Tyrosine Kinases in Renal Vasoconstrictor Signaling

描述(申请人提供)：肾小球前微血管对实现肾脏的动态平衡功能至关重要，包括调节Na+和H2O的排泄。血管紧张素II(AngII)是一种主要的钠离子保留激素，它通过AT1受体(AT1R)引起肾小球前(和肾小球后)血管收缩，从而刺激G蛋白介导的磷脂酶C(PLC)的激活和细胞内钙离子浓度([Ca2+]i)的升高，从而刺激收缩装置的激活。血管紧张素转换酶II还通过AT1R依赖的表皮生长因子受体(EGFR)的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，可形成具有多个下游分支的信号复合体，包括RAS/MAP激酶途径，用于增加促有丝分裂基因产物的表达。最近，EGFR酪氨酸激酶活性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，肾传入小动脉对血管紧张素Ⅱ的收缩反应涉及酪氨酸激酶(S)，包括EGFR酪氨酸激酶，这一过程参与了[Ca~(2+)]i反应。这项工作将解决这样的假设，即血管紧张素转换酶Ⅱ诱导的肾小球微血管平滑肌细胞(PVSMCs)的收缩涉及AT1R介导的复杂信号网络的启动，其中包括H_2O_2介导的Src家族激酶(S)的激活和随后的EGFR反式激活。我们还将研究PLC(1)和PLC(1)在血管紧张素转换酶诱导的信号事件中的作用，这些信号事件对多肽产生特征的双相[钙]i反应。我们进一步假设，在促有丝分裂状态(如血管紧张素转换酶诱导的高血压)中上调EGFR依赖的通路将促进对血管紧张素转换酶的夸大收缩反应。这些假设的有效性将通过解决下列特定目标来检验：1)确定Src家族激酶在Angii诱导的PVSMCs的信号和收缩反应中的作用；2)确定H_2O_2在Angii诱导的收缩信号中的作用；3)确定经典的AT1R/PLC(1/IP3依赖的钙动员事件是否是EGFR反式激活的先决条件)；4)确定PLC(1)是否在PVSMC对Angii的收缩反应中发挥作用，以及这种作用是否由于EGFR的反式激活而发生；以及5)评估AT1R-EGFR信号通路的慢性激活促进肾小球血管收缩前反应增强的假设。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。通过揭示PVSMCs中特异性酪氨酸激酶的活性及其在血管紧张素Ⅱ收缩反应中的作用，该项目的成功完成将促进我们对生理和病理生理条件下肾小球前微血管张力的调节的理解，肾小球微血管张力是外周阻力、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。这项工作正在探索血管紧张素II快速收缩肾脏微血管中的肌肉细胞的机制，重点放在以前被认为只发生在发育较慢的生长反应中的过程。该项目的完成将促进我们对血管紧张素II对健康和疾病中肾功能和血压调节的理解。

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.