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。本研究的总体目标是获得这些分子（尤其是MLCK）在正常功能和盐依赖性高血压模型（doca -盐模型、醋酸脱氧皮质酮和高膳食NaCl摄入量）中控制动脉收缩的新信息。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，可以直接检测分离动脉和活体动物（即体内）动脉中的MLCK活性。将通过定量肌球蛋白磷酸酶靶亚基（MYPT1）苏氨酸-855磷酸化来研究离体动脉中MLCP的调控。分离动脉的初步荧光成像研究（目的1和2）将揭示MLCK的激活和MLCP的调节与1)肌原性张力和2)某些已知在高血压中重要的g蛋白偶联受体（GPCR）有关。肌原性张力（MT）是一种关键的平滑肌功能，参与维持动脉压，并在盐性高血压初始阶段对组织过度灌注的反应。因此，目的1是量化在分离动脉中，当压力在10至150 mm Hg范围内变化时，MLCK的动态和长期（小时）激活以及MLCP的调节。目的2是量化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，2)也与G12/13强偶联的。后者尤其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了解的方式影响GPCR的收缩信号传导。目的3将建立在分离动脉中获得的知识的基础上，但将利用麻醉生物传感器动物动脉的体内成像（即活体FRET显微镜）来量化MLCK在doca盐高血压中血管收缩增加中的作用。在最后一篇文章中，我们将检验目前两种相互竞争的假设：1)doca -盐性高血压在很大程度上是由循环因子通过G12/13偶联GPCR维持的，因此涉及对MLCP的强烈抑制，而不是MLCK的唯一激活；2)盐依赖性高血压主要涉及内源性Na+泵配体（利钠因子），它通过增加[Ca2+]收缩平滑肌，因此主要通过MLCK起作用，而不是抑制MLCP。摘要：本研究旨在详细、定量、动态地描述正常和高血压动脉中MLCK和MLCP在生理刺激下的激活和调控，包括跨壁压力和GPCR信号。这将提供第一个直接证据，来自活体动物的动脉，关于MLCK在盐诱导高血压中的作用。

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.