease in uptake [3H]NA by the cells in time (4-48h)- and concentration (300-IOOOU/ml)-dependent manners. Saturation analysis of [^3H]NA uptake showed that the inhibitory effect of lFN-alpha was due to a reduction in the maximal uptake velocity (Vimax) values without altering apparent Michaelis constant (Km) values. Scatchard analysis of [^3H]desipramine binding revealed that IFN-alpha decreased the maximal … More binding (Bmax) values without any change in the dissociation constant (Kd) values. These findings suggest that IFN-alpha suppresses the function of NAT by reducing the density of the transporter in cell membranes.Ketamine (1O-1OOOmuM) inhibited desipramine-sensitive uptake of [^3H]NA (IC_<50>=97muM). Saturation analysis showed that ketamine reduced Vmax of [^3H]NA uptake without changing Km, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [^3H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Scatchard analysis of [^3H]desipramine binding revealed that ketamine increased Kd without altering Bmax, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the NAT, ketamine attenuated [^3H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT. Less

去甲肾上腺素转运蛋白（NAT）位于去甲肾上腺素能神经末梢的质膜上，通过Na^+/Cl^-依赖的共转运过程将去甲肾上腺素（NA）从细胞外环境中清除，并终止NA在突触中的作用。NAT在肾上腺髓质细胞中的药理学性质与中枢和外周去甲肾上腺素能神经元的NAT的药理学性质相似。肾上腺髓质细胞表达的NAT为研究药物对该转运体的影响提供了一个方便的模型系统，IFN-α处理培养的肾上腺髓质细胞后，可使细胞摄取[~ 3 H]NA的能力降低，并呈时间（4- 48 h）和浓度（300- 1000 U/ml）依赖性。[^3H]NA摄取的饱和分析表明，IFN-α的抑制作用是由于最大摄取速度（Vimax）值的降低而不改变表观米氏常数（Km）值。对[^3H]地昔帕明结合的Scatchard分析显示，IFN-α降低了IFN-α的最大结合强度。 ...更多信息结合（Bmax）值，而解离常数（Kd）值没有任何变化。这些结果表明IFN-α通过降低细胞膜转运蛋白的密度来抑制NAT的功能，氯胺酮（10 - 1000 μ M）抑制地昔帕明敏感的[^3H]NA摄取（IC_<50>= 97 μ M）。饱和分析表明，氯胺酮降低[^3H]NA摄取的Vmax，而不改变Km，表明非竞争性抑制。其他NAT抑制剂，即可卡因和地昔帕明，显示出对[^3 H]NA吸收的竞争性抑制，而氯胺酮的衍生物苯环利定则显示出混合类型的抑制。对[^3H]地昔帕明结合的Scatchard分析表明，氯胺酮增加了Kd，而没有改变Bmax，这表明存在竞争性抑制。在表达NAT的转染非洲爪蟾卵母细胞中，氯胺酮减弱了[^3H]NA摄取，其动力学特征与培养的肾上腺髓质细胞相似。这些发现与氯胺酮通过与NAT上与地昔帕明结合位点部分重叠的位点相互作用而非竞争性抑制NA转运的想法是一致的。