Physiological Regulation of MLCK in Intact Arteries

描述（申请人提供）：肌球蛋白轻链激酶（MLCK）和肌球蛋白轻链磷酸酶（MLCP）是血管平滑肌跨桥循环和力产生的主要调节因子。拟议研究的总体目标是获得关于这些分子（特别是MLCK）在控制正常功能的动脉收缩中的作用的新信息，以及盐依赖性高血压模型（DOCA-盐模型，醋酸脱氧皮质酮和高饮食NaCl摄入量）。通过使用表达光学（FRET）MLCK活性传感器的（转基因）“生物传感器”小鼠，将实现对分离的动脉和活动物动脉（即体内）中MLCK活性的直接检查。将通过定量肌球蛋白磷酸酶靶向亚基（MYPT 1）的苏氨酸-855磷酸化来研究离体动脉中MLCP的调节。在离体动脉中的初始荧光成像研究（目的1和2）将揭示MLCK的激活和MLCP的调节与1）肌源性张力和2）已知在高血压中重要的某些G蛋白偶联受体（GPCR）有关。肌源性张力（MT）是一种关键的平滑肌功能，参与维持动脉压，并在盐诱导的高血压的初始阶段组织过度灌注的反应。因此，目的1是量化MLCK的动态和长期（小时）激活以及MLCP的调节，因为在离体动脉中压力在10至150 mm Hg范围内变化。目的2是定量MLCK激活和MLCP抑制，这是由两类关键的GPCR完成的：1）主要与Gq/11偶联的GPCR，以及2）也与G12/13强偶联的GPCR。后者特别与盐诱导的高血压有关，除了激活MLCK外，还可能利用MLCP的强抑制作用。MT对GPCR诱导的信号传导的影响也将被研究，因为新的数据表明它以尚未完全理解的方式影响GPCR的收缩信号传导。目标3将建立在离体动脉中获得的知识基础上，但将利用麻醉生物传感器动物中动脉的体内成像（即活体FRET显微镜）来量化MLCK在DOCA盐高血压中发生的血管收缩增加中的作用。在这最后的目标，两个当前，竞争，假设将进行审查：1）DOCA-盐高血压重要地由通过G12/13偶联的GPCR起作用的循环因子维持，因此涉及MLCP的强烈抑制，而不是MLCK的唯一激活，盐依赖性高血压主要与内源性钠泵配体有关（利钠因子）通过增加[Ca 2 +]收缩平滑肌，因此主要通过MLCK发挥作用，而不是抑制MLCP。总结：该研究旨在提供对正常和高血压动脉中MLCK和MLCP的激活和调节的详细，定量，动态描述，以响应生理刺激，包括跨壁压和GPCR信号。这将提供第一个直接的证据，从动脉在活的动物，MLCK在盐诱导的高血压的作用。 公共卫生相关性：拟议的研究旨在提供有关酶（肌球蛋白轻链激酶，MLCK）的活性和调节的基本信息，该酶对正常生理学和高血压（高血压）中的动脉收缩至关重要。动脉以收缩状态存在，以维持血压。收缩的量根据神经系统和激素的活动迅速变化。这项研究将利用盐诱导的高血压小鼠模型，提供MLCK在高血压中作用的具体新信息。

DESCRIPTION (provided by applicant): Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) are the major regulators of cross-bridge cycling and force generation in vascular smooth muscle. The overall goal of the proposed research is to gain new information on the role of these molecules (particularly MLCK) in controlling arterial contraction in normal function, and in a model of salt dependent hypertension (DOCA-salt model, deoxycorticosterone acetate and high dietary NaCl intake). Direct examination of MLCK activity in isolated arteries and in the arteries of living animals (i.e. in vivo) will be achieved through the use of (transgenic) 'biosensor' mice that express an optical (FRET) MLCK activity sensor. Regulation of MLCP in isolated arteries will be studied by quantifying threonine-855 phosphorylation of myosin phosphatase targeting subunit (MYPT1). Initial fluorescence imaging studies in isolated arteries (Aims 1 & 2) will reveal the activation of MLCK and regulation of MLCP in relation to 1) myogenic tone and 2) certain G-protein coupled receptors (GPCR) that are known to be important in hypertension. Myogenic tone (MT) is a key smooth muscle function that is involved in maintenance of arterial pressure, and in the response to tissue over-perfusion in initial stages of salt-induced hypertension. Therefore, Aim 1 is to quantify the dynamic and long-term (hours) activation of MLCK and regulation of MLCP as pressure is changed over the range of 10 to 150 mm Hg in isolated arteries. Aim 2 is to quantify MLCK activation, and MLCP inhibition, accomplished by two key classes of GPCR: 1) those coupled primarily to Gq/11, and 2) those also coupled strongly to G12/13. The latter have been implicated particularly in salt-induced hypertension and may utilize strong inhibition of MLCP, in addition to activation of MLCK. The influence of MT on GPCR induced signaling will also be studied since new data indicates that it affects contractile signaling of GPCR in ways not yet fully appreciated. Aim 3 will build on the knowledge gained in the isolated arteries , but will utilize in vivo imaging (i.e. intravital FRET microscopy) of arteries in anesthetized biosensor animals to quantify the role of MLCK in the increased vasoconstriction that occurs in DOCA-salt hypertension. In this final Aim, two current, competing, hypotheses will be examined: 1) that DOCA-salt hypertension is importantly maintained by circulating factors acting through G12/13 coupled GPCR and therefore involves strong inhibition of MLCP, rather than exclusive activation of MLCK, and 2) that salt-dependent hypertension involves mainly endogenous Na+ pump ligands (natriuretic factors) that contract smooth muscle by increasing [Ca2+] and thus act mainly through MLCK, rather than inhibition of MLCP. Summary: The research is intended to provide a detailed, quantitative, dynamic description of the activation and regulation of MLCK and MLCP in normal and hypertensive arteries in response to physiological stimuli, including transmural pressure and GPCR signaling. It will provide the first direct evidence, from arteries in the living animal, on the role of MLCK in salt-induced hypertension. PUBLIC HEALTH RELEVANCE: The proposed research is intended to provide basic information on the activity and regulation of an enzyme (myosin light chain kinase, MLCK) that is critical to contraction of arteries, both in normal physiology and in high blood pressure (hypertension). Arteries exist in a contracted state in order to maintain blood pressure. The amount of contraction changes rapidly in response to activity of the nervous system and hormones. This research will utilize a mouse model of salt-induced hypertension to provide specific new information on the role of MLCK in high blood pressure.