Mechanisms of arterial myoendothelial feedback: regulation of eNOS and role of connexins

项目摘要/摘要 交感神经刺激或肾上腺素能激动剂引起的动脉收缩引起负反馈 由内皮细胞介导的减弱收缩的反应，称为肌内皮细胞反馈。这 反馈可能有助于抵消代谢综合征中可能出现的过度交感刺激， 高血压或心力衰竭。肌内皮反馈的机制始于平滑肌产生的IP3通过连接平滑肌和内皮细胞的缝隙连接通道 (称为：肌内皮细胞缝隙连接)和刺激附近肌浆网上的IP3受体 (Sr)内皮细胞。这会导致SR释放钙，从而导致eNOS激活和一氧化氮 (NO∙)的产生和钙离子的刺激 -激活钾(IKCa)通道，使超极化 血管内皮细胞，然后是通过传导的平滑肌细胞。无∙和传导超极化都会导致 平滑的肌肉放松。虽然内皮细胞钙升高会激活eNOS，但我们在之前的一项研究中发现 ENOS在Ser1176上被磷酸化，这也增加了eNOS的活性(Looft-Wilson等人，Vascul)。 药水。2013年58：112-7]。鉴于已知的肌内皮细胞反馈通路， ENOS的磷酸化机制并不明显，因此，本研究的目的是揭示这一点 路径。我们试图确定负责磷酸化的激酶/S(AKT、AMPK、PKA和/或CaMKII) ENOS(在S1176以及S632、S614和T494)和涉及的信令事件(IKCa和TRPV4 通过使用药物阻滞剂和免疫印迹(具有近红外信号检测)并通过测量功能反应，包括直径和NO∙水平(使用 分离的动脉压力肌图和无∙电极)，在完整的动脉。确定相关性有多大 肌内皮细胞的反馈是根据体内的血流水平，我们将测量血流的相对贡献 交感神经刺激通过神经刺激和管腔血流激活eNOS。 最后，我们将确定哪些缝隙连接蛋白(Cx43、Cx40、Cx37)与肌内皮细胞有关 在培养中使用siRNA处理分离的动脉进行交流。这项研究将回答重要的问题 关于这一调节动脉直径和血管直径的血管信号通路的机制的疑问 压力，以及反对交感神经过度兴奋的过度收缩。该项目还将公开 优秀的本科生基础生理学研究，包括实践经验，两者都 常用的生化技术(免疫印迹)和高级生理测量(分离的 动脉肌造影术和培养、神经刺激)。目标将是让每个学生完成一个人 项目，制作一张海报在实验生物学上展示，并为出版的手稿做出贡献。我的 实验室在这种学生培训和成就模式方面有着很好的记录。

Project Summary/Abstract Artery contraction, induced by sympathetic stimulation or adrenergic agonists, provokes a negative feedback response mediated by the endothelium that attenuates the contraction, called myoendothelial feedback. This feedback may serve to counteract excessive sympathetic stimulation which can occur in metabolic syndrome, hypertension, or heart failure. The mechanism of myoendothelial feedback begins with smooth muscle-generated IP3 passing through gap junction channels that connect the smooth muscle and endothelial cells (termed: myoendothelial gap junctions) and stimulating IP3 receptors on the nearby sarcoplasmic reticulum (SR) of endothelial cells. This leads to SR release of calcium, which results in eNOS activation and nitric oxide (NO∙) production, and stimulation of Ca++ -activated potassium (IKCa) channels, which hyperpolarizes endothelial cells, then smooth muscle cells via conduction. Both NO∙ and conducted hyperpolarization cause smooth muscle relaxation. While elevated endothelial calcium activates eNOS, we found in a previous study that eNOS is phosphorylated on Ser 1176, which also increases eNOS activity (Looft-Wilson et al., Vascul. Pharmacol. 58:112-7, 2013]. Given what is known about the myoendothelial feedback pathway, the mechanism for eNOS phosphorylation is not obvious, and it is, therefore, the goal of this study to uncover this pathway. We seek to identify the kinase/s (akt, AMPK, PKA, and/or CAMKII) responsible for phosphorylating eNOS (at S1176, as well as S632, S614, and T494), and the signaling events involved (IKCa and TRPV4 channels) during myoendothelial feedback by using pharmacological blockade and immunoblotting (with near-infrared signal detection) and by measuring the functional response, including diameter and NO∙ levels (using isolated artery pressure myography and NO∙ electrode), in intact arteries. To determine how relevant myoendothelial feedback is under in vivo levels of blood flow, we will measure the relative contribution of flow and sympathetic stimulation to eNOS activation in isolated arteries with nerve stimulation and luminal flow. Finally, we will determine which gap junction proteins (Cx43, Cx40, Cx37) are involved in myoendothelial communication using siRNA treatment of isolated arteries in culture. This study will answer important questions about the mechanism of this vascular signaling pathway essential for regulating arterial diameter and pressure, and for opposing excessive constriction with sympathetic hyperactivity. This project will also expose exceptional undergraduates to basic physiological research, including hands-on experience with both commonly used biochemical techniques (immunoblotting) and advanced physiological measurements (isolated arterial myography and culture, nerve stimulation). The goal will be for each student to complete an individual project, construct a poster to present at Experimental Biology, and contribute to a published manuscript. My laboratory has a strong track record for this model of student training and achievement.