AP4A MODULATION PC12 CELL MONOAMINE OXIDASE ACTIVITY

摘要：（Abstract） 大多数关于单胺的研究 氧化酶（MAO）强调了抑制剂的发展， 这种酶用于控制患者体内的神经递质代谢 帕金森氏症等疾病。 文学作品中充满了 关于天然和人造单胺氧化酶活性抑制剂的信息。 然而，实际上没有关于MAO调节剂的信息， 导致这种酶的活性增强。 申请人的实验室 最近证明，二腺苷四磷酸，Ap 4A，可能是 一个，只有少数，内源性细胞外刺激剂， MAO活性。 他们进行了一系列实验， 在理解与 AP4a 受体结合和增强的MAO活化。 的理解 与Ap 4A受体活化有关的转导途径可能 导致更好地理解信号转导事件 与MAO激活有关。 此外，如果Ap 4A被激活， 转导途径是MAO的一种新的活化途径， 知识 该途径可以允许开发新的 激动剂、拮抗剂或药理学试剂的类别， 控制微弧氧化 活动 Ap 4A水平异常或升高 CNS和肾上腺 腺体可能导致相关的致病条件 体内的儿茶酚胺储备耗尽，比如帕金森氏症 的 MAO在各种催化剂中的潜在重要性 异生物物质表明 Ap 4A可能具有重要的治疗作用。 本建议的具体目标是：（A）确定剂量 细胞外多磷酸二腺苷对单胺氧化酶活性的影响 在从尾壳核获得的PC 12细胞和脑突触体中， 老鼠大脑的。 多巴胺代谢物将通过使用1）HPLC进行测量 电化学检测和2）放射化学分析。 的 将确定Ap 4A应用的时间动态， 评估细胞短期和长期暴露的影响 （B）确定细胞外的影响， Ap 4A对PC 12细胞腺苷酸环化酶活性和cAMP水平的影响。 将通过酶联免疫测定法测定cAMP水平;腺苷酸 将使用[α 32 P] ATP和色谱法测定环化酶活性; (C)为了确定G蛋白参与Ap 4A的作用， 通过测量GT3酶活性引起的应答;（D） 确定酪氨酸激酶和酪氨酸 Ap 4A中的磷酸酶启动信号转导途径。 SDS-PAGE和[32 P]磷酸盐将用于标记细胞并确定 特异性磷酸化或去磷酸化的程度 proteins. (E)为了确定Ap 4A递送对 用在体微透析技术观察清醒大鼠尾壳核的超微结构 与HPLC结合。

DESCRIPTION: (from Abstract) Most studies dealing with monoamine oxidase (MAO) have emphasized the development of inhibitors of this enzyme for controlling neurotransmitter metabolism in patients with diseases such as Parkinson's. The literature is replete with information on natural and man-made inhibitors of MAO activity. However, there is virtually no information on modulators of MAO that result in enhanced activity of this enzyme. The applicants' laboratory has recently demonstrated that diadenosine tetraphosphate, Ap4A, may be one, of only a few, endogenous extracellular stimulatory agents of MAO activity. They have undertaken a series of experiments aimed at understanding the signal transduction pathway associated with Ap4A receptor binding and the enhanced activation of MAO. An understanding of the transduction pathway(s) involved with Ap4A receptor activation may result in a better understanding of the signal transduction events associated with MAO activation. In addition, if the Ap4A activated transduction pathway(s) is a novel activation pathway(s) for MAO, knowledge of this pathway(s) may allow for the development of a new class of agonists, antagonists or pharmacological agents for controlling MAO activity. Abnormal or elevated level of Ap4A in the CNS and adrenal gland may lead to pathogenic conditions associated with depleted catecholamine stores, such as Parkinson's Disease. The potential importance of MAO in the catabolism of various xenobiotics suggests that Ap4A may have an important therapeutic role. The specific aims of this proposal are: (A) To determine the dosage effects of the extracellular diadenosine polyphosphates on MAO activity in PC12 cells and brain synaptosomes obtained form the caudate putamen of rat brains. Dopamine metabolites will be measured by using 1) HPLC with electrochemical detection and 2) radiochemical analyses. The temporal dynamics of the application of Ap4A will be determined to assess the effects of short term and chronic exposure of the cells and synaptosomes; (B) To determine the effects of extracellular Ap4A on adenylate cyclase activity and cAMP levels in PC12 cells. cAMP levels will be assayed by an enzyme linked immunoassay; adenylate cyclase activity will be determined using [a32P] ATP and chromatography; (C) To determine the role of G protein involvement in the Ap4A elicited response by measuring GTPase enzymatic activity; (D) To determine the involvement of tyrosine kinases and tyrosine phosphatases in the Ap4A initiated signal transduction pathway. SDS-PAGE and [32P]phosphate will be used to label cells and determine the extent of phosphorylation or dephosphorylation of specific proteins. (E) To determine the effects of Ap4A delivery to the caudate putamen of the conscious rat with in vivo microdialysis in conjunction with HPLC.