Molecular and pharmacological analysis of the noradrenaline transporter

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）从细胞外环境中移除，并终止NA在突触中的作用。NAT在肾上腺髓细胞中的药理作用与中枢和外周去肾上腺素能神经元的药理作用相似。肾上腺髓细胞中表达的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。体外培养的肾上腺髓细胞经ifn - α处理后，细胞对[3H]NA的摄取呈时间（4-48h）和浓度（300-IOOOU/ml）依赖性减少。对[^3H]NA摄取的饱和分析表明，lfn - 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切里斯常数（Km）值。对[^3H]地西帕胺结合的Scatchard分析显示，ifn - α降低了最大的…More结合（Bmax）值，但未改变解离常数（Kd）值。这些发现表明，ifn - α通过降低细胞膜中转运体的密度来抑制NAT的功能。氯胺酮（10 ~ 10ooomum）抑制去西帕胺对[^3H]NA的敏感摄取（IC_<50>=97muM）。饱和分析表明氯胺酮降低了[^3H]NA摄取的Vmax，但没有改变Km，表明非竞争性抑制。其他NAT抑制剂，即可卡因和地西帕明，对[^3H]NA的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出混合抑制。[^3H]去西帕明结合的Scatchard分析显示氯胺酮增加Kd而不改变Bmax，表明竞争性抑制。在转染表达NAT的爪蟾卵母细胞中，氯胺酮降低了[^3H]NA的摄取，其动力学特征与培养的肾上腺髓细胞相似。这些发现与氯胺酮通过与NAT上的去西帕明结合位点部分重叠的位点相互作用而非竞争性地抑制NA转运的观点相一致

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