ROLE OF 20-HETE IN REGULATING VASCULAR OXYGEN RESPONSE

氧依赖性自身调节机制在调节 器官血流和血管阻力。虽然这些已经广泛 研究表明，负责氧气的细胞和分子“传感器” 外周循环中的感知未知。该项目将 检查假设，20-羟基二十碳四烯酸（20-HETE）， 细胞色素P450的代谢产物花生四烯酸，是一个重要的调解人 血管收缩剂对增加的氧可用性的反应 血压正常的动物，并且增加了 细胞色素P4504 A α-羟化酶，产生20-HETE或在 阻力血管对2-HETE的敏感性增强 阻力动脉对氧气增加的反应性收缩 在各种高血压模型中已经证明了这一点。 20-HETE介导血管对氧增加的反应 将使用三种方法在大鼠提睾肌中评估可用性 抑制20-HETE的形成或作用的不同方法[17- 18]。 十八碳烯酸（17-ODYA），二溴十二碳酰甲基亚磺酰亚胺（DDMS），和 19-羟基二十碳四烯酸（19-HETE）]。氧气供应量 提睾肌将增加：1）增加P0/2的 灌注液流过组织，2）灌注 变构血红蛋白效应物RSR-13，和3）增加吸入的 0/2（FI0/2）。微血管对0/2增加的反应 可用性将通过直接测量小动脉 红细胞直径和红细胞速度以及个体血流量的计算 小动脉组织中和微血管表面的P0/2将 用0/2微电极测量，以验证组织和 在0/2可用性增加期间，小动脉P0/2在P0/2范围内 其中20-HETE形成是0/2敏感的。“传感器”的位置， 20-HETE介导的微循环中的氧反应将是 通过确定负责20- HETE形成（P4504 A α-羟化酶），使用蛋白质印迹法， 用RT-PCR鉴定P4504 A α-羟化酶亚型， 小动脉和骨骼肌实质组织。20-HETE的作用 介导全身血管对增加的O/2可用性的反应， 通过以下方法在清醒动物中评价急性体积扩张 测定心输出量、总外周阻力、局部血液 增加FI 0/2期间的血流和局部血管阻力， 变构血红蛋白效应物RSR-13和急性容量扩张， 而不用DDMS阻断20-HETE的形成。的贡献 20-HETE对P0/2增加引起的血管收缩反应的增强， 高血压将在两个高血压实验模型中进行测试 先前报道显示对氧气增加的反应增强 可用性：肾质量减少（RRM）高血压大鼠和 自发性高血压大鼠（SHR）。增加的贡献 P4504 A α-羟化酶的表达对增强的 在高血压动物中，响应于升高的P0/2的小动脉将 通过使用Western印迹和竞争性RT-PCR来评估 P4504 A α-羟化酶在整个提睾肌中的表达 肌肉和孤立的小动脉和实质组织的高血压 和血压正常的动物。提高对环境的敏感性的作用 抵抗血管对20-HETE介导的增强反应， 高血压动物中升高的P0/2将通过评价 原位微循环和孤立小动脉的敏感性， 阻力动脉对外源性添加的20-HETE的反应。综上所述各项 实验应该提供一个综合的理解的作用，20- HETE介导血管对氧可用性增加的反应 分子水平，在单个阻力容器的水平上，以及 在完整的、有意识的、 动物

Oxygen dependent autoregulatory mechanisms are critical in regulating organ blood flow and vascular resistance. While these have been widely studied, the cellular and molecular "sensors" responsible for oxygen sensing in the peripheral circulation are unknown. This project will examine the hypotheses that 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 metabolite of arachidonic acid, is an important mediator of the vasoconstrictor response to increased oxygen availability in normotensive animals, and that increases either in the expression of the cytochrome P4504A alpha-hydroxylase enzymes that produce 20-HETE or in the sensitivity of resistance vessels to 2-HETE contribute to the enhanced constriction of resistance arteries in response to increased oxygen availability that has been demonstrated in various models of hypertension. The role of 20-HETE in mediating vascular response to increased oxygen availability will be assessed in the rat cremaster muscle using three different methods of inhibiting the formation or action of 20-HETE [17- octadecenoic acid (17-ODYA), dibromododeconylmethyl sulfinmide (DDMS), and 19-hydroxyeicosatetraenoic acid (19-HETE)]. Oxygen availability to the cremaster muscle will be increased by: 1) increasing the P0/2 of the superfusion solution flowing over the tissue, 2) infusion of the allosteric hemoglobin effector RSR-13, and 3) increasing the inspired fraction of 0/2 (FI0/2). Microvascular responses to increased 0/2 availability will be assessed by the direct measurement of arteriolar diameter and RBC velocity and the calculation of blood flow in individual arterioles. P0/2 in the tissue and on the surface of the microvessels will be measured with 0/2 microelectrodes to verify that changes in tissue and arteriolar P0/2 during increased 0/2 availability are in the range of P0/2 where 20-HETE formation is 0/2 sensitive. The location of the "sensor" for 20-HETE mediated oxygen responses in the microcirculation will be investigated by determining the location of the enzyme responsible for 20- HETE formation (P4504A alpha-hydroxylase) using Western blotting and by identifying the P4504A alpha-hydroxylase isoform using RT-PCR in isolated arterioles and skeletal muscle parenchymal tissue. The role of 20-HETE in mediating whole body vascular responses to increased O/2 availability and acute volume expansion will be evaluated in conscious animals by determining cardiac output, total peripheral resistance, regional blood flows and regional vascular resistance during increased FI0/2, infusion of the allosteric hemoglobin effector RSR-13, and acute volume expansion with and without blockade of 20-HETE formation with DDMS. The contribution of 20-HETE to the enhanced vasoconstrictor response to increased P0/2 in hypertension will be tested in two experimental models of hypertension previously reported to exhibit an enhanced response to increased oxygen availability: the reduced renal mass (RRM) hypertensive rat and the spontaneously hypertensive rat (SHR). The contribution of an increased expression of P4504A alpha-hydroxylase to the enhanced constriction of arterioles in response to elevated P0/2 in the hypertensive animals will be assessed by using Western blots and competitive RT-PCR to evaluate the expression of the P4504A alpha-hydroxylase enzyme in the whole cremaster muscle and in isolated arterioles and parenchymal tissue of hypertensive and normotensive animals. The role of an increased sensitivity of the resistance vessels to 20-HETE in mediating the enhanced response to elevated P0/2 in hypertensive animals will be assessed by evaluating the sensitivity of the in situ microcirculation and isolated arterioles and resistance arteries to exogenously added 20-HETE. Taken together, these experiments should provide an integrated understanding of the role of 20- HETE in mediating vascular responses to increased oxygen availability at the molecular level, at the level of the individual resistance vessel, and at the level of the peripheral circulation in the intact, conscious animal.