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首页> 外文期刊>Neurochemistry International: The International Journal for the Rapid Publication of Critical Reviews, Preliminary and Original Research Communications in Neurochemistry >Pharmacological characterisation of (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortro pane (PE2I) binding to the rat neuronal dopamine transporter expressed in COS cells.
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Pharmacological characterisation of (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortro pane (PE2I) binding to the rat neuronal dopamine transporter expressed in COS cells.

机译：（E）-N-（3-碘丙-2-烯基）-2β-羰甲氧基-3β-（4'-甲基苯基）NORT PAN（PE2I）与COS细胞中表达的大鼠神经元多巴胺转运蛋白结合的药理学表征。

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The interaction of (E)-N-(3-iodoprop-2-enyl)-2beta-Carbomethoxy-3beta-(4'-methylphenyl) nortropane (PE2I) with the rat neuronal dopamine transporter (DAT) was studied in transfected COS cells by measuring its ability to inhibit DA uptake and by measuring its affinity in radioligand binding experiments. Saturable [3H]DA uptake was measured in COS cells transiently transfected with the cDNA sequence encoding the rat DAT. Pharmacological characterisation of this uptake revealed functional properties with a V(max) value of 45.05+/-2.62 pmol/mg protein per min and a K(m) value of 2.86+/-0.28 microM. The specific [3H]DA uptake was fully inhibited by 1 microM PE2I. Concentration response curves revealed the high potency of PE2I in inhibiting DA uptake (pEC(50) value of 8.70+/-0.33), 25 times higher than that observed for the reference DAT inhibitor, GBR 12935. On crude homogenates from transfected COS cells, PE2I displaced the specific binding of [3H]GBR 12935 with a pK(i) value of 7.73+/-0.13. Accordingly, [125I]PE2I was found to specifically recognise a single binding site population which is almost completely displaced by GBR 12935 and nomifensine. Saturation experiments revealed the high affinity of [125I]PE2I (K(D) value of 3.8+/-0.63 nM) that correlates with the high potency of PE2I in inhibiting the [3H]DA uptake. This contrasts with the results obtained with GBR 12935 for which a discrepancy was found between its high affinity in binding assays (K(D) value of 0.43+/-0.04 nM) and its rather low potency in functional assays (pEC(50) value of 7.30+/-0.05). A relatively high level of [3H]GBR 12935 binding was detected in non transfected COS cells. Such nomifensine resistant binding is attributed to the interaction of GBR 12935 with cytochrome P-450 as it was displaced by cis-(Z)-flupentixol (an inhibitor of cytochrome P-450). Such interaction was not observed using PE2I. Taken together, these data demonstrate that PE2I was a highly potent inhibitor of cloned DAT compared with GBR 12935 and provided a useful tool for further investigations in cells transfected with cDNA encoding the DAT.
机译：在转染的COS细胞中研究了（E）-N-（3-碘丙-2-烯基）-2β-羧甲氧基-3β-（4'-甲基苯基）降冰片烷（PE2I）与大鼠神经元多巴胺转运蛋白（DAT）的相互作用通过测量其抑制DA摄取的能力以及通过测量其在放射性配体结合实验中的亲和力。在用编码大鼠DAT的cDNA序列瞬时转染的COS细胞中测量了饱和的[3H] DA摄取。该摄取的药理学表征揭示了功能特性，其V（max）值为每分钟45.05 +/- 2.62 pmol / mg蛋白，K（m）值为2.86 +/- 0.28 microM。 1 microM PE2I完全抑制了特定的[3H] DA摄取。浓度响应曲线表明，PE2I具有抑制DA摄取的高效力（pEC（50）值为8.70 +/- 0.33），比参考DAT抑制剂GBR 12935的观察值高25倍。在转染的COS细胞的粗匀浆中， PE2I取代了[3H] GBR 12935的特异性结合，其pK（i）值为7.73 +/- 0.13。因此，发现[125I] PE2I特异性识别单个结合位点群体，该结合位点群体几乎完全被GBR 12935和诺米芬丝胺所取代。饱和实验揭示了[125I] PE2I的高亲和力（K（D）值为3.8 +/- 0.63 nM），这与PE2I抑制[3H] DA摄取的高效力有关。这与使用GBR 12935获得的结果形成鲜明对比，在GBR 12935中发现其结合测定中的高亲和力（K（D）值为0.43 +/- 0.04 nM）与功能测定中的较低效价（pEC（50）值）之间存在差异7.30 +/- 0.05）。在未转染的COS细胞中检测到较高水平的[3H] GBR 12935结合。这种耐诺米芬的抗性结合归因于GBR 12935与细胞色素P-450的相互作用，因为它被顺式-（Z）-氟喷醇（细胞色素P-450的抑制剂）取代。使用PE2I未观察到这种相互作用。综上所述，这些数据表明，与GBR 12935相比，PE2I是一种有效的克隆DAT抑制剂，为进一步研究转染了DAT cDNA的细胞提供了有用的工具。

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