Tyrosine Kinases in Renal Vasoconstrictor Signaling

肾小球前微血管系统在实现肾脏的稳态功能方面起着至关重要的作用，包括调节Na+和H2O的排泄。主要的钠离子保留激素，血管紧张素II(AngII)，通过AT1受体(AT1R)作用于肾小球前(和肾小球后)血管收缩。 AT1R的参与刺激G蛋白介导的磷脂酶C激活，最终导致细胞内钙离子浓度([Ca~(2+)]i)升高，从而刺激钙/钙调蛋白依赖的收缩装置的激活。血管紧张素转换酶II不仅通过AT1R的参与，而且通过随后的表皮生长因子的反式激活，在血管平滑肌上发挥有丝分裂的作用 受体(EGFR)，导致多种蛋白激酶的激活，最终导致各种促有丝分裂基因产物表达的改变。我们的初步数据表明，血管紧张素转换酶抑制血管紧张素转换酶(Angii)的传入小动脉收缩反应涉及酪氨酸激酶(S)，包括EGFR酪氨酸激酶，这一过程参与了[Ca~(2+)]_i反应。拟议的工作将解决 血管紧张素转换酶诱导肾小球微血管平滑肌收缩的假说涉及AT1R介导的复杂信号网络的启动，该网络包括c-Src依赖的EGFR反式激活、钙敏感的酪氨酸激酶的激活以及参与钙离子内流和收缩反应的离子通道的磷酸化。我们进一步假设，上调EGFR依赖的通路有助于血管紧张素转换酶Ⅱ诱导的血管收缩反应的夸大。 高血压。这些假设的有效性将通过解决以下具体目标来检验：1)确定AT1R反式激活EGFR的机制，2)确定机制 3)确定EGFR反式激活相关的信号事件是否是其他通过G蛋白偶联受体(特别是精氨酸加压素)作用的肾小球前血管收缩因子的典型；以及4)确定EGFR反式激活途径是否有助于血管紧张素依赖型高血压患者对血管紧张素转换酶的过度反应。该实验策略将利用分子、药理学、生化和生理学方法来阐明酪氨酸激酶在激动剂诱导的肾小球前微血管收缩中的作用。这种方法应该揭示PVSMCs中特定的酪氨酸激酶的活性以及它们对小动脉张力调节的影响。这个项目的成功完成将促进我们对Angii调节肾脏微血管张力的机制的理解 外周阻力、Na+排泄和动脉压的重要决定因素。LAY摘要：血管紧张素II是一种强大的血压调节剂，部分通过影响肾脏器官中的微小血管发挥作用。这项工作正在探索通过什么机制 血管紧张素II迅速收缩肾脏微血管中的肌肉细胞，专注于以前被认为只在发育较慢的生长反应中发生的过程。这个项目的完成应该会推进我们的 了解血管紧张素II对健康和疾病患者肾功能和血压的调节作用。

The renal preglomerular microvasculature is critically involved in achieving the homeostatic functions of the kidneys, including the regulation of Na+ and H2O excretion. The primary Na+ retaining hormone, angiotensin II (AngII), acts via AT1 receptors (AT1R) to elicit preglomerular (and postglomerular) vasoconstriction. Engagement of the AT1R provokes G protein-mediated phospholipase C activation, eventually leading to the rise in intracellular Ca2+ concentration ([Ca2+]i) that fuels Ca2+/calmodulin-dependent activation of the contractile apparatus. AngII also exerts mitogenic effects on vascular smooth muscle through a phenomenon involving not only engagement of the AT1R but also consequent transactivation of the epidermal growth factor receptor (EGFR), leading to activation of a variety of protein kinases that ultimately result in altered expression of a variety of pro-mitogenic gene products. Signaling events downstream of the EGFR have been implicated recently in eliciting contraction of vascular smooth muscle from large arteries artery, and our preliminary data indicate that afferent arteriolar contractile responses to AngII involve tyrosine kinase(s), including the EGFR tyrosine kinase, and that this process contributes to the [Ca2+]i response. The proposed work will address the hypothesis that AngII-induced contraction of renal preglomerular microvascular smooth muscle involves AT1R-mediated initiation of a complex signaling network that includes c-Src-dependent EGFR transactivation, activation of Ca2+-sensitive tyrosine kinases, and phosphorylation of ion channels that contribute to the Ca2+ influx and the contractile response. We further postulate that up-regulation of EGFR-dependent pathways contribute to the exaggerated AngII-induced contractile responsiveness in hypertension. The validity of these postulates will be examined by addressing the following specific aims: 1) Determine the mechanism through which the AT1R transactivates the EGFR, 2) Determine the mechanism through which AngII-induced EGFR transactivation promotes Ca2+ influx, 3) Determine if EGFR transactivation-linked signaling events are prototypical for other preglomerular vasoconstrictors that act via G protein-coupled receptors (specifically, arginine vasopressin), and 4) Determine if the EGFR transactivation pathway contributes to exaggerated responsiveness to AngII in AngII-dependent hypertension. The experimental strategy will utilize molecular, pharmacological, biochemical and physiological approaches to clarify the role of tyrosine kinases in evoking agonist-induced constriction of the preglomerular microvasculature. This approach should expose the activity of specific tyrosine kinases in PVSMCs and their consequent influences on the regulation of arteriolar tone. Successful completion of this project should advance our understanding of the mechanisms through which AngII regulates renal microvascular tone, an important determinant of peripheral resistance, Na+ excretion and arterial pressure. Lay Summary: Angiotensin II is a potent regulator of blood pressure, acting in part through effects on microscopic blood vessels in the kidney organ. This work is exploring the mechanisms through which angiotensin II rapidly contracts muscle cells in kidney microvessels, focusing on processes previously thought to occur only in slower-developing growth responses. Completion of this project should advance our understanding of angiotensin II-dependent regulation of kidney function and blood pressure in health and disease.