Molecular and pharmacological analysis of the noradrenaline transporter

位于去甲肾上腺素能神经末梢质膜上的去甲肾上腺素转运体(NAT)通过依赖Na~+/Cl~-的共转运过程将去甲肾上腺素(NA)从细胞外环境中清除出去，并终止NA在突触中的作用。肾上腺髓质细胞NAT的药理特性与中枢和外周去甲肾上腺素能神经元的NAT相似。肾上腺髓质细胞表达NAT为研究药物对NAT转运体的影响提供了一个方便的模型系统。干扰素-α处理培养的肾上腺髓质细胞后，细胞摄取[~3H]NA的能力呈时间(4~48h)和浓度(300~100U/ml)依赖性下降。饱和摄取分析表明，LFN-α的抑制作用是通过降低最大摄取速度(Vimax)而不改变表观米氏常数(Km)实现的。地塞帕明结合的Scatchard分析显示干扰素-α降低了最大…更多的结合(Bmax)值，而解离常数(Kd)值没有任何变化。这些结果表明，干扰素-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(10-100muM)可抑制地昔帕明敏感的[~3H]NA摄取(IC_&lt；50&gt；=97µM)。饱和分析显示，氯胺酮降低[~(3 H)]NA摄取的Vmax而不改变Km，表明非竞争性抑制。NAT的其他抑制剂，即可卡因和地塞帕明，对[~3H]NA摄取表现出竞争性抑制，而氯胺酮的衍生物苯环利定则表现为混合型抑制。Scatchard分析显示氯胺酮增加Kd而不改变Bmax，表明竞争抑制作用。在表达NAT的非洲爪哇卵母细胞中，氯胺酮抑制[~(3 H)]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上部分重叠的地地帕明结合位点相互作用而非竞争性地抑制NA转运的想法是一致的。较少

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