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

描述（由申请人提供）：由于心血管反应（尤其是脑循环）独特的新生儿模式，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，未成熟的脑动脉在启动收缩时对 Ca 离子内流的依赖程度大于对细胞内 Ca 离子释放的依赖性，而且还表现出显着增强的肌丝 Ca 敏感性。胎儿肌丝 Ca 敏感性增强的一个可能解释是，调节性肌球蛋白轻链 (MLC20)（一种关键的收缩蛋白）的磷酸化在未成熟的脑动脉中上调。先前研究 MLC20 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 MLC20 磷酸化的快速时间过程。申请人实验室最近开发的方法已经克服了这些问题。现在，定制设计的装置可以在足以容纳胎儿脑动脉的比色皿中以毫秒分辨率同时测量完整动脉中的胞质 Ca 瞬变和 MLC20 磷酸化。现在的蛋白质表达系统可以产生适合动力学测量的数量的 MLC20 单一亚型。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，本提案旨在探索以下主要假设：与成熟脑动脉相比，由于 MLC20 磷酸化速度增加，未成熟脑动脉的肌丝 Ca 敏感性上调。由强有力的初步数据支持的推论假设预测，MLC20 在脑血管平滑肌内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，设计了三个具体目标：1) 在存在或不存在磷酸酶抑制剂的情况下，使用特定年龄来源的纯化绵羊 MLC20 和合成的非肌肉和平滑肌 MLC20 的单一亚型，确定破碎细胞制剂中 MLC20 磷酸化的底物-速度关系； 2) 确定在存在或不存在磷酸酶抑制剂的情况下，完整细胞制剂中 MLC20 磷酸化的速度，以响应自发和标准化的胞质 Ca 瞬变； 3) 确定 MLC20 与肌球蛋白轻链激酶、肌球蛋白轻链磷酸酶、平滑肌 1-肌动蛋白和肌球蛋白重链亚型相关的丰度、分布和共定位。这些实验将在足月胎儿羔羊、新生羔羊和非怀孕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为完善治疗有脑血管损伤风险的婴儿以及患有哮喘、败血症和结肠炎等许多其他疾病的婴儿的临床策略提供了可能，其中MLC20磷酸化率的改变是一个关键因素。

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.