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

动脉肌内皮反馈机制：eNOS 的调节和连接蛋白的作用

• 批准号: 10113424
• 负责人: ROBIN C LOOFT-WILSON
• 金额: $ 25.66万
• 依托单位: COLLEGE OF WILLIAM AND MARY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10113424
• 关键词: Mechanisms; arterial; myoendothelial; feedback; regulation

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.

项目总结/文摘