PHARMACOLOGY OF DOPAMINE RELEASE BY AMPHETAMINE

描述：（申请人摘要） 了解调节质膜多巴胺转运蛋白（DAT）的因素 对于全面了解几种药物的作用至关重要， 虐待安非他明（AMPH）的运动兴奋剂和强化作用， 甲基苯丙胺（METH），DAT的底物，是由于它们能够增加 多巴胺（DA）的释放。AMPH似乎引起反向转运， DAT通过交换扩散机制。我们的数据揭示了一种意想不到的成分 在AMPH介导的DA逆向转运中，我们发现AMPH介导的DA的外向转运 DA的转运需要蛋白激酶C（PKC）活性和细胞内 Ca2+。直接PKC激活模拟AMPH的释放活性。的 PKC磷酸化的底物可能是DA转运蛋白本身， AMPH和PKC激活剂都引起DAT的磷酸化。 因此，本研究拟阐明PKC对DA的作用机制 出境运输。待检验的假设是AMPH转运激活 PKC激活DAT的磷酸化，反过来， 运输具体目标是： 1.确定除AMPH以外的DAT激动剂是否增加磷酸化 的DAT。 2.验证AMPH介导的DAT磷酸化是 负责增强AMPH介导的DA释放。 3.确定TPA和AMPH诱导的磷酸化增加是否改变了 DAT插入大鼠突触体膜和hDAT转染的 cos-7细胞。 4.验证AMPH通过增加PKC活性来增加PKC活性的假设。 PKC同工酶易位至DAT附近。 在美国，可吸烟形式的甲基苯丙胺越来越受欢迎，这标志着 新一轮AMPH滥用的开始由于AMPH是DAT的底物， 其作用机制不同于作为拮抗剂的可卡因。AMPH可以 不作用于经典受体，DAT拮抗剂可卡因也是 广泛滥用。因此，一种理想的AMPH抑制剂将阻断它的能力， 释放DA。对AMPH作用机制的更完整理解可以 导致安非他明抑制剂的更合理的设计。

DESCRIPTION:(Applicant's Abstract) Knowledge of factors that regulate the plasmalemma dopamine transporter (DAT) is crucial to a complete understanding of the actions of several drugs of abuse. The motor stimulant and reinforcing effects of amphetamine (AMPH) and methamphetamine (METH), substrates of DAT, are due to their ability to increase the release of dopamine (DA). AMPHs appear to elicit reverse transport through DAT by an exchange diffusion mechanism. Our data reveal an unexpected component in AMPH-mediated reverse transport of DA. We find that AMPH-mediated outward transport of DA requires protein kinase C (PKC) activity and intracellular Ca2+. Direct PKC activation mimics the releasing activity of AMPH. The substrate for the PKC phosphorylation could be the DA transporter itself since both AMPH and PKC activators elicit phosphorylation of DAT. The focus of this proposal is to elucidate the mechanism by which PKC exerts its effects on DA outward transport. The hypothesis to be tested is that AMPH transport activates PKC which elicits a phosphorylation of DAT that, in turn, enhances outward transport. The specific aims are: 1. Determine whether DAT agonists other than AMPH increase the phosphorylation of DAT. 2. Test the hypothesis that an AMPH-mediated phosphorylation of DAT is responsible for enhanced AMPH-mediated DA release. 3. Determine whether TPA and the AMPH-induced increase in phosphorylation alter insertion of DAT into the membranes of rat synaptosomes and of hDAT-transfected cos-7 cells. 4. Test the hypothesis that AMPH increases PKC activity by increasing translocation of a PKC isozyme proximal to DAT. Smokable forms of METH are becoming more popular in America, signalling the start of a new wave of AMPH abuse. Since AMPH is a substrate for DAT, its mechanism of action is different from that of cocaine, an antagonist. AMPH does not act at a classical receptor, and the DAT antagonist, cocaine, is also widely abused. Thus, an ideal inhibitor of AMPH would block its ability to release DA. A more complete understanding of the mechanism of AMPH action can lead to a more rational design of inhibitors of the amphetamines.