Physiological Regulation of MLCK in Intact Arteries

完整动脉中 MLCK 的生理调节

基本信息

批准号：
8432821
负责人：
Withrow Gil Wier
金额：
$ 35.34万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-22 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8432821
关键词：
3-Dimensional Accounting Acetates Adrenergic Receptor Affect Animals Arteries Biosensor Blood Pressure Caliber Calmodulin Contracts Coupled DOCA Data Deoxycorticosterone Diet Endothelin-1 Enzymes Fluo 4 Fluorescence Resonance Energy Transfer Fluorescent Dyes Fura-2 Future G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Generations Goals Hormones Hour Hypertension Image Intake Knowledge Life Ligands Maintenance Measurement Measures Mediating Mesentery Microscope Microscopy Modeling Molecular Mus Muscle function Myosin Light Chain Kinase Myosin Light Chains Na(+)-K(+)-Exchanging ATPase Natriuretic Factors Nervous system structure Optics Pattern Perfusion Peripheral Resistance Phosphorylation Physiological Physiology Process Receptor Activation Regulation Relative (related person)Reporting Research Rho-associated kinase Role Signal Transduction Smooth Muscle Sodium Chloride Speed Staging Stimulus Testing Threonine Time Tissues Transgenic Organisms Vascular Smooth Muscle argipressin receptor base design fluorescence imaging in vivo mouse model myosin phosphatase pressure public health relevance receptor research study response salt intake sensor vasoconstriction

项目摘要

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.

描述（由申请人提供）：肌球蛋白轻链激酶（MLCK）和肌球蛋白轻链磷酸酶（MLCP）是血管平滑肌中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有关这些分子（特别是 MLCK）在控制正常功能中的动脉收缩以及在盐依赖性高血压模型（DOCA-盐模型、醋酸脱氧皮质酮和高膳食氯化钠）中的作用的新信息。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，可以直接检查离体动脉和活体动物动脉（即体内）的 MLCK 活性。将通过量化肌球蛋白磷酸酶靶向亚基 (MYPT1) 的苏氨酸 855 磷酸化来研究离体动脉中 MLCP 的调节。离体动脉的初始荧光成像研究（目标 1 和 2）将揭示 MLCK 的激活和 MLCP 的调节与 1) 肌原张力和 2) 某些已知在高血压中很重要的 G 蛋白偶联受体 (GPCR) 的关系。肌源性张力（MT）是一种关键的平滑肌功能，参与维持动脉压以及在盐诱发高血压的初始阶段对组织过度灌注的反应。因此，目标 1 是量化当离体动脉中压力在 10 至 150 mm Hg 范围内变化时 MLCK 的动态和长期（小时）激活和 MLCP 的调节。目标 2 是量化 MLCK 激活和 MLCP 抑制，通过两类关键的 GPCR 实现：1) 主要与 Gq/11 偶联的那些，2) 也与 G12/13 强烈偶联的那些。后者尤其与盐诱发的高血压有关，除了激活 MLCK 之外，还可能利用 MLCP 的强抑制作用。还将研究 MT 对 GPCR 诱导信号传导的影响，因为新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。目标 3 将建立在离体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的动脉体内成像（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。在这个最终目标中，将检验两个当前的、相互竞争的假设：1) DOCA-盐高血压主要是通过 G12/13 耦合 GPCR 作用的循环因子维持的，因此涉及 MLCP 的强烈抑制，而不是仅激活 MLCK， 2) 盐依赖性高血压主要涉及内源性 Na+ 泵配体（利尿钠因子），其通过增加 [Ca2+] 收缩平滑肌，因此主要通过 MLCK 发挥作用，而不是抑制MLCP。摘要：该研究旨在提供正常和高血压动脉中 MLCK 和 MLCP 响应生理刺激（包括跨壁压力和 GPCR 信号传导）的激活和调节的详细、定量、动态描述。它将提供来自活体动物动脉的第一个直接证据，证明 MLCK 在盐诱发的高血压中的作用。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

High vascular tone of mouse femoral arteries in vivo is determined by sympathetic nerve activity via α1A- and α1D-adrenoceptor subtypes.

体内小鼠股动脉的高血管张力是由交感神经活动通过α1A-和α1D-肾上腺素受体亚型决定的。

DOI：
发表时间：
2013
期刊：
影响因子：
3.7
作者：
Zacharia, Joseph;Mauban, Joseph R H;Raina, Hema;Fisher, Steven A;Wier, Withrow G
通讯作者：
Wier, Withrow G

Intravital Förster resonance energy transfer imaging reveals elevated [Ca2+]i and enhanced sympathetic tone in femoral arteries of angiotensin II-infused hypertensive biosensor mice.

活体 Förster 共振能量转移成像显示，注射血管紧张素 II 的高血压生物传感器小鼠的股动脉 [Ca2]i 升高，交感张力增强。

DOI：
发表时间：
2013-11-01
期刊：
影响因子：
0
作者：
Wang, Youhua;Chen, Ling;Wier, W Gil;Zhang, Jin
通讯作者：
Zhang, Jin

PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries.

PC-PLC/鞘磷脂合酶活性在小鼠抗性动脉的肌原性张力的发展中起着核心作用。

DOI：
发表时间：
2015-06-15
期刊：
影响因子：
0
作者：
Mauban, Joseph R H;Zacharia, Joseph;Fairfax, Seth;Wier, Withrow Gil
通讯作者：
Wier, Withrow Gil

More in vivo experimentation is needed in cardiovascular physiology.

心血管生理学需要更多的体内实验。

DOI：
发表时间：
2014-07-15
期刊：
影响因子：
0
作者：
Wier; W Gil
通讯作者：
W Gil

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats.

米兰高血压大鼠动脉平滑肌 Ca2 信号传导、血管收缩和肌源性反应性增加。

DOI：
10.1152/ajpheart.00950.2011
发表时间：
2012-02-01
期刊：
American journal of physiology. Heart and circulatory physiology
影响因子：
0
作者：
Cristina I. Linde;E. Karashima;H. Raina;A. Zulian;W. Wier;J. Hamlyn;P. Ferrari;M. Blaustein;V. Golovina
通讯作者：
V. Golovina