BRAIN SYNTHESIS OF ENDOGENOUS VASOACTIVE CP450 PRODUCTS

描述（研究者摘要）：大脑代谢花生四烯酸 通过三种不同的酶， 途径，即环氧合酶、脂氧合酶和细胞色素P450 （cP450）。 就资料而言，大部分集中在环氧化酶的生理学上 和脂氧合酶产物。 然而，我们实验室最近的工作和 其他人已经证明花生四烯酸的cP 450产物是 生物过程的有效控制剂，包括调节因子 细胞离子转运系统和血管肌肉张力。 我们有 初步数据表明C450环氧合酶和ω羟化酶 大脑皮层和皮层的花生四烯酸产物形成 微血管组织的猫，其作用在nM浓度，以改变 脑小动脉肌细胞静息K+通道活性。 蛋白质印迹分析证实了cP 450 4A Ω的存在 脑微血管组织内的羟化酶。 初步 数据表明，cP 450产物的形成对氧敏感 在组织PO 2的生理范围内（即在50和30之间 （.） 本提案中概述的研究将分离cP 450 猫脑实质中花生四烯酸代谢产物的研究 皮质、大脑微血管和内皮细胞， 与[14 C]花生四烯酸孵育并使用rpHPLC分离。 产品将通过与已知的 标准品，并与GC/MS确认。我们将确定的能力， cP 450抑制剂阻断产物形成的特异性 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仪测量脑血流量， 颅窗，并在体外使用肌电描记器研究 分离的加压脑动脉对cP 450代谢物的反应。 产品 形成和cP 450酶活性将被确定为以下的函数： P02（在100和20 ℃之间）以及大脑的能力 cP 450抑制前后血管对缺氧的反应 产物形成 我们将定义细胞和离子机制， cP 450代谢产物对脑小动脉肌细胞的作用 通过测定cP 450产物对K+和 使用膜片钳技术测量[Ca]i， 荧光探针。 这些研究的独特之处在于， 生物化学、细胞、分子和功能方法来确定 cP 450产物在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 了解一种相对未被研究但重要的生物化学 大脑中的通路，以及与适应性 以及缺氧损伤和中风的病理学意义。

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.