Johnson R A, Eshleman A J, Meyers T, Neve K A, Janowsky A
Research Service, Veterans Affairs Medical Center, Portland, Oregon 97201, USA.
Synapse. 1998 Sep;30(1):97-106. doi: 10.1002/(SICI)1098-2396(199809)30:1<97::AID-SYN12>3.0.CO;2-M.
Drug-induced efflux of substrates was characterized in C6 rat glioma cells stably expressing a recombinant human dopamine (DA) or serotonin (5-HT) transporter (C6-hDAT and C6-hSERT, respectively). In the absence of Ca2+, these cells spontaneously and rapidly released preloaded [3H]DA or [3H]5-HT, respectively, but maintained constant levels of [3H]N-methy-4-phenylpyridinium (MPP+) for up to 90 minutes. In C6-hSERT cells, transporter substrates such as methamphetamine, amphetamine, and dopamine induced relatively rapid release of [3H]MPP+, with t1/2 values of approximately 15 minutes, while the t1/2 value for serotonin was about 30 minutes. Similar results were obtained with C6-hDAT cells. Uptake blockers that are not substrates at the transporters had considerably greater t1/2 values, as compared to substrates, suggesting different mechanisms for altering transporter function. Dose-response curves for each drug, conducted at each drug's t1/2, indicated considerable differences in potency (EC50) at stimulating [3H]MPP+ release from C6-hSERT cells [3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester (RTI-55) > imipramine > 1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine (GBR-12935) threo-(+/-)-methylphenidate > cocaine > mazindol > 2-beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) > (+)methamphetamine > amphetamine > DA > fenfluramine > norepinephrine (NE) > 5-HT]. A different rank order of potency was observed for the effects of drugs on [3H]MPP+ release from C6-hDAT cells [imipramine > RTI-55 > cocaine > mazindol > CFT > GBR-12935 > threo-(+/-)-methylphenidate > amphetamine > (+)methamphetamine > fenfluramine > DA > NE > 5-HT]. Based on efficacies for stimulating [3H]MPP+ release from C6-hDAT cells, drugs could be grouped into three categories, with substrates causing release of approximately 75% of loaded [3H]MPP+, cocaine analogues causing approximately 50% release, and other drugs causing an average release of approximately 25% of loaded [3H]MPP+. The results, taken together with results from previous reports, suggest that the transfected cell type contributes to the characteristics of transporter-mediated release, that drugs interact with different sites on the transporters in the uptake and release process, and that the mechanism of transporter-mediated release may not be a simple reversal of substrate uptake.
在稳定表达重组人多巴胺（DA）或5-羟色胺（5-HT）转运体（分别为C6-hDAT和C6-hSERT）的C6大鼠胶质瘤细胞中，对药物诱导的底物外排进行了表征。在无Ca2+的情况下，这些细胞分别自发且快速地释放预先加载的[3H]DA或[3H]5-HT，但[3H]N-甲基-4-苯基吡啶鎓（MPP+）的水平在长达90分钟内保持恒定。在C6-hSERT细胞中，转运体底物如甲基苯丙胺、苯丙胺和多巴胺诱导[3H]MPP+相对快速地释放，t1/2值约为15分钟，而5-羟色胺的t1/2值约为30分钟。C6-hDAT细胞也得到了类似结果。与底物相比，转运体上不是底物的摄取阻断剂具有大得多的t1/2值，这表明改变转运体功能的机制不同。在每种药物的t1/2时进行的每种药物的剂量-反应曲线表明，刺激C6-hSERT细胞释放[3H]MPP+的效力（EC50）存在显著差异[3β-（4-碘苯基）托烷-2β-羧酸甲酯（RTI-55）>丙咪嗪>1-[2-二苯甲氧基]乙基-4-（3-苯基丙基）-哌嗪（GBR-12935）苏式-（±）-哌醋甲酯>可卡因>马吲哚>2-β-甲氧基羰基-3β-（4-氟苯基）托烷（CFT）>（+）甲基苯丙胺>苯丙胺>DA>芬氟拉明>去甲肾上腺素（NE）>5-HT]。观察到药物对C6-hDAT细胞释放[3H]MPP+的作用具有不同的效力排序[丙咪嗪>RTI-55>可卡因>马吲哚>CFT>GBR-12935>苏式-（±）-哌醋甲酯>苯丙胺>（+）甲基苯丙胺>芬氟拉明>DA>NE>5-HT]。基于刺激C6-hDAT细胞释放[3H]MPP+的效力，药物可分为三类，底物导致约75%的加载[3H]MPP+释放，可卡因类似物导致约50%释放，其他药物导致加载[3H]MPP+平均约25%释放。这些结果与先前报告的结果一起表明，转染的细胞类型有助于转运体介导的释放特性，药物在摄取和释放过程中与转运体上的不同位点相互作用，并且转运体介导的释放机制可能不是底物摄取的简单逆转。
