Mechanosensation and the basis of myogenic tone development

在动态条件下，动脉张力受到多种刺激的调节，包括神经元活动、组织代谢和血管内压。一个世纪前，贝利斯首次推测动脉可以对血压的变化做出反应。这种“肌源性反应”可在多种血管床上观察到，并依赖于平滑肌[Ca~(2+)]升高。压力升高可通过以下两种途径增加平滑肌细胞内钙离子浓度：1)通过动脉去极化驱动的钙通道活性；2)肌浆网(SR)内钙释放。虽然过去的研究已经弄清楚了哪些离子通道和离子储存库参与了这一过程，但关于平滑肌细胞实际上是如何“感觉”血管内压力以促进胞浆[Ca~(2+)]升高的问题仍然存在。为了与这个关键问题保持一致，NSERC的这项建议将解决黏附复合蛋白及其下游信号通路是否使平滑肌细胞能够感知机械力并提高胞浆[Ca~(2+)]。在接下来的5年里，申请者将考察以下角色： 1.整合素受体对细胞内钙动力学和肌源性张力的影响； 2.粘着斑激酶(FAK)、Src酪氨酸激酶和磷脂酶C-γ(Plcy1)对钙离子动力学和肌源性张力的影响。 我们的实验方法将综合使用：1)血管肌图评估血管功能；2)蛋白质分析确定信号蛋白的磷酸化状态；3)共聚焦显微镜测量钙动力学；4)膜片钳电生理学监测离子通道活性。整合素受体和下游信号蛋白将使用现有的药理学工具或siRNA方法进行主动操纵。我们对压力敏感和黏附复合体蛋白的关注将促进对肌源性反应及其对心血管功能的生物学意义的更深层次的机制评价。

Under dynamic conditions, arterial tone is regulated by multiple stimuli including neuronal activity, tissue metabolism and intravascular pressure. A century ago, Bayliss first surmised that arteries could respond to changes in blood pressure. The "myogenic response" is observable in a variety of vascular beds and is dependent upon a rise in smooth muscle [Ca2+]. Elevated pressure augments smooth muscle [Ca2+] by augmenting: 1) Ca2+ channel activity through a mechanism driven by arterial depolarization; and 2) internal Ca2+ release from the sarcoplasmic reticulum (SR). While past studies have clarified which ion channels and stores participate, questions remains as to how smooth muscle cells actually "sense" intravascular pressure to facilitate the cytosolic [Ca2+] rise. In keeping with this key question, this NSERC proposal will address whether adhesion complex proteins and their downstream signaling pathways enable smooth muscle cells to sense mechanical forces and elevate cytosolic [Ca2+]. Over the next 5 years, the applicant will examine the role of: 1. Integrin receptors on cytosolic Ca2+ dynamics and myogenic tone; and 2. Focal adhesion kinase (FAK), Src tyrosine kinases and phospholipase C gamma (PLCy1) on Ca2+ dynamics and myogenic tone. Our experimental approach will synergistically employ: 1) vessel myography to assess vessel function; 2) western analysis to determine the phosphorylation state of signaling proteins; 3) confocal microscopy to measure Ca2+ dynamics; and 4) patch clamp electrophysiology to monitor ion channel activity. Integrin receptors and downstream signaling proteins will be actively manipulated using established pharmacological tools or a siRNA approach. Our focus on pressure-sensing and adhesion complex proteins will foster a deeper mechanistic appreciation of the myogenic response and its biological significance to cardiovascular function.