Tyrosine Kinases in Renal Vasoconstrictor Signaling

描述（由申请人提供）：肾肾小球前微血管在实现肾脏稳态功能中起关键作用，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AT1受体（AT1R）的参与引起肾小球前（和肾小球后）血管收缩，从而引起G蛋白介导的磷脂酶C （PLC）的激活和细胞内Ca2+浓度（[Ca2+]i）的升高，从而激活收缩装置。AngII还通过at1r依赖性表皮生长因子受体（EGFR）的转激活对血管平滑肌产生有丝分裂作用。EGFR是一种受体酪氨酸激酶，可以与多个下游分支形成信号复合物，包括Ras/MAP激酶途径，以增加促有丝分裂基因产物的表达。最近，EGFR酪氨酸激酶活性与诱导大动脉血管平滑肌收缩有关。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(s)，包括EGFR酪氨酸激酶，这一过程有助于[Ca2+]i反应。本研究提出的假设是，血管素诱导的肾肾小球前微血管平滑肌细胞（PVSMCs）收缩涉及at1r介导的复杂信号网络的启动，包括h2o2介导的Src家族激酶激活和随后的EGFR反激活。我们还将研究PLC(1)和PLC(1)在血管诱导的信号事件中的作用，这些事件产生对肽的特征性双相[Ca2+]i反应。我们进一步假设，促有丝分裂状态下egfr依赖通路的上调（如血管增生诱导的高血压）将促进对血管增生的过度收缩反应。这些假设的有效性将通过解决以下具体目标来检验：1)建立Src家族激酶在血管内皮细胞诱导的信号传导和PVSMCs收缩反应中的作用；2)确定H2O2在血管收缩信号传导中的作用；3)确定经典的AT1R / PLC（1 / ip3依赖的Ca2+动员事件是否是EGFR反激活的先决条件；4）确定PLC(1)是否在PVSMC对AngII的收缩反应中起作用，以及这是否作为EGFR反激活的结果发生；5)评估AT1R-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。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活激动剂诱导的肾小球前微血管收缩中的作用。通过揭示PVSMCs中特定酪氨酸激酶的活性及其对AngII收缩反应的贡献，该项目的成功完成将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+排泄和动脉压力的重要决定因素。

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.