ROLE OF P450 MONOOXYGENASES IN RENAL HEMODYNAMICS

长期的研究目标是阐明通过什么机制 花生四烯酸代谢产物对肾血流和肾小球的影响 过滤。肾血流量和肾小球滤过有效 由传入和传出小动脉调节，这些小动脉协同反应 荷尔蒙和旁分泌的影响。肾血流调节出现了 来自肾小球前和肾小球后使用的不同信号机制 小动脉。我们已经证明了外源应用 花生四烯酸血管收缩传入小动脉，但不 影响传出的菊花色调。这根传入小动脉 血管收缩作用部分是通过花生四烯酸的新陈代谢来实现的。 到P450代谢物。此外，花生四烯酸的激活 代谢物调节肾脏和血管对激素和 旁分泌激动剂。我们的假设是P450代谢物是 重要参与肾微血管对旁分泌的反应和 激素激动剂。肾小球前血管对P450的扩张作用 代谢物11，12-EET和14，15-EET和血管收缩为20-HETE和 5，6-EET。P450代谢物对肾小球后的直接作用 小动脉是未知的。因此，P450代谢物可能影响传入 小动脉对旁分泌和激素激动剂的反应 比传出微动脉反应的范围更大。上述假设 将利用体外灌流的肾血管床进行研究 新鲜分离和培养的肾血管平滑肌制剂 细胞。实验将利用体外灌流的 可直接接触的延髓旁肾单位制剂 肾小球前小动脉和肾小球后小动脉。我们将评估 肾小球后对P450代谢物的反应并确定 P450代谢物对小动脉传入和传出的作用 血管活性激动剂引起的反应。这些研究将采用新的 开发稳定的P450代谢物类似物，异构体特异性P450 抑制剂，以及具有特定肾脏P450酶的小鼠被剔除。 此外，分离的灌流血管床和血管平滑肌 细胞实验将确定细胞信号机制，通过它 P450代谢物具有扩张和收缩肾微血管的作用。 P450代谢物作为细胞内信号转导因子的作用 血管活性激动剂也将被确定。我们将评估其能力 P450代谢产物对激动剂诱导的细胞内钙的调节作用 新鲜分离和培养的肾微血管平滑肌的反应 肌肉细胞。特定P450酶异构体的表达载体将 将其植入培养的肾微血管平滑肌细胞中， 立体选择性EETs和HETEs的过度表达对激动剂的影响 诱导肾微血管平滑肌细胞的钙反应 要下定决心。这些垂直整合的研究将提供一个改进 细胞色素P450花生四烯酸作用机制的研究 代谢产物影响肾血流和肾小球滤过。

The long term research goal is the elucidation of mechanisms by which arachidonic acid metabolites influence renal blood flow and glomerular filtration. Renal blood flow and glomerular filtration are efficiently regulated by afferent and efferent arterioles which respond in concert to hormonal and paracrine influences. Renal blood flow regulation arises from disparate signaling mechanisms utilized by pre- and postglomerular arterioles. We have demonstrated that exogenous applications of arachidonic acid vasoconstricts the afferent arteriole and does not influence efferent artiolar tone. This afferent arteriolar vasoconstriction is mediated, in part, by metabolism of arachidonic acid to P450 metabolites. Additionally, activation of arachidonic acid metabolites modulate renal and vascular responses to hormonal and paracrine agonists. Our hypothesis is that P450 metabolites are importantly involved in the renal microvascular responses to paracrine and hormonal agonists. The pre-glomerular vascular vasodilates to the P450 metabolites 11, 12-EET and 14,15-EET and vasoconstricts to 20-HETE and 5,6-EET. The direct actions of P450 metabolites on the postglomerular arterioles are not known. Thus, P450 metabolites may influence afferent arteriolar responses to paracrine and hormonal agonists to a greater extent than efferent arteriolar responses. The above stated hypothesis will be investigated utilizing an in vitro perfused renal vascular bed preparations, freshly isolated and cultured renal vascular smooth muscle cells. Experiments will be performed utilizing the in vitro perfused juxtamedullary nephron preparation which provides direct accessibility to both the pre- and postglomerular arterioles. We will evaluate the postglomerular responses to P450 metabolites and determine the contribution of P450 metabolites to the afferent and efferent arteriolar responses evoked by vasoactive agonists. These studies will employ newly developed stable analogues of P450 metabolites, isoform specific P450 inhibitors, and mice with specific kidney P450 enzymes knocked out. Additionally, isolated perfused vascular bed and vascular smooth muscle cell experiments will determine the cellular-signaling mechanisms by which P450 metabolites vasodilate and vasoconstrict the renal microvasculature. The role of P450 metabolites as intracellular signaling messengers for vasoactive agonists will also be determined. We will evaluate the ability of P450 metabolites to regulate agonist-induced intracellular calcium responses in freshly isolated and cultured renal microvascular smooth muscle cells. Expression vectors of specific P450 enzymatic isoforms will be inserted into cultured renal microvascular smooth muscle cells and the effect of over expression of stereoselective EETs and HETEs on agonist- induced calcium responses in renal microvascular smooth muscle cells will be determined. These vertically integrated studies will provide an improve understanding of the mechanisms by which cytochrome P450 arachidonic acid metabolites influence renal blood flow and glomerular filtration.