Role of Arachidonic Isoforms in Nerve Cell Function

花生四烯酸异构体在神经细胞功能中的作用

基本信息

批准号：
7422313
负责人：
Ann R. Rittenhouse
金额：
$ 17.77万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7422313
关键词：
Action Potentials Agonist Apoptosis Arachidonic Acids Blood Platelets Blood flow Brain Brain Diseases Cell Death Cell Survival Cell physiology Cells Cessation of life Charge Chemical Structure Chemicals Chronic Condition Disease Endothelial Cells Epilepsy Fire - disasters Ganglia Gas Chromatography Growth Human Individual Inflammatory Inflammatory Response Injury Ischemia L Cells Location Mass Chromatography Mass Spectrum Analysis Measures Mediating Membrane Metabolic Methodology Molecular Conformation Muscarinic Agonists Muscarinic M1 Receptor Muscarinics Myxoid cyst Nerve Degeneration Nerve Tissue Nervous system structure Neurons Nitric Oxide Synthase Nonesterified Fatty Acids Normal Cell Object Attachment Oxidative Stress Pattern Peripheral Phospholipase A2 Phospholipids Physiological Physiological reperfusion Play Process Property Protein Isoforms Range Recombinants Reperfusion Injury Reperfusion Therapy Reporting Role Seizures Signal Transduction Signaling Molecule Site Spectrum Analysis Structure Structure of superior cervical ganglion Testing Thrombospondin 1 Tissue Sample Tissues Traumatic Brain Injury Vascular Endothelium Weight cell growth clinically relevant extracellular in vivo ionization neuron loss novel novel therapeutics peroxidation therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Inappropriate electrical activity gives rise to devastating brain disorders including, epilepsies and cell death following ischemia. These diseases trigger damaging inflammatory responses that elevate free arachidonic acid (AA) levels. Under these pathological conditions, excessive Ca2+ influx leads to cell death. By understanding the role that AA serves in modulating nerve cell excitability and survival, we hope to identify therapeutic targets that will minimize nerve cell death due to inflammatory signaling. My lab has found that AA inhibits two Ca2+ currents called L- and N-currents in superior cervical ganglion (SCG) neurons by promoting channel closing. AA also increases N-current amplitude by acting at a distinct site. As with inhibition, enhancement is mimicked by stimulating M1 muscarinic receptors and requires phospholipase A2, indicating functional relevance of AA's dual actions. We have measured increased AA release from individual SCG following muscarinic stimulation using gas chromatography (GC) and mass spectroscopy (MS). Three additional, distinct peaks with a mass/charge ratio (m/z) equal to that of AA are also released suggesting that SCGs acutely synthesize AA isoforms with novel double bond patterns. AA normally has four cis double bonds. Our findings appear to be the first study documenting AA isoforms in neurons. However a recent report described a peroxidation process in endothelial cells that generates AAs with one of the bonds in the trans- conformation (TAAs). Moreover TTAs appear to mediate oxidative stress-induced microvascular degeneration. These findings raise many questions surrounding the roles of AA versus its isoforms in normal and pathological nerve cell functioning. Because of the possible clinical relevance of AA isoforms as therapeutic targets for the treatment of ischemia, the following specific aims are proposed: Aim I. Define the chemical structures of the AA isoforms released from SCG using GC-MS and nanospray ionization MS-MS. Characterizing the changes in AA double bond geometry and/or location will allow us to identify new potential proinflammatory signaling molecules. Aim II. Separate, collect and test the four AA isoforms found in SCG neurons for their ability to modulate whole-cell Ca2+ currents of SCG neurons and recombinant channels transiently transfected into HEK293 cells. Aim III. Determine whether any of the AA isoforms alter action potential firing. Whether particular AA isoforms promote nerve cell growth or death will also be tested. These studies will document at the cellular level the cumulative effect of AA (or AA isoforms) on membrane excitability and viability. If one or more of the isoforms modulates channel activity, action potential firing and/or cell survival, we will have identified a new signaling molecule(s) that may serve as a novel therapeutic target for treating ischemia. Inappropriate electrical activity gives rise to devastating brain disorders including, epilepsies and cell death following ischemia. These diseases trigger damaging inflammatory responses that elevate free arachidonic acid (AA) levels. We have discovered what appear to be novel isoforms of AA in a peripheral ganglion that regulates blood flow to the brain. By understanding the roles that AA and the novel AA isoforms serve in modulating nerve cell excitability, growth and survival, we hope to identify therapeutic targets that will minimize nerve cell death due to inflammatory signaling.

描述（由申请人提供）：不适当的电活动会引起毁灭性的脑部疾病，包括癫痫和缺血后的细胞死亡。这些疾病会引发破坏性炎症反应，从而提高游离花生四烯酸 (AA) 水平。在这些病理条件下，过量的 Ca2+ 流入会导致细胞死亡。通过了解 AA 在调节神经细胞兴奋性和存活中的作用，我们希望找到能够最大限度减少炎症信号导致的神经细胞死亡的治疗靶点。我的实验室发现，AA 通过促进通道关闭来抑制颈上神经节 (SCG) 神经元中的两种 Ca2+ 电流（称为 L 电流和 N 电流）。 AA 还通过作用于不同的位点来增加 N 电流幅度。与抑制一样，增强作用是通过刺激 M1 毒蕈碱受体来模拟的，并且需要磷脂酶 A2，表明 AA 双重作用的功能相关性。我们使用气相色谱 (GC) 和质谱 (MS) 测量了毒蕈碱刺激后单个 SCG 中 AA 释放的增加。还释放了三个额外的、质荷比 (m/z) 等于 AA 的不同峰，表明 SCG 能够快速合成具有新型双键模式的 AA 亚型。 AA通常有四个顺式双键。我们的研究结果似乎是第一个记录神经元中 AA 亚型的研究。然而，最近的一份报告描述了内皮细胞中的过氧化过程，该过程会生成带有反式构象键之一的 AA (TAA)。此外，TTA 似乎可以介导氧化应激诱导的微血管变性。这些发现提出了许多围绕 AA 及其亚型在正常和病理神经细胞功能中的作用的问题。由于 AA 同工型作为治疗缺血的治疗靶点可能具有临床相关性，因此提出以下具体目标：目标 I. 使用 GC-MS 和纳喷雾电离 MS-MS 定义从 SCG 释放的 AA 同工型的化学结构。表征 AA 双键几何形状和/或位置的变化将使我们能够识别新的潜在促炎信号分子。目标二。分离、收集并测试 SCG 神经元中发现的四种 AA 亚型，了解它们调节瞬时转染至 HEK293 细胞的 SCG 神经元和重组通道的全细胞 Ca2+ 电流的能力。目标三。确定任何 AA 同工型是否会改变动作电位放电。特定的 AA 同工型是否促进神经细胞生长或死亡也将得到测试。这些研究将在细胞水平记录 AA（或 AA 亚型）对膜兴奋性和活力的累积效应。如果一种或多种同种型调节通道活性、动作电位放电和/或细胞存活，我们将鉴定出一种新的信号分子，其可以作为治疗缺血的新治疗靶点。不适当的电活动会引起毁灭性的脑部疾病，包括癫痫和缺血后的细胞死亡。这些疾病会引发破坏性炎症反应，从而提高游离花生四烯酸 (AA) 水平。我们在调节大脑血流的外周神经节中发现了似乎是新型 AA 亚型。通过了解 AA 和新型 AA 异构体在调节神经细胞兴奋性、生长和存活中的作用，我们希望找到能够最大限度减少炎症信号导致的神经细胞死亡的治疗靶点。