ROLE OF P450 MONOOXYGENASES IN RENAL HEMODYNAMICS

长期的研究目标是阐明 花生四烯酸代谢物影响肾血流量和肾小球 过滤肾血流量和肾小球滤过有效地 由传入和传出小动脉调节， 激素和旁分泌的影响。肾血流调节出现 来自肾小球前和肾小球后细胞利用的不同信号传导机制， 小动脉我们已经证明， 花生四烯酸使传入小动脉血管收缩， 影响传出心房张力。这条传入小动脉 血管收缩部分由花生四烯酸的代谢介导 P450代谢物。此外，花生四烯酸的活化 代谢物调节肾和血管对激素的反应， 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， 激素激动剂肾小球前血管扩张至P450 代谢物11，12-EET和14，15-EET，血管收缩为20-HETE， 5，6-EET。P450代谢产物对肾小球后膜的直接作用 小动脉未知。因此，P450代谢产物可能影响传入神经元的功能。 小动脉对旁分泌和激素激动剂的反应， 比传出小动脉的反应程度。上述假设 将利用体外灌注肾血管床进行研究 制备，新鲜分离和培养的肾血管平滑肌 细胞实验将利用体外灌注的 可直接进入骨髓的肾单位制剂 肾小球前和肾小球后小动脉我们将评估 肾小球后反应P450代谢产物，并确定 P450代谢产物对传入和传出小动脉的贡献 血管活性激动剂引起的反应。这些研究将采用新的 开发了P450代谢物的稳定类似物，亚型特异性P450 抑制剂和特定肾脏P450酶敲除的小鼠。 此外，离体灌流血管床和血管平滑肌 细胞实验将确定细胞信号机制， P450代谢物使肾微血管舒张和收缩。 P450代谢产物作为细胞内信号信使的作用 还将测定血管活性激动剂。我们将评估 调节激动剂诱导的细胞内钙离子 在新鲜分离和培养的肾微血管平滑肌中的反应 肌肉细胞特异性P450酶同工型的表达载体将 插入培养的肾微血管平滑肌细胞， 过表达立体选择性ETEs和HETEs对激动剂- 在肾微血管平滑肌细胞中诱导的钙反应将 下定决心。这些垂直整合的研究将提供一个改善 了解细胞色素P450花生四烯酸 代谢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.