AP4A MODULATION PC12 CELL MONOAMINE OXIDASE ACTIVITY

描述：(摘自摘要)大多数研究涉及单胺 氧化物酶(MAO)强调了酶抑制剂的发展。 这种控制患者神经递质代谢的酶 像帕金森氏症这样的疾病。文献中充斥着 关于天然和人为的MAO活性抑制物的信息。 然而，几乎没有关于MAO调节器的信息 导致这种酶的活性增强。申请人的实验室 最近证明了二腺苷四磷酸，Ap4A，可能是 仅有的几种内源性细胞外刺激剂之一 MAO活动。他们进行了一系列旨在 在理解与Ap4A相关的信号转导途径方面 受体结合和MAO的增强激活。一种理解 参与Ap4A受体激活的信号转导通路(S)可能 从而更好地理解信号转导事件 与MAO激活有关。此外，如果Ap4A被激活 转导途径(S)是MAO的一种新的激活途径(S)， 对这一途径的了解(S)可能允许开发一种新的 药物激动剂、拮抗剂或药剂类别 控制MAO活动。载脂蛋白4A水平异常或升高 中枢神经系统和肾上腺可能导致相关的致病条件 儿茶酚胺商店耗尽，如帕金森氏症。这个 MAO在不同物种分解代谢中的潜在重要性 外源生物研究表明，Ap4A可能具有重要的治疗作用。 这项建议的具体目标是：(A)确定剂量 胞外多聚磷酸二腺苷对MAO活性的影响 在PC12细胞和从尾壳核获得的脑突触体中 老鼠的大脑。多巴胺代谢物将使用高效液相色谱法进行测定 有电化学检测和2)放射化学分析。这个 Ap4A应用的时间动力学将被确定为 评估细胞短期和长期暴露的影响 和突触体；(B)确定细胞外的影响 Ap4A对PC12细胞腺苷环化酶活性和cAMP水平的影响 CAMP水平将通过酶联免疫测定法进行测定；腺苷 环化酶活性将用[a32P]ATP和层析法测定； (C)确定G蛋白参与Ap4A的作用 通过测定GTP酶活性引起的反应；(D) 确定酪氨酸激酶和酪氨酸的参与 Ap4A中的磷酸酶启动了信号转导途径。 将使用SDS-PAGE和[32P]磷酸标记细胞并确定 特定蛋白的磷酸化或去磷酸化的程度 蛋白质。(E)确定将Ap4A交付给 清醒大鼠在体微透析尾壳核的实验研究 与高效液相色谱联用。

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.