New roles of Src tyrosine kinases in vascular tone

描述（由申请人提供）：血管张力受多种信号控制，包括强效血管收缩剂和血栓形成剂，如5-羟基曲普他明（5-HT）和血栓烷A2（TXA 2）。5-HT和TXA 2受体在血管生理学和病理生理学中的关键作用得到了使用5-HT和TXA 2受体拮抗剂的抗血栓形成模型的支持。因此，这项研究计划的长期目标是确定血栓形成事件的血管（如主动脉、冠状动脉和脑动脉）平滑肌细胞中TXA 2和5-HT 2A受体信号级联的机制和蛋白质。Src酪氨酸激酶（细胞迁移和分化的经典调节剂）作为血管收缩中的新信号出现，因为它们的抑制减少血管紧张素（AngII）和5-HT诱导的血管收缩。然而，Src收缩级联的机制仍然难以捉摸。在这方面，我们最近发现在人冠状动脉和大鼠主动脉中，5-HTAngII-和苯丙氨酸（Phe）-Src酪氨酸激酶收缩途径中的一个机制是K+通道抑制。我们现在将测试的假设，Src酪氨酸激酶可能是强制性的步骤，在5-HT和TXA 2诱导的收缩，可能差异刺激不同的信号级联反应，这取决于膜受体被激活，因此，可能位于专门的膜隔室形成大分子复合物与他们的信号合作伙伴。我们对大鼠主动脉的初步研究结果表明：（1）Src是5-HT和TXA_2诱导的收缩的绝对必需，而不是Phe诱导的收缩的必需;（2）5-HT诱导的收缩可能是c-Src激活的结果;（3）c-Src和5-HT受体可能在细胞膜小窝中形成大分子信号复合物。在本研究中，我们将主要利用来自大鼠髂动脉的天然组织和多学科的方法来进行以下特定目的的研究：1）Src激活是否是5-HT、TXA 2（U46619）和Phe诱导的血管收缩的必要步骤，并研究下游途径; 2）血管Src酪氨酸激酶的特性、它们在单个细胞中的定位以及它们对5-HT、TXA_2和Phe的反应活性; 3）5-HT-、TXA 2和Phe-Src-收缩途径的分子机制及其在大分子复合物中的潜在组织; 4）受体-Src复合物在脂筏中的定位和激动剂刺激后的可能重组; 5）静息条件下和5-HT收缩血管后的c-Src亚蛋白质组。阐明参与5-HT和TXA 2平滑肌刺激的主要步骤应有助于设计新的治疗方法来控制心血管疾病。

DESCRIPTION (provided by applicant): Vascular tone is controlled by a variety of signals including potent vasoconstrictors and thrombotic agents like 5-hydroxytriptamine (5-HT) and Thromboxane A2 (TXA2). The critical role of 5-HT and TXA2 receptors in vascular physiology and pathophysiology is supported by anti-thrombotic models that use both 5-HT- and TXA2-receptor antagonists. Thus, the long-term goal of this research program is to identify the mechanisms and proteins underlying the signaling cascades of TXA2 and 5-HT2A receptors in smooth muscle cells from blood vessels subject to thrombotic episodes, like aorta, coronary, and cerebral arteries. Src tyrosine kinases (classical regulators of cell migration and differentiation) are emerging as new signals in vascular contraction, as their inhibition diminishes Angiotensin (Angll)- and 5-HT-induced vasoconstriction. However, mechanisms of the Src-constricting cascade remained elusive. In this regard, we recently discovered that one mechanism in 5-HTAngll- and Phenylephrine (Phe)-Src tyrosine kinase constricting pathway is K+ channel inhibition in human coronary arteries and rat aorta. We will now test the hypothesis that Src tyrosine kinases may be obligatory steps in 5-HT- and TXA2-induced contraction, may differentially stimulate distinct signaling cascades depending on the membrane receptor being activated, and thus, may be located in specialized membrane compartments forming macromolecular complexes with their signaling partners. Our preliminary data using rat aorta indicate that: 1) Src seems to be an absolute requirement for both 5-HT- and TXA2-induced contractions but not for Phe-induced contraction; 2) 5-HT-induced contraction may result from activation of c-Src; and 3) c-Src and 5-HT receptors may be organized in caveolae forming a macromolecular signaling complex. In this proposal, we will use native tissue mainly from rat aortas and a multidisciplinary approach to perform the following Specific Aims to determine: 1) if Src activation is an obligatory step in 5-HT-, TXA2 (U46619)-, and Phe-induced vasoconstriction, and investigate downstream pathways; 2) the identity of vascular Src tyrosine kinases, their localization in single cells, and their activity in response to 5-HT, TXA2, and Phe; 3) the molecular mechanisms of 5-HT-, TXA2, and Phe-Src-constricting pathways, and their potential organization in macromolecular complexes; 4) the localization of receptor-Src complexes in lipid rafts and possible reorganization upon agonist stimulation; and 5) the c-Src subproteome in resting conditions, and after vasoconstriction with 5-HT. The elucidation of primary steps involved in 5-HT and TXA2 smooth muscle stimulation should help in the design of new therapeutic ways to control cardiovascular disease.