BRAIN SYNTHESIS OF ENDOGENOUS VASOACTIVE CP450 PRODUCTS

内源性血管活性 CP450 产品的脑合成

• 批准号: 2270409
• 负责人: David Rae Harder
• 金额: $ 23.1万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2270409
• 关键词: arachidonate; brain metabolism; calcium flux; cats; cerebrovascular system; cytochrome P450; eicosanoid metabolism; high performance liquid chromatography; microcirculation; oxygen tension; potassium channel; tissue /cell culture; ultrasound blood flow measurement; vascular endothelium; vascular smooth muscle; voltage /patch clamp

DESCRIPTION (Investigator's Abstract): The brain metabolizes arachidonic acid into biologically active products via three distinct enzymatic pathways, namely, cyclooxygenase, lipoxygenase, and cytochrome-P450 (cP450). To data, most has focused on the physiology of cyclooxgenase and lipoxygenase products. However, recent work from our laboratory and others has demonstrated that cP450 products of arachidonic acid are potent controllers of biological processes including regulation f cellular ion transport systems and vascular muscle tone. We have preliminary data demonstrating c450 epoxygenase and omega hydroxylase arachidonic acid product formation form cerebral cortex and cortical microvascular tissue of cats which act in nM concentrations to alter resting K+ channel activity in muscle cells form cerebral arterioles. Western blot analysis demonstrates the presence of cP450 4A omega hydroxylase enzymes within cerebral microvascular tissue. Preliminary data demonstrates that cP450 product formation is sensitive to oxygen within the physiological range of tissue PO2 (i.e. between 50 and 30 torr.) the studies outlined in this proposal will isolate cP450 metabolites of arachidonic acid from parenchymal tissue of cat cerebral cortex, the cerebral microvasculature, and endothelial cells by incubation with [14C] arachidonic acid and separation using rpHPLC. Products will be identified via identified via co-elution with known standards and confirmed with GC/MS. We will determine the ability and specificity of inhibitors of cP450 to block product formation in our system. The physiological action of these products will be determined in vivo using laser-doppler flowmetry to measure cerebral blood flow via a cranial window, and in vitro using a myograph to study the response of isolated, pressurized cerebral arteries to cP450 metabolites. Product formation and cP450 enzyme activity will be determined as a function of P02 (between 100 and 20 torr) as will the ability of the cerebral vasculature to respond to hypoxia before and after inhibition of cP450 product formation. We will define the cellular and ionic mechanism of action of cP450 metabolites on arteriolar muscle cells form the cerebral microvasculature by determining the effect of cP450 products on K+ and Ca2+ currents using the patch-clamp technique and measuring [Ca]i with fluorescent probes. These studies are unique in that they incorporate biochemical, cellular, molecular, and functional approaches to determine the importance of cP450 products in brain function and control of "nutritive" cerebral blood flow. These are vital studies with respect to understanding a relatively unstudied but important biochemical pathway in the brain, and the mechanisms associated with the adaptive and pathological implications of hypoxic insult and stroke.

描述（研究者摘要）：大脑代谢花生四烯酸 通过三种不同的酶促将酸转化为生物活性产物 途径，即环氧合酶、脂氧合酶和细胞色素-P450 （cP450）。 就数据而言，大多数都集中在环加氧酶的生理学上 和脂氧合酶产品。 然而，我们实验室最近的工作和 其他人已经证明花生四烯酸的 CP450 产品是 生物过程的有效控制器，包括调节 细胞离子传输系统和血管肌张力。 我们有 初步数据证明 c450 环氧化酶和 omega 羟化酶 大脑皮层和皮质的花生四烯酸产物形成 猫的微血管组织在 nM 浓度下起作用以改变 肌肉细胞中的静息 K+ 通道活性形成脑小动脉。 蛋白质印迹分析表明 cP450 4A omega 的存在 脑微血管组织内的羟化酶。 初步的 数据表明 cP450 产物的形成对氧气敏感 在组织 PO2 的生理范围内（即 50 至 30 torr.）本提案中概述的研究将分离 cP450 猫脑实质组织中花生四烯酸的代谢物 皮质、脑微血管和内皮细胞 与[14C]花生四烯酸一起孵育并使用rpHPLC分离。 产品将通过与已知的共洗脱来识别 标准并通过 GC/MS 进行确认。我们将确定能力和 cP450 抑制剂在我们的研究中阻止产物形成的特异性 系统。 这些产品的生理作用将在体内确定 使用激光多普勒血流计通过 颅窗，并在体外使用肌动描记器来研究 分离、加压脑动脉以产生 cP450 代谢物。 产品 形成和 cP450 酶活性将被确定为函数 P02（100 到 20 托之间）以及大脑的能力 cP450 抑制前后脉管系统对缺氧的反应 产品形成。 我们将定义细胞和离子机制 cP450代谢物对脑小动脉肌细胞的作用 通过确定 cP450 产品对 K+ 和 K+ 的影响来研究微血管 使用膜片钳技术并测量 [Ca]i 的 Ca2+ 电流 荧光探针。 这些研究的独特之处在于它们结合了 生物化学、细胞、分子和功能方法来确定 cP450 产品在大脑功能和控制中的重要性 “营养”脑血流量。 这些都是至关重要的研究 了解相对未被研究但重要的生化 大脑中的通路以及与适应性相关的机制 以及缺氧和中风的病理学影响。