Characterization of Ap4A Induced Release of Nitric Oxide From Endothelial Cells

Ap4A 诱导内皮细胞释放一氧化氮的表征

• 批准号: 9816681
• 负责人: Richard Hilderman
• 金额: $ 21.51万
• 依托单位: Clemson University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9816681&HistoricalAwards=false
• 关键词: Characterization; Ap4A; Induced; Release; Nitric

The adenylated dinucleotide Ap4A (P1,P4-diadenosine 5'-teptraphosphate) has been termed an "alarmone" to denote that it is synthesized by cells in response to metabolic challenges and stress and acts as a hormonal signal on other cells. Specifically, Ap4A is stored in the dense granules of platelets and, when released, induces surrounding endothelial cells to release nitric oxide (NO), an important vasodilator. The goal of this project is to define the biochemical mechanism of action of Ap4A in the target endothelial cells. Two fundamental questions will be addressed. (1) What are the receptors that bind Ap4A to induce NO release? (2) Which signaling pathways and intracellular mediators are activated in response to receptor binding? The working hypothesis is that extracellular Ap4A binds to a membrane receptor that is specific for adenylated dinucleotides. A transient elevation of intracellular calcium, triggered by receptor activation, promotes binding of calcium to calmodulin which in turn activates endothelial nitric oxide synthase. The receptor will be characterized by studying the effects of various adenylated dinucleotides and purino-receptor antagonists and agonists of Ap4A induced NO release. Rational drug design will be used to synthesize analogs of Ap4A. These analogs should provide insight into the molecular architecture of the receptor binding site and will enhance efforts to design new agonists and antagonists specific for this receptor. The role of intracellular calcium in signaling will be studied in cell populations and individual cells. Specific inhibitors will be used to determine the possible roles of inositol phosphate activation of calcium release and calmodulin participation in NO synthesis. The biochemical characterization of this important signal transduction cascade will provide fundamental new understanding of how adenylated dinucleotides act as important extracellular vasoregulators.

腺苷化二核苷酸Ap 4A（P1，P4-二腺苷5 '-四磷酸）被称为“alarmone”，以表示它是由细胞响应代谢挑战和应激而合成的，并作为激素信号作用于其他细胞。 具体而言，Ap 4A储存在血小板的致密颗粒中，当释放时，诱导周围的内皮细胞释放一氧化氮（NO），一种重要的血管扩张剂。 本项目的目标是确定Ap 4A在靶内皮细胞中的生化作用机制。将讨论两个基本问题。 (1)什么是结合Ap 4A诱导NO释放的受体？ (2)哪些信号通路和细胞内介质被激活以响应受体结合？ 工作假设是细胞外Ap 4A结合到对腺苷二核苷酸特异的膜受体。 由受体激活触发的细胞内钙的瞬时升高促进钙与钙调蛋白的结合，钙调蛋白反过来激活内皮一氧化氮合酶。 该受体将通过研究各种腺苷二核苷酸和嘌呤受体拮抗剂和激动剂的影响，Ap 4A诱导的NO释放的特点。 合理的药物设计将用于合成Ap 4A的类似物。 这些类似物应提供深入了解受体结合位点的分子结构，并将加强设计这种受体特异性的新激动剂和拮抗剂的努力。 将在细胞群和单个细胞中研究细胞内钙在信号传导中的作用。 特异性抑制剂将被用来确定磷酸肌醇激活钙释放和钙调素参与NO合成的可能作用。 这个重要的信号转导级联的生化特性将提供腺苷二核苷酸如何作为重要的细胞外血管调节剂的基本新的理解。