Pharmacology of Dopamine Release by Amphetamine

描述（由申请人提供）：该提案将填补我们对多巴胺转运蛋白（DAT）的正常和苯丙胺（AMPH）诱导调节的理解中的空白，这可能导致新的治疗方式。AMPH的增强特性取决于细胞外多巴胺（DA）的水平，其由DA释放、DAT和DA自身受体（D2 S）调节。我们发现PKC β调节DAT和D2 S的功能及其相互作用。抑制或删除PKC β可减少AMPH刺激的DA外排和AMPH刺激的运动和奖励行为，并增强D2 S对胞吐的直接抑制。DAT和D2 S都是PKC β的底物，但尚不清楚PKC β的磷酸化如何调节这些活性。我们建议，抑制PKC β减少AMPH刺激的细胞外DA的增加，从而加强AMPH的作用，这表明一个潜在的治疗目标AMPH滥用。我们的目标是：a。检查PKC β调节AMPH作用和D2 S-DAT功能相互作用的分子机制，提供关于该关键系统的调节的重要新信息; B.整合机制研究中学到的原理，以测试PKC β抑制剂是否会减少胞吐和AMPH诱发的DA释放以及体内药物摄取行为。为了实现这些目标，将测试以下假设：1。PKC β激活通过磷酸化DAT N-末端丝氨酸增强AMPH刺激的DA外排。将测定PKC β-磷酸化丝氨酸，并合成相关的非PKC β-磷酸化DAT突变体，并检测神经母细胞瘤N2 A细胞中AMPH刺激的DA流出、DA摄取和内向和外向转运的Michaelis-Menton动力学。2. D2 S激动剂需要PKC β刺激的DAT或D2 S或两者的磷酸化以增加表面DAT。将合成非PKC β磷酸化的DAT和D2 S突变体，并测试DAT功能的D2 S刺激、D2 S运输和D2 S对DA释放的影响。3.抑制PKC β将降低电和AMPH诱发的细胞外DA水平，从而减轻AMPH的增强作用。我们预测：a. PKC β抑制会钝化细胞外DA对电刺激和AMPH的反应，因为D2 S对DA胞吐的抑制增强，通过DAT的向外转运减少，而DA再摄取没有减少，和B。细胞外DA的减少将导致在自我给药过程中AMPH的药物摄取和药物寻求行为减少。将使用循环伏安法检查电刺激DA释放后PKC β抑制对细胞外DA的功能后果，同时评估DA释放和再摄取参数。为了检查PKC β是否是AMPH滥用的潜在治疗靶点，将评价PKC β抑制对吸毒行为、药物引发的恢复和自我施用AMPH的动机的影响。将获得对调节突触DA的因素的更大的机械理解，推进我们对设计有效的未满足的需求，非强化治疗AMPH滥用。

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.