Price David A, Sorkin Alexander, Zahniser Nancy R
Department of Pharmacology, University of Colorado Denver, Mail Stop 8303, RC1-North Tower, P18-6402K, 12800 East 19th Ave., Aurora, CO 80045, USA.
Mol Pharmacol. 2009 Oct;76(4):812-23. doi: 10.1124/mol.109.056978. Epub 2009 Jul 23.
Cyclin-dependent kinase (Cdk) 5 reduces the rewarding properties of psychostimulants by dampening postsynaptic dopamine (DA) receptor signaling. Cdk5 is also present in midbrain DA neurons, where the DA transporter (DAT) is localized and limits DA neurotransmission by removing extracellular DA. Here, we tested the hypothesis that Cdk5 could also affect the disposition of DA by regulating DAT activity. Incubation of rat dorsal striatal (dSTR) synaptosomes with the Cdk5 inhibitors roscovitine, olomoucine, and 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(2-pyridyl)benzenesulfonamide (GW8510) or the inactive congener iso-olomoucine resulted in a rapid, concentration-dependent inhibition of specific [3H]DA uptake. However, roscovitine was the only inhibitor that did not also decrease [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (WIN35,428) binding to dSTR DATs. Roscovitine-induced inhibition of dSTR [3H]DA uptake was explained by decreased maximal uptake velocity, without a change in cell-surface DAT levels. Roscovitine did not enhance [3H]DA release mediated by either DAT reverse-transport or Ca(2+) channels in dSTR slices. Instead, roscovitine enhanced spontaneous [3H]DA outflow and inhibited DAT-mediated [3H]DA reaccumulation into dSTR slices. To explore the involvement of Cdk5 in roscovitine-induced down-regulation of DAT activity, Cdk5 protein was knocked down via Cdk5-small interfering RNA by as much as 86% in porcine aortic endothelial cells stably expressing human (h)DATs. However, Cdk5 depletion did not alter hDAT activity. Taken together, our results suggest that roscovitine inhibits DAT activity independently of Cdk5; therefore, results obtained with such inhibitors should be interpreted with caution. Our study is the first to demonstrate that Cdk5 inhibitors reduce brain DAT activity via a mechanism that is independent of DAT trafficking and reverse-transport.
细胞周期蛋白依赖性激酶（Cdk）5通过抑制突触后多巴胺（DA）受体信号传导来降低精神兴奋剂的奖赏特性。Cdk5也存在于中脑DA神经元中，多巴胺转运体（DAT）定位于此处，并通过清除细胞外DA来限制DA神经传递。在此，我们测试了一个假设，即Cdk5也可能通过调节DAT活性来影响DA的处置。用Cdk5抑制剂罗可辛、olomoucine和4-{[(7-氧代-6,7-二氢-8H-[1,3]噻唑并[5,4-e]吲哚-8-亚基)甲基]氨基}-N-(2-吡啶基)苯磺酰胺（GW8510）或无活性的同系物异olomoucine孵育大鼠背侧纹状体（dSTR）突触体，导致特异性[3H]DA摄取迅速、浓度依赖性抑制。然而，罗可辛是唯一一种不会同时降低[3H]2β-甲氧基羰基-3β-(4-氟苯基)托烷（WIN35,428）与dSTR DATs结合的抑制剂。罗可辛诱导的dSTR [3H]DA摄取抑制是由于最大摄取速度降低，而细胞表面DAT水平没有变化。罗可辛不会增强由DAT反向转运或dSTR切片中的Ca(2+)通道介导的[3H]DA释放。相反，罗可辛增强了自发的[3H]DA流出，并抑制了DAT介导的[3H]DA重新积累到dSTR切片中。为了探究Cdk5在罗可辛诱导的DAT活性下调中的作用，通过Cdk5小干扰RNA在稳定表达人（h）DATs的猪主动脉内皮细胞中敲低Cdk5蛋白多达86%。然而，Cdk5的缺失并没有改变hDAT的活性。综上所述，我们的结果表明罗可辛独立于Cdk5抑制DAT活性；因此，使用此类抑制剂获得的结果应谨慎解释。我们的研究首次证明Cdk5抑制剂通过一种独立于DAT转运和反向转运的机制降低脑DAT活性。
