ROLE OF 20-HETE IN REGULATING VASCULAR OXYGEN RESPONSE

氧依赖的自动调节机制在调节 器官血流和血管阻力。虽然这些已经被广泛地 研究表明，负责氧气的细胞和分子“传感器” 外周循环中的感觉是未知的。这个项目将 检验假设20-羟基二十碳四烯酸(20-HETE)，a 花生四烯酸的细胞色素P450代谢物，是一种重要的介质 血管收缩反应对氧气供应增加的影响 血压正常的动物，而这要么增加了 细胞色素P4504Aα-羟基酶产生20-HETE或在 阻力血管对2-HETE的敏感性有助于增强 阻力动脉对氧气增加的反应 在高血压的各种模型中已经证明了这一点。 20-HETE在介导血管对高氧反应中的作用 将使用三种方法评估大鼠提睾肌的利用率 抑制20-HETE形成或作用的不同方法[17- 十八碳烯酸(17-ODYA)、二溴十二烷基甲基亚磺胺(DDMS)和 19-羟基二十碳四烯酸(19-HETE)]。氧气对人体的利用率 提睾肌将增加：1)增加P0/2 流过组织的灌注液，2)输注 变构血红蛋白效应器RSR-13，以及3)增加灵感 0/2的分数(FI0/2)。微血管对0/2增加的反应 利用度将通过直接测量小动脉来评估 个体的直径、红细胞流速与血流量的计算 小动脉。P0/2在组织和微血管表面将 用0/2微电极测量，以验证组织和 微动脉P0/2在0/2可用性增加期间在P0/2范围内 其中20-HETE的形成是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 通过确定负责20-的酶的位置进行调查 HETE形成(P4504Aα-羟基酶)的Western blotting和 分离株P4504Aα-羟基酶亚型的RT-PCR鉴定 小动脉和骨骼肌实质组织。20-HETE在植物生长发育中的作用 调节全身血管对增加的氧/氧供应和 有意识的动物的急性体积扩张将通过以下方式进行评估 测定心输出量、总外周阻力、局部血流量 FI0/2增加时的血流和局部血管阻力 变构血红蛋白效应器RSR-13和急性容量膨胀 不阻断20-HETE与DDMS的形成。的贡献 20-HETE对P0/2升高的血管收缩反应的增强作用 高血压将在两个高血压实验模型中进行测试 以前的报道显示对增加的氧气有增强的反应 可用性：降低肾质量(RRM)高血压大鼠和 自发性高血压大鼠(SHR)。增加的贡献 P4504Aα-羟基酶在血管增强收缩中的表达 高血压动物微动脉对P0/2升高的反应 用Western blotts和竞争性RT-PCR方法评价 P4504Aα-羟基酶在整个生殖器中的表达 高血压病患者的肌肉及离体微动脉和实质组织 以及血压正常的动物。经济增长的敏感性所起的作用 血管对20-HETE的拮抗作用 高血压动物的P0/2升高将通过评估 原位微循环和离体微动脉的敏感性及 对外源性添加20-HETE的阻力动脉。这些加在一起， 实验应该提供对20- HETE在介导血管对氧气供应增加的反应中的作用 分子水平，在单个阻力血管水平，以及 在外周循环的水平上，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.