New roles of Src tyrosine kinases in vascular tone

描述(申请人提供)：血管张力由多种信号控制，包括有效的血管收缩药和血栓形成药，如5-羟色胺(5-羟色胺)和血栓素A2(TXA2)。5-羟色胺和血栓素A2受体在血管生理学和病理生理学中的关键作用被同时使用5-羟色胺和血栓素A2受体拮抗剂的抗血栓模型所支持。因此，这项研究计划的长期目标是确定动脉、冠状动脉和脑动脉等血栓形成血管的平滑肌细胞中TXA2和5-HT2A受体信号级联的机制和蛋白质。SRC酪氨酸激酶(经典的细胞迁移和分化调节因子)正在成为血管收缩的新信号，因为它们的抑制作用减弱了血管紧张素(Ang11)和5-羟色胺(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，5-HTAng11和苯肾上腺素(Phe)-Src酪氨酸激酶收缩通路的一个机制是抑制人冠状动脉和大鼠主动脉的K+通道。我们现在将检验这一假说，即Src酪氨酸激酶可能是5-HT和TXA2诱导的收缩的必经步骤，可能根据被激活的膜受体而不同地刺激不同的信号级联，因此可能位于特殊的膜间隔中，与其信号伙伴形成大分子复合体。我们用大鼠主动脉的初步数据表明：1)5-羟色胺和血栓素A_2诱导的收缩对Src似乎是绝对必需的，但对苯丙氨酸诱导的收缩不是；2)5-羟色胺诱导的收缩可能是c-Src激活的结果；3)c-Src和5-羟色胺受体可能排列在小窝中，形成一个大分子信号复合体。在这个方案中，我们将主要使用来自大鼠主动脉的天然组织和多学科方法来确定：1)Src激活是否是5-HT-、TXA2(U46619)-和Phe诱导的血管收缩的必需步骤，并研究下游通路；2)血管Src酪氨酸激酶的身份、它们在单个细胞中的定位以及它们对5-HT、TXA2和Phe的反应活性；3)5-HT-、TXA2和Phe-Src-收缩通路的分子机制，以及它们在大分子复合体中的潜在组织；4)受体-Src复合体在脂筏中的定位和激动剂刺激后可能的重组；5)静息状态下和5-羟色胺收缩血管后的c-Src亚蛋白质组。阐明刺激5-羟色胺和TXA2的主要步骤有助于设计控制心血管疾病的新的治疗方法。

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.