Lipid Mediators of Signal Transduction in Smooth Muscle

平滑肌信号转导的脂质介质

基本信息

批准号：
7391740
负责人：
RICHARD W GROSS
金额：
$ 38万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-07-01 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7391740
关键词：
Ablation Active Sites Acyl CoA Hydrolase Acyl Coenzyme A Acylation Adjuvant Affect Affinity Agonist Animals Argipressin Binding Biochemical Pathway Biology Blood Vessels Blood flow Bradykinin Calcium Caliber Calmodulin Catalysis Catalytic Domain Cells Cellular biology Chemicals Chemistry Cholinergic Agents Complex Condition Constriction procedure Cross-Linking Reagents Cultured Cells Cyclic AMP-Dependent Protein Kinases Eicosanoids Electrospray Ionization Endothelial Cells Endothelium-Dependent Relaxing Factors Engineering Enzymes Epoprostenol Generations Genetic Grant Hormonal Hydrolysis In Vitro Individual Infection Infusion procedures Kinetics Knock-out Knockout Mice Lipase Lipids Mass Spectrum Analysis Measurement Mediating Mesentery Metabolic Metabolic Pathway Metabolic syndrome Methods Microscopy Modeling Molecular Mus Nitric Oxide Pathway Nitric Oxide Synthase Nutrient Oleic Acid Oleic Acids Oxygen PLA2G2A gene Pathway interactions Phenotype Phenylephrine Phospholipase Phospholipase A2 Phosphoproteins Phosphorylation Physiological Physiology Preparation Production Property Prostaglandins I Protein Isoforms Proteins Rate Recombinants Regulation Relaxation Research Personnel Resistance Role Second Messenger Systems Series Shotguns Signal Transduction Site-Directed Mutagenesis Smooth Muscle Smooth Muscle Myocytes Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Stable Isotope Labeling System Testing Tissues Vascular Capacitance Vascular System Vasodilation Viral Physiology base cell type crosslink digital human PLA2G2A protein human PLA2G6 protein in vivo insight interdisciplinary approach knockout animal lipid mediator lipid metabolism mouse Smc1l1 protein mouse Smc1l2 protein mutant programs release of sequestered calcium ion into cytoplasm research study response second messenger vasoconstriction

项目摘要

DESCRIPTION (provided by applicant): Biologic tissues depend on the precise regulation of vascular tone and blood flow to maintain the appropriate delivery of oxygen, nutrients, and hormonal signals. Central to the regulation of vascular tone, capacitance and vascular cell biology are the generation of lipid 2nd messengers of signal transduction such as eicosanoids and lysolipids that collectively orchestrate vascular responses. In most cell types, including endothelial cells and smooth muscle cells, the rate-determining step in the production of lipid 2nd messengers is the activity of intracellular phospholipases. During the current grant interval, we have demonstrated the important role of iPLA2-3 in vascular contraction and relaxation using protein, chemical, pharmacologic and genetic approaches. In the vasculature, iPLA2p is inhibited by calcium-activated calmodulin and we have demonstrated that this inhibition can be reversed by acyl-CoA. Moreover, we have demonstrated that iPLA2p is also an acyl-CoA hydrolase and that iPLA2p forms a stable acyl-enzyme at a second active site distinct from the S465 catalytic site. Collectively, these results integrate phospholipase activation and lipid 2nd messenger generation with cellular lipid metabolism to provide an interactive metabolic network regulating vascular tone and vascular biology. These pathways are modulated by acylation, calcium flux and phosphorylation and converge at the level of the iPLA2p protein itself. Accordingly, in Specific Aim 1, we will first determine the molecular mechanisms regulating calcium-independent phospholipase A2 activity by acyl-CoA. Next, we will determine the chemical and physiologic sequelae of covalent acylation of iPLA2p and explore covalent alterations in iPLA2 that affect the kinetic properties and physiologic function of the enzyme in vascular tissues. Multiple complementary approaches will be pursued including tryptic footprinting and cross-linking of signaling complexes with bifunctional cross-linking reagents with analysis by mass spectrometry. In Specific Aim 2, we will identify the intracellular phospholipases A2 responsible for eicosanoid and lysolipid production in the vascular system using genetically altered mice and mass spectrometric identification of alterations in eicosanoid and lipid messengers produced after agonist stimulation. Shotgun lipidomics will facilitate identification of the role of each phospholipase in generating signaling metabolites and modulating specific lipid metabolic fluxes in vascular cells. In Specific Aim 3, physiologic experiments employing in vitro study of mesenteric resistance vessels will determine the mechanisms through which each phospholipase regulates agonist-induced vasoconstriction and vasodilation. Through this multidisciplinary approach, the roles of intracellular phospholipases in vascular function can be determined from detailed chemical mechanisms to in vivo physiology clarifying the functional significance of each intracellular phospholipase in vascular regulation.

描述（由申请人提供）：生物组织依赖于血管张力和血流的精确调节来维持氧气、营养物质和激素信号的适当输送。血管张力、电容和血管细胞生物学调节的核心是信号转导脂质第二信使的产生，例如类二十烷酸和溶血脂，它们共同协调血管反应。在大多数细胞类型中，包括内皮细胞和平滑肌细胞，脂质第二信使产生的速率决定步骤是细胞内磷脂酶的活性。在当前的资助期间，我们使用蛋白质、化学、药理学和遗传方法证明了 iPLA2-3 在血管收缩和舒张中的重要作用。在脉管系统中，iPLA2p 被钙激活的钙调蛋白抑制，我们已经证明这种抑制可以被酰基辅酶 A 逆转。此外，我们还证明 iPLA2p 也是一种酰基辅酶 A 水解酶，并且 iPLA2p 在不同于 S465 催化位点的第二个活性位点形成稳定的酰基酶。总的来说，这些结果将磷脂酶激活和脂质第二信使生成与细胞脂质代谢结合起来，提供调节血管张力和血管生物学的交互式代谢网络。这些途径受到酰化、钙流和磷酸化的调节，并汇聚在 iPLA2p 蛋白本身的水平。因此，在具体目标 1 中，我们将首先确定酰基辅酶 A 调节钙非依赖性磷脂酶 A2 活性的分子机制。接下来，我们将确定 iPLA2p 共价酰化的化学和生理后遗症，并探索 iPLA2 中影响血管组织中酶的动力学特性和生理功能的共价改变。将寻求多种互补方法，包括胰蛋白酶足迹法、信号复合物与双功能交联试剂的交联以及质谱分析。在具体目标 2 中，我们将使用基因改造小鼠来鉴定负责血管系统中类二十烷酸和溶血脂产生的细胞内磷脂酶 A2，并通过质谱鉴定激动剂刺激后产生的类二十烷酸和脂质信使的变化。鸟枪法脂质组学将有助于识别每种磷脂酶在产生信号代谢物和调节血管细胞中特定脂质代谢通量中的作用。在具体目标 3 中，采用肠系膜阻力血管体外研究的生理实验将确定每种磷脂酶调节激动剂诱导的血管收缩和血管舒张的机制。通过这种多学科方法，可以从详细的化学机制到体内生理学来确定细胞内磷脂酶在血管功能中的作用，从而阐明每种细胞内磷脂酶在血管调节中的功能意义。