NITRIC OXIDE AND 20-HETE INTERACTION IN CONTROL OF RENAL FUNCTION

我们实验室最近的研究表明， 平滑肌细胞通过P4504 A依赖性代谢花生四烯酸 20-HETE通路，并且该系统作为一种新的信号 在肾脏的调节中起核心作用的转导途径 通过调节Ca ~（++）激活的K ~（++）的开放状态概率来调节血管张力 渠道这项建议将检验一氧化氮（NO） 抑制肾脏中20-HETE的产生，这有助于 对肾血管张力和肾小管肾小球的抑制作用 反馈一氧化氮对花生四烯酸肾代谢的影响 将使用肾微粒体、完整肾微血管和 小管可见光光谱将用于研究结合 用杆状病毒和Sf 9表达的重组P4504 A亚型的NO 昆虫细胞一氧化氮对肾血管钾通道活性的影响 使用膜片钳技术表征平滑肌细胞， 以及20-HETE与cGMGP对这些反应的相对贡献 将通过改变细胞内20-HETE的水平来确定。 鸟苷酸环化酶和cGMP依赖性抑制剂的存在和不存在 蛋白激酶平行研究将确定以下方面的贡献： 20-HETE与cGMP的产生变化对NO对 离体灌流肾细胞膜电位和血管张力 小动脉P4504 A抑制剂对大鼠动脉粥样硬化性心脏病血管病变的影响 NO供体和合成酶产生的压力和肾血流量 将在大鼠中研究抑制剂以评估NO-20- HETE在肾脏和外周血管张力调节中的相互作用 将进行体内和肾小管灌注研究，以确定 20-HETE对NO抑制作用的贡献 管球反馈最后，盐摄入量变化的影响 AII和循环水平对P4504 A mRNA、蛋白、 和酶活性沿着肾单位将测量使用竞争性 RT-PCR、蛋白质印迹和放射化学分析，以确定是否 这一系统表达的变化可能有助于 调节肾血管张力和肾小管肾小球反馈相关 盐摄入量的变化。

Recent studies from our laboratory have indicated that renal vascular smooth muscle cells metabolize arachidonic acid via a P4504A dependent pathway to 20-HETE, and that this system serves as a novel signal transduction pathway that plays a central role in the regulation of renal vascular tone by regulate the open state probability of Ca++-activated K+ channels. This proposal will examine the hypothesis that nitric oxide (NO) inhibits the production of 20-HETE in the kidney and that this contributes to its inhibitory effects on renal vascular tone and tubuloglomerular feedback. The influence of NO on the renal metabolism of arachidonic acid will be evaluated using renal microsomes, intact renal microvessels and tubules. Visible light spectroscopy will be utilized to study the binding of NO to recombinant P4504A isoforms expressed with a baculovirus and Sf9 insect cells. The effects of NO on K+ channel activity in renal vascular smooth muscle cells will be characterized using patch clamp techniques., and the relative contribution of 20-HETE versus cGMGP to these responses will be determined by altering intracellular levels of 20-HETE in the presence and absence of inhibitors of guanylyl cyclase and cGMP-dependent protein kinase. Parallel studies will determine the contribution of changes in the production of 20-HETE versus cGMP to the effects of NO on membrane potential and vascular tone in isolated perfused renal arterioles. The effects of P4504A inhibitors on the changes in arterial pressure and renal blood flow produced by NO donors and synthase inhibitors will be studied in rats to evaluate the significance of NO-20- HETE interactions in the regulation of renal and peripheral vascular tone in vivo, and tubular perfusion studies will be performed to determine the contribution of 20-HETE to the inhibitory effects of NO on tubuloglomerular feedback. Finally, the effects of changes in salt intake and circulating levels of AII on the distribution of P4504A mRNA, protein and enzyme activity along the nephron will be measured using competitive RT-PCR, Western blots and radio-chemical assays to determine whether changes in the expression of this system might contribute to the modulation of renal vascular tone and tubuloglomerular feedback associated with changes in salt intake.