NADPH oxidase signaling, blood pressure sensing and the development of myogenic tone

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，贝利斯首次观察到动脉可以对血压的变化做出反应。这种“肌源性反应”在各种血管床上都有记载，并且依赖于平滑肌[Ca~(2+)]的升高。压力升高通过激活信号转导通路来增加平滑肌[Ca~(2+)]：1)使平滑肌去极化，提高Ca~(2+)通道活性；2)动员内钙储存。虽然一些驱动肌源性张力发育的离子通道已经被发现，但能够实现压力调节的信号级联还没有被发现。目前的工作主要集中在标准的G蛋白偶联机制上，很少有人尝试“在这个信号盒之外”考虑新的分子。这包括NADPH氧化酶(NOX)可以促进其产生的活性氧物种，NOX是已知与机械敏感蛋白相关的酶。 与我们对肌源性反应的长期兴趣一致，NSERC的这项提案将探索血管内压、NOX信号和脑循环中肌源性张力之间的联系。这三个目标将： 1)确定NOX是否促进肌张力。 2)确定NOX是否通过机电耦合促进肌张力。 3)确定NOX是否对机械刺激敏感的离子通道有调节作用。 我们的主要假设是，NOX(尤其是NOX1)通过产生过氧化氢和激活PKC来增强血管平滑肌中TRPC6和/或TRPM4的活性。这种去极化电流的增加将促进压力诱导的去极化，这一事件将提高胞浆[Ca~(2+)]和肌源性张力。我们的实验方法将综合使用：1)血管肌图评估张力、膜电位和胞浆[Ca~(2+)]，2)Q-PCR和免疫组织化学检测NOX的表达和定位，以及3)电生理学来确定离子通道的活性。实验将在从大鼠、NOX1-/-小鼠和人类分离出来的大脑动脉上进行。我们对压力传感和NOX信号的关注将促进对肌源性反应及其对血压和血流调节的生物学意义的更深层次的机械理解。

Under dynamic conditions, arterial tone is regulated by multiple stimuli including neuronal activity, tissue metabolism and intravascular pressure. A century ago, Bayliss first observed that arteries could respond to changes in blood pressure. The “myogenic response” has been documented in various vascular beds and is dependent upon a rise in smooth muscle [Ca2+]. Elevated pressure augments smooth muscle [Ca2+] by activating signal transduction pathways that: 1) depolarize smooth muscle and elevate Ca2+ channel activity; and 2) mobilize internal Ca2+ stores. While some of the ion channels driving myogenic tone development have have been identified, the signaling cascades which enable pressure modulation have not. Present day work typically focuses on standard G-protein coupled mechanisms and few venture “outside this signaling box” to consider novel molecules. This includes reactive oxygen species whose production could be facilitated by NADPH oxidases (NOX), enzymes known to associate with mechanosensitive proteins. Consistent with our long term interests in the myogenic response, this NSERC proposal will explore the linkage between intravascular pressure, NOX signaling and myogenic tone in the cerebral circulation. The three aims will: 1) Determine whether NOX facilitates myogenic tone. 2) Determine whether NOX facilitates myogenic tone via electromechanical coupling. 3) Determine whether NOX modulates ion channels sensitive to mechanical stimuli. Our overarching hypothesis states that NOX (in particular NOX1) augments TRPC6 and/or TRPM4 activity in vascular smooth muscle by generating H2O2 and activating PKC. This increase in depolarizing current will facilitate pressure-induced depolarization, an event that will elevate cytosolic [Ca2+] and myogenic tone. Our experimental approach will synergistically employ: 1) vessel myography to assess tone, membrane potential, and cytosolic [Ca2+], 2) Q-PCR and immunohistochemistry to determine NOX expression and localization; and 3) electrophysiology to ascertain ion channel activity. Experiments will be performed in cerebral arteries isolated from rat, NOX1-/- mice and humans. Our focus on pressure-sensing and NOX signaling will foster a deeper mechanistic appreciation of the myogenic response and its biological significance to blood pressure and blood flow regulation.