Sharif N A, Nunes J L, Michel A D, Whiting R L
Institute of Pharmacology, Syntex Research, 3401 Hillview Ave, Palo Alto, CA 94303, U.S.A.
Neurochem Int. 1989;15(3):325-32. doi: 10.1016/0197-0186(89)90140-x.
The biochemical and pharmacological characteristics of sodium-dependent [(3)H]GBR12935 ([(3)H]GBR) binding to homogenates of rat, mouse, guinea-pig and dog striatum were determined at 23 degrees C. In mouse striatum, [(3)H]GBR binding was also compared with [(3)H]dopamine ([(3)H]DA) uptake. Specific [(3)H]GBR binding to the dopamine (DA) transport complex (DTC) represented ? 70% of the total binding and was saturable, reversible and involved a single class of non-interacting sites in the rodent and dog striatal homogenates. The dog DTC exhibited a significantly higher affinity (mean +/- SEM) (K(d) = 1.8 +/- 0.6 nM, P < 0.02-0.01) than the guinea-pig and a lower binding capacity (B(max) = 0.6 +/- 0.1 pmol/mg protein; P < 0.05-0.01) than the rat (K(d) = 2.3 +/- 0.2 nM; B(max) = 2.1 +/- 0.17 pmol/mg) and guinea-pig (K(d) = 4.9 +/- 0.49 nM; B(max) = 1.9 +/- 0.35 pmol/mg) (n = 3-4) striatum. [(3)H]GBR binding to the striatal DTC in the rodents and dog was competitively inhibited by unlabelled GBR 12909 (K(i) = 6-12 nM), mazindol (K(i) = 106-216 nM), benzotropin (K(i) = 113-335 nM), nomifensine (K(i) = 0.7-2.6 ?M), cocaine (K(i) = 1-2.3 ?M), phencyclidine (K(i) = 1.6-2.6 ?M) and amfonelic acid (1-6 ?M). Compounds with K(i) ? 6-10 ?M included dopamine, d, l -amphetamine and spiperone. In general, the majority of the compounds appeared to be more active competitors of [(3)H]GBR binding in the dog striatum as compared to the rodent tissues, but the rank order of activity was identical in the dog and all the rodents. Numerous other compounds, such as desipramine, norepinephrine, epinephrine, amitriptyline, propranolol, histamine, etc. were essentially inactive at 1 ?M against [(3)H]GBR binding. [(3)H]DA uptake into mouse striatal synaptosomes was competitively inhibited by amfonelic acid (IC(50) = 25 nM), GBR12909 (IC(50) = 35 nM), DA (IC(50) = 106 nM), benzotropin (IC(50) = 175 nM), mazindol (IC(50) = 252 nM), cocaine (IC(50) = 0.8 ?M) and phencyclidine (IC(50) = 1.2 ?M). Apart from amfonelic acid and DA, there was a good correlation (R = 0.88) between the relative potency of the above compounds at inhibiting [(3)H]DA uptake and competing for [(3)H]GBR binding in the mouse striatum. These studies have therefore shown that the biochemical and pharmacological properties of [(3)H]GBR binding to the rodent and dog DTC are similar, and that [(3)H]GBR is a good marker for the [(3)H]DA uptake inhibitor site.
在23摄氏度下测定了钠依赖性[³H]GBR12935（[³H]GBR）与大鼠、小鼠、豚鼠和犬纹状体匀浆结合的生化和药理学特性。在小鼠纹状体中，还将[³H]GBR结合与[³H]多巴胺（[³H]DA）摄取进行了比较。与多巴胺（DA）转运复合物（DTC）的特异性[³H]GBR结合占总结合的约70%，具有饱和性、可逆性，并且在啮齿动物和犬纹状体匀浆中涉及一类非相互作用位点。犬DTC表现出比豚鼠显著更高的亲和力（平均值±标准误）（Kd = 1.8±0.6 nM，P < 0.02 - 0.01），并且与大鼠（Kd = 2.3±0.2 nM；Bmax = 2.1±0.17 pmol/mg）和豚鼠（Kd = 4.9±0.49 nM；Bmax = 1.9±0.35 pmol/mg）（n = 3 - 4）纹状体相比，结合能力更低（Bmax = 0.6±0.1 pmol/mg蛋白质；P < 0.05 - 0.01）。在啮齿动物和犬中，[³H]GBR与纹状体DTC的结合被未标记的GBR 12909（Ki = 6 - 12 nM）、马吲哚（Ki = 106 - 216 nM）、苄托品（Ki = 113 - 335 nM）、诺米芬辛（Ki = 0.7 - 2.6 μM）、可卡因（Ki = 1 - 2.3 μM）、苯环己哌啶（Ki = 1.6 - 2.6 μM）和氨苯蝶啶（1 - 6 μM）竞争性抑制。Ki≤6 - 10 μM的化合物包括多巴胺、d，l - 苯丙胺和螺哌隆。一般来说，与啮齿动物组织相比，大多数化合物在犬纹状体中似乎是[³H]GBR结合的更活跃竞争者，但在犬和所有啮齿动物中活性顺序相同。许多其他化合物，如地昔帕明、去甲肾上腺素、肾上腺素、阿米替林、普萘洛尔、组胺等在1 μM时对[³H]GBR结合基本无活性。氨苯蝶啶（IC50 = 25 nM）、GBR12909（IC50 = 35 nM）、DA（IC50 = 106 nM）、苄托品（IC50 = 175 nM）、马吲哚（IC50 = 252 nM）、可卡因（IC50 = 0.8 μM）和苯环己哌啶（IC50 = 1.2 μM）竞争性抑制[³H]DA摄取到小鼠纹状体突触体中。除了氨苯蝶啶和DA外，上述化合物在抑制[³H]DA摄取和竞争小鼠纹状体中[³H]GBR结合的相对效力之间存在良好的相关性（R = 0.88）。因此，这些研究表明[³H]GBR与啮齿动物和犬DTC结合的生化和药理学特性相似，并且[³H]GBR是[³H]DA摄取抑制剂位点的良好标志物。
