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

我们实验室最近的研究表明，肾血管 平滑肌细胞通过依赖P4504A代谢花生四烯酸 20-HETE途径，该系统是一个新的信号 在肾脏调节中起核心作用的信号转导途径 调节Ca~(++)激活K~+开放状态概率的血管张力 频道。这项提议将检验一氧化氮(NO) 抑制肾脏中20-HETE的产生，这有助于 对肾血管张力和肾小球球蛋白的抑制作用 反馈。一氧化氮对花生四烯酸肾脏代谢的影响 将使用肾微粒体、完整的肾微血管和 小管。将利用可见光光谱学来研究结合 杆状病毒和Sf9表达的重组P4504A亚型的NO 昆虫细胞。一氧化氮对肾血管K+通道活性的影响 将使用膜片钳技术对平滑肌细胞进行表征。 以及20-HETE和cGMGP对这些反应的相对贡献 将通过改变细胞内20-HETE水平来确定 鸟苷酸环化酶和cGMP依赖抑制剂的存在与不存在 蛋白激酶。平行研究将确定 NO对20-HETE和cGMP产生的影响 离体灌流肾的膜电位和血管张力 小动脉。P4504A抑制剂对动脉粥样硬化的影响 一氧化氮供体和合酶产生的血压和肾血流量 将在大鼠身上研究抑制剂，以评估NO-20-2的意义 HETE相互作用在调节肾脏和外周血管张力中的作用 在体内，将进行肾小管灌注研究，以确定 20-HETE在一氧化氮抑制作用中的作用 肾小管球状反馈。最后，盐摄入量变化的影响 循环AII水平对P4504A mRNA、蛋白分布的影响 沿肾单位的酶活性将用竞争性的 RT-PCR、Western blotts和放化分析 这一系统的表达方式的变化可能有助于 肾血管张力调节与肾小球反馈相关 随着盐摄入量的变化。

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.