BRAIN SYNTHESIS OF ENDOGENOUS VASOACTIVE CP450 PRODUCTS

描述(研究人员摘要)：大脑代谢花生四烯酸 酸通过三种不同的酶转化为生物活性产物 环氧合酶、脂氧合酶和细胞色素-P450途径 (CP450)。从数据来看，大多数都集中在环氧合酶的生理学上。 和脂氧合酶产物。然而，我们实验室最近的工作和 其他人则证明了花生四烯酸的cP450产物是 生物过程的强有力的控制器，包括规则f 细胞离子转运系统和血管肌张力。我们有 C450环氧合酶和omega羟基酶的初步数据 大脑皮层和皮质花生四烯酸产物的形成 猫的微血管组织在NM浓度作用下改变 大脑小动脉形成肌细胞的静息K+通道活动。 Western印迹分析表明cP450 4A omega的存在 脑微血管组织中的羟基酶。初步 数据表明，cp450产物的形成对氧气很敏感。 在组织PO2的生理范围内(即在50和30之间 Torr.)这项提案中概述的研究将分离出cP450 猫脑实质组织中花生四烯酸代谢产物的研究 皮质，大脑微血管系统，和内皮细胞，通过 与[14C]花生四烯酸孵育，用反相高效液相色谱分离。 产品将通过与已知的共同洗脱进行鉴定 标准，并与GC/MS确认。我们将确定能力和 CP450抑制剂阻断OUR细胞产物形成的特异性 系统。 这些产品的生理作用将在体内确定。 应用激光多谱勒血流仪测量脑血流 颅窗，并在体外使用肌电图仪研究反应。 分离的，加压的大脑动脉，以cP450代谢物。产品 形成和cP450酶活性将被确定为以下的函数 P02(在100到20托之间)以及大脑的能力 抑制cP450前后血管系统对低氧的反应 产品形成。我们将定义细胞和离子机制 CP450代谢物对大脑小动脉肌细胞的作用 通过测定cP450产物对K~+和K~+的影响实现微血管生成 膜片钳技术测定[Ca]i及钙电流的研究 荧光探头。这些研究的独特之处在于它们包含了 生化、细胞、分子和功能方法来确定 CP450产物在脑功能中的重要性及其调控 “营养”的脑部血液流动。这些都是值得尊敬的重要研究 去理解一种相对未被研究但很重要的生物化学 大脑中的通路，以及与适应性相关的机制 以及缺氧性侮辱和中风的病理含义。

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.