New roles of Src tyrosine kinases in vascular tone

描述（由申请人提供）：血管张力由多种信号控制，包括有效的血管收缩剂和血栓形成剂，如5-羟雷丁胺（5-HT）和血栓素A2 （TXA2）。使用5-HT和TXA2受体拮抗剂的抗血栓模型支持5-HT和TXA2受体在血管生理和病理生理中的关键作用。因此，本研究项目的长期目标是确定血栓发作血管（如主动脉、冠状动脉和脑动脉）平滑肌细胞中TXA2和5-HT2A受体信号级联反应的机制和蛋白质。Src酪氨酸激酶（细胞迁移和分化的经典调节因子）正在成为血管收缩的新信号，因为它们的抑制作用减少了血管紧张素(Angll)-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5-HTAngll-和Phenylephrine (Phe)- src酪氨酸激酶收缩途径中的一种机制是在人冠状动脉和大鼠主动脉中抑制K+通道。我们现在将验证以下假设：Src酪氨酸激酶可能是5-HT和txa2诱导的收缩的必要步骤，可能根据被激活的膜受体不同地刺激不同的信号级联反应，因此，可能位于与信号伙伴形成大分子复合物的特殊膜室中。我们使用大鼠主动脉的初步数据表明：1)Src似乎是5-HT-和txa2诱导的收缩的绝对需要，而不是phe诱导的收缩；2) 5- ht诱导的收缩可能是c-Src活化所致；3) c-Src和5-HT受体可能组织在小泡中形成大分子信号复合物。在本研究中，我们将使用主要来自大鼠主动脉的天然组织和多学科方法来实现以下具体目的：1)Src激活是否是5-HT-、TXA2 (U46619)-和ph诱导的血管收缩的必要步骤，并研究下游途径；2)血管Src酪氨酸激酶的身份、在单细胞中的定位及其对5-HT、TXA2和Phe的反应活性；3) 5-HT-、TXA2和ph - src收缩通路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以及激动剂刺激下可能的重组；5)静息状态和5- ht血管收缩后的c-Src亚蛋白质组。阐明5-HT和TXA2平滑肌刺激的主要步骤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.