Tyrosine Kinases in Renal Vasoconstrictor Signaling

肾小球前微血管系统在维持肾脏稳态功能中起着重要作用，包括Na+和H2O排泄的调节。主要的Na+保留激素，血管紧张素II（AngII），通过AT 1受体（AT 1 R）引起肾小球前（和肾小球后）血管收缩。 AT 1 R的参与引起G蛋白介导的磷脂酶C活化，最终导致细胞内Ca 2+浓度（[Ca 2 +]i）升高，从而促进收缩器的Ca 2 +/钙调蛋白依赖性活化。AngII还通过一种不仅涉及AT 1 R参与而且随后涉及表皮生长因子反式激活的现象对血管平滑肌发挥促有丝分裂作用 受体（EGFR），导致多种蛋白激酶的活化，最终导致多种促有丝分裂基因产物的表达改变。EGFR下游的信号传导事件最近被认为与引起大动脉血管平滑肌收缩有关，我们的初步数据表明，对AngII的传入小动脉收缩反应涉及酪氨酸激酶，包括EGFR酪氨酸激酶，并且该过程有助于[Ca 2 +]i反应。拟议的工作将涉及 假设AngII诱导的肾小球前微血管平滑肌收缩涉及AT 1 R介导的复杂信号网络的启动，该网络包括c-Src依赖性EGFR反式激活、Ca 2+敏感性酪氨酸激酶的激活和有助于Ca 2+内流和收缩反应的离子通道的磷酸化。我们进一步假设EGFR依赖性通路的上调有助于血管紧张素II诱导的收缩反应性的增强， 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AT 1 R反式激活EGFR的机制，2）确定AT 1 R反式激活EGFR的机制。 通过该途径AngII诱导的EGFR反式激活促进Ca 2+内流，3）确定EGFR反式激活相关的信号传导事件是否是通过G蛋白偶联受体（特别是精氨酸加压素）起作用的其他肾小球前血管收缩剂的原型，和4）确定EGFR反式激活途径是否有助于AngII依赖性高血压中对AngII的过度反应。实验策略将利用分子，药理学，生物化学和生理学的方法，以澄清酪氨酸激酶在引起激动剂诱导的肾小球前微血管收缩的作用。这种方法应暴露PVSMC中特定酪氨酸激酶的活性及其对小动脉张力调节的影响。本项目的成功完成将促进我们对AngII调节肾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。血管紧张素II是一种有效的血压调节剂，部分通过影响肾脏器官中的微观血管起作用。这项工作正在探索的机制， 血管紧张素II迅速收缩肾脏微血管中的肌肉细胞，集中在以前认为只发生在缓慢发育的生长反应中的过程。这个项目的完成将促进我们的 了解血管紧张素II依赖性调节肾功能和血压在健康和疾病。

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.