Role of EETs in Vascular Response to Neural Activation

EET 在神经激活的血管反应中的作用

• 批准号: 6967918
• 负责人: RAYMOND Charles KOEHLER
• 金额: $ 54.59万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967918
• 关键词: astrocytes; brain circulation; brain metabolism; calcium flux; clinical research; cytochrome P450; eicosanoid metabolism; eicosanoids; epoxide hydrolase; genetically modified animals; glutamate receptor; heme oxygenase; hemodynamics; laboratory mouse; laboratory rat; nitric oxide; potassium channel; purinergic receptor; receptor expression; tissue /cell culture; vascular smooth muscle nervous control; vasodilation

Although it has long been known that increases in neuronal activity evoke localized increases in cerebral blood flow (CBF), the mechanisms involved in this functional hyperemia have not been fully explained. Because CBF is used as a surrogate for mapping human brain function in health and disease, it is critical to understand the neurovascular coupling mechanisms, which could be altered by vascular or neurological diseases. Previous work done in this project demonstrated that astrocytes produce EETs which hyperpolarize and dilate cerebral vascular smooth muscle (VSM) and that inhibitors of this pathway markedly attenuate the increase in CBF evoked by NMDA, whisker or forepaw stimulation. More recently, we found that stimulation of metabotropic glutamate receptors (mGluR) activates a novel Kca channel in astrocytes by an epoxygenase-dependent mechanism. Glutamate also activates Kca channels in VSM via a mechanism that is dependent on the release of EETs by astrocytes. However, the mechanism by which activation of glutamate receptors promote the synthesis and release of EETs by astrocytes is unknown and is one of the major focuses of this proposal. The working hypothesis is that astrocytes couple CBF to neuronal activity through activation of mGluR in astrocytes leading to localized increases in Ca 2+ and synthesis of EETs, which together open Kca channels and lead to astrocyte hyperpolarization and further capacitive Ca 2+ influx. This sequence activates phospholipases and promotes astrocyte release of EETs which, in turn, diffuse to nearby VSM to open Kca channels and produce vasodilation. In Specific Aim 1, the mechanisms by which mGtuR activation promotes the release of EETs through Ca 2+ signaling pathways will be investigated. In Specific Aim 2, the interaction of EETs with other potential vasodilatory mechanisms involving nitric oxide, heine oxygenase, and 2A and 2B adenosine receptors in mediating functional hyperemia will be investigated using both pharmacological and gene knockout strategies. In Specific Aim 3, the effect of increasing EETs availability on functional hyperemia will be examined by reducing EETs breakdown using both pharmacological inhibitors and epoxide hydrolase knockout strain of mice. In Specific Aim 4, the effect of acute and chronic hypoxic exposure on altering the control mechanisms that match blood flow to metabolic activity in the brain will be investigated. Experiments will focus on P450 epoxygenase activity and expression which, in preliminary data, are found to be upregulated by hypoxia. Parallel studies will be performed in cultured astrocytes to determine the effect of hypoxia on the coupling of mGluR activation to Ca 2+ signaling, Kca channel function, and the release of EETs. Therefore, the proposed interdisciplinary studies will offer a more comprehensive view at both the cellular and integrated level on the mechanisms by which CBF is coupled to neuronal activity and how neurotransmitters promote the release of EETs from astrocytes.

尽管长期以来已经知道，神经元活性的增加引起了脑血流（CBF）的局部局部增加，但尚未完全解释这种功能性充血的机制。由于CBF被用作绘制人脑在健康和疾病中的功能的替代物，因此了解神经血管偶联机制至关重要，这可以通过血管或神经系统疾病改变。该项目中所做的先前工作表明，星形胶质细胞产生的EET超极化和扩张脑血管平滑肌（VSM），并且该途径的抑制剂显着减轻了NMDA，Whisker或前爪刺激引起的CBF的增加。最近，我们发现刺激代谢型谷氨酸受体（MGLUR）通过环氧酶依赖性机制激活星形胶质细胞中的新型KCA通道。谷氨酸还激活VSM中的KCA通道 一种取决于星形胶质细胞释放EET的机制。但是，谷氨酸受体激活促进星形胶质细胞促进EET的合成和释放的机制尚不清楚，并且是该提案的主要重点之一。有效的假设是，星形胶质细胞通过在星形胶质细胞中激活MGLUR，导致Ca 2+的局部增加和EET的合成，从而共同开放KCA通道并导致星形胶质细胞超极化和进一步的CA 2+膨胀。该序列激活磷脂酶并促进Eets的星形胶质细胞释放，而Eets又可以扩散到附近的VSM以打开KCA通道并产生血管舒张。在特定的目标1中，MGTUR激活促进Eets的机制通过 将研究Ca 2+信号通路。在特定的目标2中，将使用药理学和基因敲除策略研究EET与涉及一氧化氮，海苷氧酶以及2A和2B腺苷受体的其他潜在血管舒张机制的相互作用。在特定的目标3中，将通过减少EET分解来检查EET可用性对功能充血的影响 使用小鼠的药理学抑制剂和环氧水解酶基因敲除菌株。在特定的目标4中，将研究急性和慢性缺氧暴露对改变血液流动与大脑代谢活性相匹配的控制机制的影响。实验将集中于P450环氧酶活性和表达，在初步数据中，发现缺氧上调。平行研究将在培养的星形胶质细胞中进行，以确定缺氧对MGLUR激活与Ca 2+信号传导，KCA通道功能和EET的释放的影响。因此，拟议的跨学科研究将在CBF与神经元活性耦合的机制以及神经递质如何促进EET从星形胶质细胞中释放出来的机制，从而在细胞和综合水平上提供更全面的视图。