Cyclic adenosine-diphosphate-ribose (cADPr) is a calcium-mobilizing second messenger that was first discovered and characterize in sea urchin eggs, but which has since been shown to modulate calcium homeostasis in a wide range of mammalian tissue types. cADPr appears to regulate release from intracellular ryanodine-sensitive calcium stores. However, the mechanism by which cADPr regulates intracellular calcium release is unknown. Moreover, although cADPr-induced Ca2+ release has been shown to be modulated by Ca2+ and other agents known to modulate ryanodine receptor-channels (RyRCs), the mechanism by which they do this is unclear. Intracellular calcium signals control a diverse assortment of processes in a wide range of cells. The recognition that cADPr may participate in calcium signaling in many of these cell types reinforces the need for understanding ho the pathway targeted by this second messenger operates. The long-term goal of this project is to elucidate the mechanism by which cADP regulates calcium release and to understand how modulators of this process exert their action. We have shown that cADPr is strongly [Ca2+]-dependent. Indeed, it is essentially inactive at normal resting [Ca2+]s. Because of this calcium dependence, it is essential to have control over the free [Ca2+] in assays of cADPr activity. Toward this goal, we will assess the ability of cADP to release Ca2+ from microsomes under conditions where the [Ca2+] is tightly controlled. We will examine how the free [Ca2+] can modulate this release, and assess its site of action. We will assess whether modulation by calcium alters cADPr binding. Using similar techniques we will examine how modulators of cADP release, such as caffeine and ryanodine, exert their action. Finally, we will study the response in whole eggs to photolysis of caged cADPr and caged Ca2+ t explore the spatial and kinetic relation between the Ca2+ release triggered by these two agents.
