Rothman R B, Cadet J L, Akunne H C, Silverthorn M L, Baumann M H, Carroll F I, Rice K C, de Costa B R, Partilla J S, Wang J B
Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse/NIH Addiction Research Center, Baltimore, Maryland.
J Pharmacol Exp Ther. 1994 Jul;270(1):296-309.
The drug 3 beta-[4'-iodophenyl]tropan-2 beta-carboxylic acid methyl ester (RTI-55) is a cocaine congener with high affinity for the dopamine transporter (Kd < 1 nM). The present study characterized [125I]RTI-55 binding to membranes prepared from rat, monkey and human caudates and COS cells transiently expressing the cloned rat dopamine (DA) transporter. Using the method of binding surface analysis, two binding sites were resolved in rat caudate: a high-capacity binding site (site 1, Bmax = 11,900 fmol/mg of protein) and a low-capacity site (site 2, Bmax = 846 fmol/mg of protein). The Kd (or Ki) values of selected drugs at the two sites were as follows: (Ki for high-capacity site and Ki for low-capacity site, respectively): RTI-55 (0.76 and 0.21 nM), 1-[2-diphenyl-methoxy)ethyl]-4-(3-phenylpropyl)piperazine (0.79 and 358 nM), mazindol (37.6 and 631 nM), 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (45.0 and 540 nM) and cocaine (341 and 129 nM). Nisoxetine, a selective noradrenergic uptake blocker, had low affinity for both sites. Serotonergic uptake blockers had a high degree of selectivity and high affinity for the low-capacity binding site (Ki of citalopram = 0.38 nM; Ki of paroxetine = 0.033 nM). The i.c.v. administration of 5,7-dihydroxytryptamine to rats pretreated with nomifensine (to protect dopaminergic and noradrenergic nerve terminals) selectively decreased the Bmax of site 2, strongly supporting the idea that site 2 is a binding site on the serotonin (5-HT) transporter. This serotonergic lesion also increased the affinity of [125I]RTI-55 for the DA transporter by 10-fold. The ligand selectivity of the caudate 5-HT transporter was different from the [I125]RTI-55 binding site on the 5-HT transporter present in membranes prepared from whole rat brain minus caudate. The [125I]RTI-55 binding to the DA transporter was further resolved into two components, termed sites 1a and 1b, by using human and monkey (Macaca mulatta) caudate membranes but not the membranes prepared from rat caudate or COS cells that transiently expressed the cloned cocaine-sensitive DA transporter complementary DNA. Similar experiments also resolved two components of the caudate 5-HT transporter. Viewed collectively, these data provide evidence that [125I]RTI-55 labels multiple binding sites associated with the DA and 5-HT transporters.
药物3β-[4'-碘苯基]托烷-2β-羧酸甲酯（RTI-55）是一种对多巴胺转运体具有高亲和力（Kd < 1 nM）的可卡因类似物。本研究对[125I]RTI-55与大鼠、猴和人尾状核以及瞬时表达克隆大鼠多巴胺（DA）转运体的COS细胞制备的膜的结合特性进行了表征。使用结合表面分析方法，在大鼠尾状核中分辨出两个结合位点：一个高容量结合位点（位点1，Bmax = 11,900 fmol/mg蛋白质）和一个低容量位点（位点2，Bmax = 846 fmol/mg蛋白质）。所选药物在这两个位点的Kd（或Ki）值如下：（分别为高容量位点的Ki和低容量位点的Ki）：RTI-55（0.76和0.21 nM）、1-[2-二苯基甲氧基）乙基]-4-（3-苯基丙基）哌嗪（0.79和358 nM）、马吲哚（37.6和631 nM）、2β-甲氧羰基-3β-（4-氟苯基）托烷（45.0和540 nM）以及可卡因（341和129 nM）。去甲替林，一种选择性去甲肾上腺素摄取阻滞剂，对这两个位点的亲和力都很低。5-羟色胺摄取阻滞剂对低容量结合位点具有高度选择性和高亲和力（西酞普兰的Ki = 0.38 nM；帕罗西汀的Ki = 0.033 nM）。向用诺米芬辛预处理过的大鼠（以保护多巴胺能和去甲肾上腺素能神经末梢）脑室内注射5,7-二羟基色胺，选择性地降低了位点2的Bmax，有力地支持了位点2是5-羟色胺（5-HT）转运体上的一个结合位点的观点。这种5-羟色胺能损伤还使[125I]RTI-55对DA转运体的亲和力增加了10倍。尾状核5-HT转运体的配体选择性与从大鼠全脑减去尾状核制备的膜中存在的5-HT转运体上的[I125]RTI-55结合位点不同。通过使用人类和猴（猕猴）尾状核膜，但不使用大鼠尾状核或瞬时表达克隆可卡因敏感DA转运体互补DNA的COS细胞制备的膜，[125I]RTI-55与DA转运体的结合进一步分辨为两个成分，称为位点1a和1b。类似的实验也分辨出了尾状核5-HT转运体的两个成分。总体来看，这些数据提供了证据，表明[125I]RTI-55标记了与DA和5-HT转运体相关的多个结合位点。
