Physiological Regulation of MLCK in Intact Arteries

完整动脉中 MLCK 的生理调节

• 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
• 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; 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.

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剂。拟议研究的总体目标是获取有关这些分子（尤其是MLCK）在正常功能中控制动脉收缩的作用的新信息，以及在盐依赖性高血压的模型中（DOCA-salt模型，乙酸脱氧乙酸乙酸脱氧乙酸乙酸脱氧甲酸盐和高饮食NACL摄入量）。通过使用（转基因）“生物传感器”小鼠，可以直接检查分离的动脉和活体动物（即体内）中的MLCK活性（即体内）。通过量化肌球蛋白磷酸酶靶向亚基（MYPT1）的苏氨酸-855磷酸化（MYPT1），将研究MLCP的调节。在分离的动脉中的初始荧光成像研究（AIMS 1和2）将揭示MLCK的激活和MLCP的调节，相对于1）肌原构和2）某些G蛋白偶联受体（GPCR）在高血压中很重要。肌原态（MT）是一种关键的平滑肌功能，涉及维持动脉压，以及在盐引起的高血压初始阶段对组织过度灌注的反应。因此，目标1是量化MLCK的动态和长期（小时）激活和MLCP的调节，因为压力在孤立的动脉中的10至150 mm Hg范围内发生了变化。 AIM 2是通过两个关键类别的GPCR来量化MLCK激活和MLCP抑制：1）主要耦合到GQ/11，以及2）那些也与G12/13耦合的抑制作用。后者尤其与盐诱导的高血压有关，除了激活MLCK外，还可以使用强烈的MLCP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理解的方式影响GPCR的收缩信号传导。 AIM 3将建立在孤立的动脉中获得的知识，但将利用麻醉生物传感器动物中动脉的体内成像（即插入式FRET显微镜）来量化MLCK在增加DOCA-SALT高血压中发生的增加血管收缩中的作用。在这一最终目标中，将检查两个当前的竞争，假设：1）通过通过G12/13耦合GPCR作用的循环因素来维持DOCA-SALT高血压，因此，MLCP的强烈抑制，涉及MLCK的强烈抑制，而不是抗盐依赖性的（依赖盐）的特定因素（2）NAIN依赖性（2）+2）通过增加[Ca2+]并主要通过MLCK起作用，而不是抑制MLCP来平滑肌肉。摘要：这项研究旨在提供针对生理刺激的正常和高血压动脉中MLCK和MLCP激活和调节的详细，定量，动态描述，包括透射压力和GPCR信号。它将提供有关MLCK在盐引起的高血压中的作用的第一个直接证据，该证据来自活动物的动脉。