Tyrosine Kinases in Renal Vasoconstrictor Signaling

描述（由申请方提供）：肾小球前微血管系统在实现肾脏稳态功能（包括Na+和H2O排泄的调节）中发挥关键作用。 血管紧张素II（AngII）是一种主要的钠离子保留激素，通过与血管紧张素1受体（AT 1 R）的结合，引起G蛋白介导的磷脂酶C（PLC）激活和细胞内Ca ~（2+）浓度（[Ca ~（2+）]i）升高，从而激活收缩器官，从而引起肾小球前（和肾小球后）血管收缩。 AngII还通过表皮生长因子受体（EGFR）的AT 1 R依赖性反式激活对血管平滑肌发挥促有丝分裂作用。EGFR是一种受体酪氨酸激酶，可与多个下游分支形成信号传导复合物，包括用于增加促有丝分裂基因产物表达的Ras/MAP激酶途径。 最近，EGFR酪氨酸激酶活性已被牵连在引发收缩的血管平滑肌从大动脉。我们的初步数据表明肾传入小动脉对AngII的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。 拟议的工作将解决的假设，血管紧张素II诱导的收缩肾小球前微血管平滑肌细胞（PVSMC）涉及AT 1 R介导的启动一个复杂的信号网络，包括H2 O2介导的激活Src家族激酶（S）和随后的EGFR反式激活。 我们还将研究PLC（1）和PLC（1）在AngII诱导的信号传导事件中的作用，这些信号传导事件产生对肽的特征性双相[Ca 2 +]i应答。我们进一步假设，促有丝分裂状态（如AngII诱导的高血压）中EGFR依赖性通路的上调将促进对AngII的过度收缩反应。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Src家族激酶在AngII诱导的信号传导和PVSMC收缩反应中的作用; 2）确定H2 O2在AngII诱导的收缩信号传导中的作用; 3）确定经典的AT 1 R/ PLC是否在血管紧张素Ⅱ诱导的收缩反应中起作用。（1 /IP 3依赖性Ca 2+动员事件是EGFR反式激活的先决条件; 4）确定PLC是否（1）在PVSMC对AngII的收缩反应中起作用，如果这是EGFR反式激活的结果;和5）评估AT 1 R-EGFR信号通路的慢性激活促进肾小球前血管收缩剂对AngII的反应性增强的假设。 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。 通过暴露PVSMCs中特定酪氨酸激酶的活性及其对AngII收缩反应的贡献，该项目的成功完成应促进我们对肾小球前微血管张力调节的理解，这是生理和病理生理条件下外周阻力、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。这项工作正在探索血管紧张素II快速收缩肾脏微血管中肌肉细胞的机制，重点关注以前认为仅发生在发育缓慢的生长反应中的过程。这个项目的完成将促进我们对血管紧张素II依赖性调节健康和疾病中肾功能和血压的理解。

DESCRIPTION (provided by applicant): 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) elicits preglomerular (and postglomerular) vasoconstriction via engagement of AT1 receptors (AT1R), which provoke G protein-mediated phospholipase C (PLC) activation and the rise in intracellular Ca2+ concentration ([Ca2+]i) that fuels activation of the contractile apparatus. AngII also exerts mitogenic effects on vascular smooth muscle through AT1R-dependent transactivation of the epidermal growth factor receptor (EGFR). The EGFR is a receptor tyrosine kinase that can form a signaling complex with multiple downstream branches, including the Ras/MAP kinase pathway for increasing expression of pro-mitogenic gene products. Recently, EGFR tyrosine kinase activity has been implicated in eliciting contraction of vascular smooth muscle from large arteries. Our preliminary data indicate that renal 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 cells (PVSMCs) involves AT1R-mediated initiation of a complex signaling network that includes H2O2-mediated activation of Src family kinase(s) and subsequent EGFR transactivation. We will also investigate the roles of PLC(1 and PLC(1 in the AngII-induced signaling events that generate the characteristic bi-phasic [Ca2+]i response to the peptide. We further postulate that up-regulation of EGFR-dependent pathways in pro-mitogenic states (such as AngII-induced hypertension) will promote exaggerated contractile responsiveness to AngII. The validity of these postulates will be examined by addressing the following specific aims: 1) Establish the role of Src family kinases in AngII-induced signaling and contractile responses of PVSMCs; 2) Determine the role of H2O2 in AngII-induced contractile signaling; 3) Determine if the classical AT1R / PLC(1 / IP3-dependent Ca2+ mobilization event is a prerequisite for EGFR transactivation; 4) Determine if PLC(1 plays a role in the PVSMC contractile response to AngII, and if this occurs as a consequence of EGFR transactivation; and 5) Evaluate the postulate that chronic activation of AT1R-EGFR signaling pathways promotes accentuated preglomerular vasoconstrictor responsiveness to AngII. 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. By exposing the activity of specific tyrosine kinases in PVSMCs and their contributions to the contractile response to AngII, the successful completion of this project should advance our understanding of the regulation of preglomerular microvascular tone, an important determinant of peripheral resistance, Na+ excretion and arterial pressure under physiological and pathophysiological conditions. 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.