Zhu Jun, Mactutus Charles F, Wallace David R, Booze Rosemarie M
Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina, 1512 Pendleton St., Columbia, SC 29208, USA.
J Pharmacol Exp Ther. 2009 Jun;329(3):1071-83. doi: 10.1124/jpet.108.150144. Epub 2009 Mar 26.
Human immunodeficiency virus (HIV)-1 Tat protein plays a key role in the pathogenesis of both HIV-1-associated cognitive-motor disorder and drug abuse. Dopamine (DA) transporter (DAT) function is strikingly altered in patients with HIV-1-associated dementia and a history of chronic drug abuse. This study is the first in vitro evaluation of potential mechanisms underlying the effects of Tat protein on DAT function. Rat striatal synaptosomes were incubated with recombinant Tat(1-86) protein, and [(3)H]DA uptake and the binding of [(3)H]2beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (WIN 35,428) and [(3)H]1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine (GBR 12935) were determined. Tat decreased [(3)H]DA uptake, [(3)H]WIN 35,428 binding, and [(3)H]GBR 12935 binding in a time-dependent manner. The potency of Tat for inhibiting [(3)H]DA uptake (K(i) = 1.2 microM) was the same as that for inhibiting [(3)H]GBR 12935 binding but 3-fold less than that for inhibiting [(3)H]WIN 35,428 binding. Mutant Tat proteins did not alter [(3)H]DA uptake. Kinetic analysis of [(3)H]DA uptake revealed that Tat (1 or 10 microM) decreased the V(max) value and increased the K(m) value in a dose-dependent manner. The V(max) value, decreased by Tat (1 microM), returned to the control level after washout of Tat, indicating that the inhibitory effect of Tat on DA uptake was reversible. Saturation studies revealed that Tat decreased the B(max) value and increased the K(d) value of [(3)H]WIN 35,428 binding, whereas Tat decreased the B(max) value of [(3)H]GBR 12935 binding, without a change in the K(d) value. These findings provide new insight into understanding the pharmacological mechanisms of Tat-induced dysfunction of the DAT in the dopaminergic system in HIV-infected patients.
人类免疫缺陷病毒1型（HIV-1）反式激活蛋白（Tat）在HIV-1相关的认知运动障碍和药物滥用的发病机制中均起关键作用。在患有HIV-1相关痴呆症且有慢性药物滥用史的患者中，多巴胺（DA）转运体（DAT）的功能发生了显著改变。本研究首次在体外评估了Tat蛋白对DAT功能影响的潜在机制。将大鼠纹状体突触体与重组Tat(1-86)蛋白一起孵育，然后测定[³H]DA摄取以及[³H]2β-甲氧羰基-3-β-(4-氟苯基)托烷（WIN 35,428）和[³H]1-[2-(二苯甲氧基)乙基]-4-(3-苯基丙基)-哌嗪（GBR 12935）的结合情况。Tat以时间依赖性方式降低了[³H]DA摄取、[³H]WIN 35,428结合和[³H]GBR 12935结合。Tat抑制[³H]DA摄取的效力（K(i)=1.2 microM）与抑制[³H]GBR 12935结合的效力相同，但比抑制[³H]WIN 35,428结合的效力低3倍。突变型Tat蛋白未改变[³H]DA摄取。对[³H]DA摄取的动力学分析表明，Tat（1或10 microM）以剂量依赖性方式降低了V(max)值并增加了K(m)值。Tat（1 microM）降低的V(max)值在洗脱Tat后恢复到对照水平，这表明Tat对DA摄取的抑制作用是可逆的。饱和研究表明，Tat降低了[³H]WIN 35,428结合的B(max)值并增加了K(d)值，而Tat降低了[³H]GBR 12935结合的B(max)值，K(d)值未改变。这些发现为理解Tat诱导HIV感染患者多巴胺能系统中DAT功能障碍的药理学机制提供了新的见解。
