Cerebrovascular Myosin Light Chain Phosphorylation in Fetus, Newborn, and Adult

胎儿、新生儿和成人的脑血管肌球蛋白轻链磷酸化

• 批准号: 8448654
• 负责人: William J. Pearce
• 金额: $ 28.1万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8448654
• 关键词: Actins; Address; Adult; Age; Antihypertensive Agents; Arteries; Asthma; Birth; Blood Pressure; Blood Vessels; Blood flow; Brain; Calcium; Cardiovascular system; Cells; Cerebrovascular Circulation; Cerebrum; Characteristics; Clinical; Clinical Management; Colitis; Contractile Proteins; Custom; Data; Development; Diagnosis; Disease; Enzymatic Biochemistry; Equilibrium; Exhibits; Fetus; Hand; Homeostasis; In Situ; Individual; Infant; Injury; Ischemic-Hypoxic Encephalopathy; Kinetics; Laboratories; Lead; Life; Measurement; Measures; Methods; Microfilaments; Muscle; Myosin Heavy Chains; Myosin Light Chain Kinase; Myosin Light Chains; Myosin Regulatory Light Chains; Neonatal; Newborn Infant; Outcome; Pattern; Phosphorylation; Preparation; Protein Isoforms; Proteins; Regulation; Relative (related person); Reliance; Research; Resolution; Risk; Sepsis; Sheep; Smooth Muscle; Source; Structure; System; Techniques; Thick Filament; Thin Filament; Time; alpha Actin; base; cell preparation; cerebral artery; cerebrovascular; design; fetal; improved; in vivo; intraventricular hemorrhage; middle cerebral artery; millisecond; myosin phosphatase; neonate; older patient; patient population; phosphatase inhibitor; postnatal; programs; protein expression; protein protein interaction; research study; response; tool

DESCRIPTION (provided by applicant): Cardiovascular instability is one of the most difficult challenges in clinical management of pre-term infants due to unique neonatal patterns of cardiovascular reactivity, particularly in the cerebral circulation. Compared to adult arteries, immature cerebral arteries exhibit greater reliance on Ca++ influx than on intracellular Ca++ release for initiation of contraction, but also exhibit markedly enhanced myofilament Ca++ sensitivity. One possible explanation for this enhanced fetal myofilament Ca++ sensitivity is that phosphorylation of regulatory myosin light chain (MLC20), a key contractile protein, is upregulated in immature cerebral arteries. Previous attempts to study phosphorylation of MLC20 in situ have been limited by technical barriers, including the inability to measure the rapid time course of MLC20 phosphorylation in intact arteries. Methods recently developed in the applicant's laboratory have surmounted these problems. A custom designed apparatus now can simultaneously measure cytosolic Ca++ transients and MLC20 phosphorylation with millisecond resolution in intact arteries in a cuvette small enough to accommodate fetal cerebral arteries. Protein expression systems are now available that can produce single isoforms of MLC20 in quantities suitable for kinetic measurements. New confocal colocalization methods are capable of resolving interactions among multiple different contractile proteins in these small arteries. Based on these exciting new methods, this proposal was designed to explore the main hypothesis that myofilament Ca++ sensitivity is upregulated in immature compared to mature cerebral arteries due to an increased velocity of MLC20 phosphorylation. A corollary hypothesis supported by strong preliminary data predicts that the ultra-structural organization of MLC20 within cerebrovascular smooth muscle is an important determinant of its ability to serve as a substrate for phosphorylation. To address these ideas, three specific aims have been designed: 1) determine substrate- velocity relations for MLC20 phosphorylation in broken cell preparations in the presence and absence of phosphatase inhibitors with age-specific sources of purified ovine MLC20 and synthesized single isoforms of Non-Muscle and Smooth-Muscle MLC20; 2) determine the velocities of MLC20 phosphorylation in intact cell preparations in the presence and absence of phosphatase inhibitors, in response to both spontaneous and standardized cytosolic Ca++ transients; and 3) determine the abundance, distribution, and co-localization of MLC20 in relation to myosin light chain kinase, myosin light chain phosphatase, smooth muscle 1-actin, and myosin heavy chain isoforms. These experiments will be conducted in middle cerebral arteries from term fetal lambs, newborn lambs, and non-pregnant adult sheep to define the effects of postnatal age on these regulatory mechanisms. Further understanding of these mechanisms offers potential to refine clinical strategies for management of infants at risk for cerebrovascular injury, as well as those afflicted by numerous other diseases such as asthma, sepsis, and colitis, in which altered rates of MLC20 phosphorylation are a key factor.

描述（由申请方提供）：由于新生儿心血管反应性的独特模式，特别是在脑循环中，心血管不稳定是早产儿临床管理中最困难的挑战之一。与成人动脉相比，未成熟的脑动脉表现出对Ca++内流的依赖性大于对细胞内Ca++释放的依赖性，但也表现出明显增强的肌丝Ca++敏感性。这种增强的胎儿肌丝Ca++敏感性的一个可能的解释是，调节肌球蛋白轻链（MLC 20），一个关键的收缩蛋白，磷酸化在未成熟的脑动脉上调。先前研究MLC 20原位磷酸化的尝试受到技术障碍的限制，包括无法测量完整动脉中MLC 20磷酸化的快速时程。最近在申请人的实验室中开发的方法已经克服了这些问题。定制设计的装置现在可以同时测量细胞溶质Ca++瞬变和MLC 20磷酸化，在一个小到足以容纳胎儿脑动脉的比色皿中的完整动脉中具有毫秒级分辨率。蛋白质表达系统现在可用于生产适合于动力学测量的MLC 20的单一同种型。新的共聚焦共定位方法能够解决这些小动脉中多种不同收缩蛋白之间的相互作用。基于这些令人兴奋的新方法，该提议旨在探索主要假设，即与成熟脑动脉相比，由于MLC 20磷酸化速度增加，未成熟脑动脉中肌丝Ca++敏感性上调。由强有力的初步数据支持的推论假设预测，脑血管平滑肌内MLC 20的超微结构组织是其作为磷酸化底物的能力的重要决定因素。为了解决这些想法，设计了三个具体的目标：1）在存在和不存在磷酸酶抑制剂的情况下，用年龄特异性来源的纯化的绵羊MLC 20和合成的非肌肉和平滑肌MLC 20的单一同种型确定破碎细胞制备物中MLC 20磷酸化的底物-速度关系; 2）在存在和不存在磷酸酶抑制剂的情况下，测定完整细胞制备物中响应自发和标准化胞质Ca++瞬变的MLC 20磷酸化速度;和3）确定MLC 20相对于肌球蛋白轻链激酶、肌球蛋白轻链磷酸酶、平滑肌1-肌动蛋白和肌球蛋白重链同种型的丰度、分布和共定位。这些实验将在足月胎羊、新生羔羊和非妊娠成年绵羊的大脑中动脉中进行，以确定出生后年龄对这些调节机制的影响。对这些机制的进一步了解提供了改进临床策略的潜力，用于管理有脑血管损伤风险的婴儿，以及那些患有许多其他疾病如哮喘，败血症和结肠炎的婴儿，其中MLC 20磷酸化速率的改变是一个关键因素。