Excitatory Amino Acid Release in Ischemia

缺血时的兴奋性氨基酸释放

• 批准号: 6539875
• 负责人: Harold K Kimelberg
• 金额: $ 39.5万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6539875
• 关键词: astrocytes; brain disorder chemotherapy; calmodulin; caveolas; cerebral ischemia /hypoxia; chemoprevention; dosage; excitatory aminoacid; glutamate transporter; immunocytochemistry; ion transport; laboratory rat; membrane channels; membrane transport proteins; neuropharmacology; neuroprotectants; neurotransmitter transport; nonhuman therapy evaluation; stroke therapy; tamoxifen; tissue /cell culture

DESCRIPTION (Adapted from applicant's abstract): Our major hypothesis for the last funded period was that reversal of EAA transporters and activation of Volume-Regulated Anion Channels (YRACs) are major sources of EAAs in rat cerebral ischemia and that inhibition of these routes of release would be neuroprotective. In support of the first part of this hypothesis, we found that elevated extracellular [K+] induced EAA release due to both reversal of the EAA transporter and activation of VRACs in primary asfrocyte cultures. In vivo, microdialysis studies in a rat temporary global ischemia model established that application via a microdialysis probe of dihydrokainaxe, an inhibitor of the astrocyte-specific EAA transporter GLT-1, or DNDS an anion channel inhibitor, led to potent suppression of EAA levels during the ischemic episode. If applied together these compounds reduced EAA levels in ischemia by over 80 percent. To check whether inhibition of VRACs is neuroprotective, we chose, on the basis of its high blood-brain barrier permeability, the estrogen receptor antagonist/agonist tamoxifen (TAM) that is also an efficient inhibitor of VRACs in vitro. In the rat middle cerebral artery occlusion model (rMCAO), 5 mg/kg TAM reduced infarction volume by up to 80 percent if applied just before the 2 hour ischemic episode or 3 h after initiation of ischemia. We propose to continue these studies along two lines. One will be devoted to molecular identification of VRACs and intracellular signalling events involved in the volume-dependent release of EAAs in primary astrocyte cultures. Our hypotheses for this part of the project are that more than one VRAC is involved in volume-dependent amino acid release, one or more of these channels are incorporated in calveolae signaling complexes and calmodulin and tyrosine kinases are involved in their volume-dependent activation. The second line of the study will be to explore the molecular mechanisms of TAM neuroprotection and evaluation of its therapeutic window with different dosages and with different durations of reversible middle cerebral artery occlusion. Our hypothesis here is that TAM is highly neuroprotective in rMCAo because it has multiple protective effects. These include inhibition of VRACs, suppression of Ca24 about/calmodulin-dependent nitric oxide production, and/or antioxidant action. We also cannot exclude that some portion of the protection may also be mediated by brain estrogen receptors. This second part of the project will test all these possibilities in animal studies. Both aspects of the project will add new basic knowledge on VRACs, with the potential for understanding their functions in the brain. The second half of the project that deals with neuroprotection has direct potential clinical implications, as TAM is known to be well tolerated in humans being widely used for breast cancer treatment.

描述(改编自申请者的摘要)：我们对 上一个资助期是EAA转运体逆转和激活 容量调节阴离子通道(YRAC)是大鼠EaAs的主要来源 脑缺血和这些释放途径的抑制将是 具有神经保护作用。为了支持这一假设的第一部分，我们发现 细胞外[K+]升高引起的EAA释放 VRAC在原代无泡细胞培养中的转运和激活。在体内， 在大鼠暂时性全脑缺血模型中进行的微透析研究证实 通过微渗析探头应用二氢海人藻氨酸抑制物 星形胶质细胞特异性的EAA转运体GLT-1，或DNDS，一种阴离子通道抑制剂， 导致EAA水平在缺血发作期间受到有效抑制。如果适用 这些化合物总共降低了缺血时的EAA水平超过80%。至 检查VRAC的抑制是否具有神经保护作用，我们选择了，基于 它的高血脑屏障通透性，雌激素受体 拮抗剂/激动剂三苯氧胺()，也是一种有效的VRAC抑制剂 在试管中。大鼠大脑中动脉阻塞模型(RMCAO)，5 mg/kg 如果在2个月前使用，最高可减少80%的脑梗塞体积 缺血发作1小时或缺血开始后3小时。我们建议 沿着两条路线继续这些研究。其中一个将致力于分子 VRAC的识别和参与的细胞内信号事件 原代星形胶质细胞培养中EaAs的体积依赖性释放。我们的假设 对于该项目的这一部分，不止一个vrac参与 依赖于体积的氨基酸释放，其中一个或多个通道是 结合在小脑信号复合体和钙调蛋白和酪氨酸中 激酶参与了它们的体积依赖性激活。第二行 本研究旨在探讨神经保护的分子机制 评价不同剂量和不同剂量对其治疗窗的影响。 可逆性大脑中动脉闭塞持续时间不同。我们的 这里的假设是在rMCAO中具有高度的神经保护作用，因为它 多重保护作用。这些包括抑制VRAC、抑制 CA24关于钙调素依赖的一氧化氮和/或抗氧化剂的产生 行动。我们也不能排除保护的某一部分也可能是 由大脑雌激素受体介导。该项目的第二部分将测试 所有这些在动物研究中的可能性。 该项目的两个方面都将增加关于VRAC的新的基本知识， 有可能了解它们在大脑中的功能。年的下半年 这个涉及神经保护的项目具有直接的临床潜力。 含义，因为众所周知，在人类被广泛使用时是很容易被容忍的 用于乳腺癌的治疗。