Pharmacology of Dopamine Release by Amphetamine

描述(由申请人提供)：这项建议将填补我们对正常和苯丙胺(AMPH)诱导的多巴胺转运体(DAT)调节的理解空白，这可能导致新的治疗方式。AMPH的增强作用依赖于细胞外多巴胺(DA)水平，DA释放、DAT和DA自身受体(D2S)调节细胞外多巴胺(DA)水平。我们找到PKC了吗？调节DAT和D2S的功能及其相互作用。PKC的抑制或缺失？减少Amph刺激的DA外流和Amph刺激的运动和奖励行为，并增强D2S对胞吐的直接抑制。DAT和D2S都是PKC？底物，但目前尚不清楚PKC是如何磷酸化的？将规范这些活动。我们认为抑制PKC？减少Amph刺激的细胞外DA增加，从而减少Amph的强化作用，提示Amph滥用的潜在治疗靶点。我们的目标是：a.研究PKC通过什么分子机制？调节amph作用和D2S-DAT功能相互作用，为调节这一关键系统提供重要的新信息；B.整合在机制研究中学到的原理，以测试PKC？抑制将减少胞吐和amph诱发的DA释放和体内的药物行为。为了达到这些目标，将检验以下假设：1.PKC？激活通过磷酸化DAT N-末端丝氨酸来增强Amph刺激的DA外流。将测定PKC？磷酸化的丝氨酸，并合成相关的非PKC？磷酸化的DAT突变体，并测试AMH刺激的DA外流、DA摄取以及神经母细胞瘤N2A细胞内外转运的米氏动力学。2.D2S激动剂需要PKC？刺激的DAT和/或D2S的磷酸化才能增加表面DAT。将合成非PKC？-磷酸化的DAT和D2S突变体，并测试D2S刺激DAT功能、D2S转运和D2S对DA释放的影响。3.抑制PKC？会降低电刺激和Amph诱发的细胞外多巴胺水平，从而减弱Amph的强化作用。我们预测：A.那个PKC？抑制将钝化细胞外DA对电刺激和AMPH的反应，因为D2S增强了对DA胞吐的抑制，并减少了通过DAT的向外转运，而不减少DA的再摄取，以及B.细胞外DA的减少将导致AMPH在自我给药过程中的药物摄取和寻药行为的减少。PKC的功能后果是什么？用循环伏安法检测电刺激DA释放后对细胞外DA的抑制作用，同时评估DA释放和再摄取参数。以检验PKC？PKC的作用是AMPH滥用的潜在治疗靶点吗？对吸毒行为的抑制，药物诱导的恢复，以及自我给药的动机将被评估。对调节突触DA的因素将获得更大的机械性理解，将我们推向设计有效的、非强化的AMPH滥用治疗的未得到满足的需要。

DESCRIPTION (provided by applicant): This proposal will fill a gap in our understanding of normal and amphetamine (AMPH)-induced regulation of the dopamine transporter (DAT) which may lead to new therapeutic modalities. Reinforcing properties of AMPHs depend on the level of extracellular dopamine (DA), which is regulated by DA release, DAT and DA autoreceptors (D2S). We find that PKC? regulates the functions of DAT and D2S and their interaction. Inhibition or deletion of PKC? reduces AMPH-stimulated DA efflux and AMPH-stimulated locomotor and rewarded behaviors, and enhances direct D2S inhibition of exocytosis. Both DAT and D2S are PKC? substrates but it is unknown how phosphorylation by PKC? will regulate these activities. We propose that inhibition of PKC? reduces AMPH-stimulated increases in extracellular DA and thus the reinforcing effects of AMPH, suggesting a potential therapeutic target for AMPH abuse. Our objectives are to: a. examine molecular mechanisms by which PKC? regulates AMPH action and D2S-DAT functional interactions, providing significant new information on regulation of this crucial system; b. integrate the principles learned in mechanisti studies to test if PKC? inhibition will reduce exocytotic and AMPH-evoked DA release and drug-taking behaviors in vivo. To meet these objectives, the following hypotheses will be tested: 1. PKC? activation enhances AMPH-stimulated DA efflux by phosphorylating DAT N-terminal serines. PKC?-phosphorylated serines will be determined and relevant non-PKC?-phosphorylatable DAT mutants will be synthesized and tested for AMPH-stimulated DA efflux, DA uptake, and Michaelis-Menton kinetics of inward and outward transport in neuroblastoma N2A cells. 2. PKC?-stimulated phosphorylation of DAT or D2S or both is required for D2S agonists to increase surface DAT. Non-PKC?-phosphorylatable DAT and D2S mutants will be synthesized and tested for D2S-stimulation of DAT function, D2S trafficking and D2S effects on DA release. 3. Inhibition of PKC? will reduce electrical- and AMPH-evoked levels of extracellular DA thereby lessening the reinforcing effects of AMPH. We predict: a. that PKC? inhibition will blunt extracellular DA in response to electrical stimulation and AMPH because of enhanced D2S inhibition of DA exocytosis and reduced outward transport through DAT with no reduction in DA reuptake, and b. the reduction in extracellular DA will lead to reduced drug-taking and drug- seeking behavior for AMPH in a self-administration procedure. The functional consequences of PKC? inhibition on extracellular DA following electrically-stimulated DA release will be examined using cyclic voltammetry, giving simultaneous assessment of DA release and reuptake parameters. To examine if PKC? is a potential therapeutic target for AMPH abuse, the effect of PKC? inhibition on drug-taking behavior, drug-primed reinstatement, and motivation to self-administer AMPH will be evaluated. A greater mechanistic understanding of factors regulating synaptic DA will be attained, advancing us toward the unmet need of designing an effective, non-reinforcing treatment for AMPH abuse.