Physiological Regulation of MLCK in Intact Arteries

完整动脉中 MLCK 的生理调节

• 批准号: 8235851
• 负责人: Withrow Gil Wier
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-22 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8235851
• 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

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.

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。 公共健康相关性：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，该酶对于正常生理和高血压（高血压）中的动脉收缩至关重要。动脉处于收缩状态以维持血压。收缩量随着神经系统和激素的活动而迅速变化。这项研究将利用盐诱发高血压的小鼠模型来提供有关 MLCK 在高血压中的作用的具体新信息。