Characterization of Ap4A Induced Release of Nitric Oxide From Endothelial Cells

腺苷化二核苷酸Ap4A （P1， p4 -二腺苷5'-四磷酸）被称为“警报器”，表明它是由细胞在应对代谢挑战和应激时合成的，并作为其他细胞的激素信号。具体来说，Ap4A储存在血小板的致密颗粒中，当释放时，诱导周围内皮细胞释放一氧化氮（NO），这是一种重要的血管扩张剂。本项目的目的是明确Ap4A在靶内皮细胞中的作用生化机制。将解决两个基本问题。(1)结合Ap4A诱导NO释放的受体有哪些？(2)哪些信号通路和细胞内介质在受体结合时被激活？目前的假设是细胞外Ap4A与腺苷化二核苷酸特异性的膜受体结合。细胞内钙的短暂升高，由受体激活触发，促进钙与钙调素的结合，钙调素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化二核苷酸和嘌呤受体拮抗剂和激动剂对Ap4A诱导NO释放的作用来表征。合理的药物设计将用于合成Ap4A的类似物。这些类似物将有助于深入了解受体结合位点的分子结构，并将加强设计针对该受体的新型激动剂和拮抗剂的努力。细胞内钙在信号传导中的作用将在细胞群和单个细胞中进行研究。特异性抑制剂将用于确定肌醇磷酸激活钙释放和钙调素参与NO合成的可能作用。这一重要信号转导级联的生化表征将为腺苷化二核苷酸如何作为重要的细胞外血管调节剂提供基本的新理解。

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.