Analytical Methods for Investigating Peptide Transport

描述(申请人提供)：神经肽在体内扮演多种角色，包括神经递质、神经调节剂和激素。与谷氨酸和多巴胺等小分子神经递质不同，神经肽在释放后不会被神经元重新吸收，而是在细胞外空间代谢成更小的肽片段。这些碎片可以直接影响周围区域细胞外空间的细胞，穿过血脑屏障(BBB)，并在远离其初始释放部位的位置发挥作用。本研究采用LC-MS和在线微渗析-芯片电泳法，对强啡肽A(Dyn A)在体内和体外的转运、代谢和神经药理学进行了研究。Dyn A是kappa阿片受体的天然配体。强啡肽及其代谢物的类似物已被研究用于治疗外周疼痛、药物成瘾和情感障碍。矛盾的是，Dyn A也被证明具有神经毒性。中枢神经系统中这种多肽水平的升高与神经病理性疼痛和阿尔茨海默病有关。 这项提议有三个主要目标。第一个是研究Dyn A1-17、其代谢物和合成类似物在血脑屏障中的运输和代谢。通过这些实验获得的信息将有助于更好地了解转运机制，并将有可能生产出针对kappa阿片受体的更好的治疗药物。二是开发基于分离的传感器，可用于监测Dyn A及其代谢物对中枢神经系统神经递质释放和神经肽的影响。第三个目标是开发一种基于分离的传感器，可以放在动物身上，这样就可以利用清醒的、自由漫游的动物同时测量神经递质和行为。这些技术将被用于研究Dyn A在血脑屏障和中枢神经系统的药理作用。这里开发的方法学将普遍适用于所有需要测量血脑屏障转运或神经递质的神经化学研究，特别是那些监测行为也是感兴趣的情况。强啡肽是一种重要的神经肽，与疼痛、可卡因成瘾、抑郁症和阿尔茨海默病有关。更好地了解这种肽在血脑屏障中的运输和代谢将有助于科学家开发出更好的治疗这些疾病的方法。本提案中开发的分析方法也将对参与中枢神经系统研究的科学家具有普遍意义。

DESCRIPTION (provided by applicant): Neuropeptides play multiple roles in vivo including that of a neurotransmitter, neuromodulator, and hormone. In contrast to small molecule neurotransmitters such as glutamate and dopamine, neuropeptides are not taken back up by neurons following their release but are metabolized in the extracellular space into smaller peptide fragments. These fragments can directly affect the cells in the extracellular space in the surrounding area, pass through the blood brain barrier (BBB), and exert effects at sites far removed from the site of their initial release. In this proposal, the transport, metabolism and neuropharmacology of dynorphin A (Dyn A) is investigated both in vivo and in vitro using LC-MS and on-line microdialysis-microchip electrophoresis. Dyn A is a natural ligand of the kappa opioid receptor. Analogs of dynorphin and its metabolites have been investigated for the treatment of peripheral pain, drug addiction, and affective disorders. Paradoxically, Dyn A has also been shown to be neurotoxic. An elevated level of this peptide in the CNS has been associated with neuropathic pain and Alzheimer's disease. There are three main goals of this proposal. The first is to investigate the transport and metabolism of Dyn A 1-17, its metabolites, and synthetic analogs across the BBB. Information acquired through these experiments will lead to a better understanding of the mechanism of transport and will make it possible to produce better therapeutics targeted at the kappa opioid receptor. The second is to develop separation-based sensors that can be used to monitor the effects of Dyn A and its metabolites on neurotransmitter release and neuropeptides in the CNS. The third objective is to develop a separation-based sensor that can be placed on-animal so that measurements of neurotransmitters and behavior can be made simultaneously, using awake, freely roaming animals. These techniques will be employed to study the pharmacological actions of Dyn A at the BBB and in the CNS. The methodology developed here will be generally applicable to all neurochemical studies in which the measurement of blood brain barrier transport or neurotransmitters is desired, especially those cases where monitoring behavior is also of interest. PUBLIC HEALTH RELEVANCE Dynorphin is an important neuropeptide that is involved in pain, cocaine addiction, depression, and Alzheimer's disease. A better understanding of the transport and metabolism of this peptide across the BBB will assist scientists in producing better treatments for these diseases. The analytical methods developed in this proposal will also be of general utility to scientists involved in CNS research.