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。这项研究的总体目标是获得关于这些分子(特别是MLCK)在正常功能和盐依赖高血压模型(DOCA-盐模型、醋酸脱氧皮质酮和高食盐摄入量)控制动脉收缩中所起作用的新信息。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，将实现对分离动脉和活体动物动脉中MLCK活性的直接检测。通过量化肌球蛋白磷酸酶靶向亚基(MYPT1)的苏氨酸-855磷酸化来研究MLCP在分离动脉中的调节。对分离动脉的初步荧光成像研究(AIMS 1和2)将揭示MLCK的激活和MLCP的调节与1)肌源性张力和2)某些已知在高血压中起重要作用的G蛋白偶联受体(GPCR)有关。肌张力(MT)是一种关键的平滑肌功能，它参与维持动脉压，并在盐诱导高血压的早期阶段对组织过度灌流做出反应。因此，目标1是量化MLCK的动态和长期(小时)激活，以及当压力在10-150毫米汞范围内变化时对MLCP的调节。目的2是为了定量MLCK的激活和MLCP的抑制，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)与G12/13强偶联的GPCR。后者尤其与盐诱导的高血压有关，除了MLCK的激活外，可能还利用MLCP的强烈抑制。我们还将研究MT对GPCR诱导的信号转导的影响，因为新的数据表明，MT对GPCRs收缩信号的影响方式尚未完全被认识。目的3将建立在分离动脉获得的知识的基础上，但将利用麻醉生物传感器动物的动脉的活体成像(即活体FRET显微镜)来量化MLCK在DOCA-盐性高血压发生的血管收缩增加中的作用。在这个最终目标中，将检验两个目前相互竞争的假说：1)DOCA-盐高血压是由通过G12/13偶联GPCR作用的循环因子维持的，因此涉及对MLCP的强烈抑制，而不是唯一地激活MLCK；2)盐依赖型高血压主要涉及内源性钠泵配体(利钠因子)，它通过增加[钙]来收缩平滑肌，因此主要通过MLCK起作用，而不是抑制MLCP。摘要：本研究旨在提供一个详细的、定量的、动态的描述MLCK和MLCP在正常动脉和高血压动脉对生理刺激的反应的激活和调节，包括跨壁压力和GPCR信号。这将提供来自活体动物动脉的第一个关于MLCK在盐诱导的高血压中的作用的直接证据。 公共卫生相关性：这项拟议的研究旨在提供一种酶(肌球蛋白轻链激酶，MLCK)的活性和调节的基本信息，这种酶在正常生理和高血压(高血压)中对动脉收缩至关重要。动脉处于收缩状态是为了维持血压。收缩的量会随着神经系统和荷尔蒙的活动而迅速变化。这项研究将利用盐诱导高血压的小鼠模型来提供关于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.