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.
