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。我们发现，PKC？调节DAT和D2S的功能及其相互作用。抑制或缺失PKC？可减少Amph刺激的DA外流和Amph刺激的运动和奖赏行为，并增强D2S对胞吐的直接抑制。DAT和D2S都是PKC？底物，但尚不清楚PKC？的磷酸化如何调节这些活性。我们认为，抑制PKCç可减少Amph刺激的细胞外DA的增加，从而减少Amph的强化作用，这表明Amph滥用的潜在治疗靶点。我们的目标是：A.研究PKCü调节amph作用和D2S-DAT功能相互作用的分子机制，为调控这一关键系统提供重要的新信息；B.整合机制研究中学到的原理，测试抑制PKC？是否会减少体内胞吐和amph诱发的DA释放和药物行为。为了达到这些目标，我们将检验以下假设：1.通过磷酸化DAT N-末端丝氨酸，PKC？激活增强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细胞内向外转运的米氏动力学。2.D2S激动剂需要PKC？刺激的DAT或D2S磷酸化或两者兼而有之才能增加表面DAT。将合成非PKC？磷酸化的DAT和D2S突变体，并测试D2S对DAT功能的刺激、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DA水平，从而减弱AMPH的增强作用。我们预测：A.由于D2S增强了对DA胞吐的抑制作用，减少了通过DAT的向外转运，而不减少DA的再摄取，PKC？抑制将钝化细胞外DA对电刺激和AMPH的反应；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药物寻找行为的减少。我们将使用循环伏安法来研究在电刺激DA释放后，PKC？抑制对细胞外DA的功能影响，从而同时评估DA释放和再摄取参数。为了检验PKC？是否是AMPH滥用的潜在治疗靶点，将评估PKC？抑制对吸毒行为、药物诱导的恢复以及自我给药的动机的影响。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 PKCß regulates the functions of DAT and D2S and their interaction. Inhibition or deletion of PKCß reduces AMPH-stimulated DA efflux and AMPH-stimulated locomotor and rewarded behaviors, and enhances direct D2S inhibition of exocytosis. Both DAT and D2S are PKCß substrates but it is unknown how phosphorylation by PKCß will regulate these activities. We propose that inhibition of PKCß 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 PKCß 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 PKCß 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. PKCß activation enhances AMPH-stimulated DA efflux by phosphorylating DAT N-terminal serines. PKCß-phosphorylated serines will be determined and relevant non-PKCß-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. PKCß-stimulated phosphorylation of DAT or D2S or both is required for D2S agonists to increase surface DAT. Non-PKCß-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 PKCß will reduce electrical- and AMPH-evoked levels of extracellular DA thereby lessening the reinforcing effects of AMPH. We predict: a. that PKCß 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 PKCß 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 PKCß is a potential therapeutic target for AMPH abuse, the effect of PKCß 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.