The overall objective of the proposed research is to define the role of cyclic ADP-ribose (cADPR) in the regulation of calcium homeostasis in NG108 cells, a neuronal cell line. cADPR is a naturally occurring nucleotide found to be a potent mediator of calcium release from intracellular stores. The specific aims of the proposal are: (1) to characterize intracellular calcium pools in NG 108 cells, (2) to characterize the cADPR synthetic and degradative enzymes in NG108 cells, (3) to characterize the cADPR binding protein(s) in NG108 cells, and (4) to examine the regulation of the endogenous level of cADPR in NG108 cells. The intracellular pools of calcium in NG108 cells will be probed using detergent permeabilized cells or purified microsomes and characterized in terms of the ability of cADPR, caffeine and inositol trisphosphate to release stored calcium. The characterization of the proteins that synthesize, degrade and bind cADPR will be achieved through studies on their subcellular localization, regulation, and purification. The regulation of the intracellular concentration of cADPR will be examined by measuring cADPR under a variety of conditions known to raise intracellular calcium. The regulation of the intracellular concentration of calcium is a critical process in all cells . In neuronal cells, changes in calcium play an important role in a number of neuronal processes including neurotransmitter release, changes in the cytoskeleton, gene expression and a number of metabolic events. Recent evidence suggests that cADPR may be the endogenous modulator of calcium-induced calcium release and act through a mechanism similar to caffeine. Caffeine is a powerful stimulant of the central nervous system, and probably because of this action, is one of the most widely used drugs in the world. The mechanism by which caffeine stimulates the central nervous system is not completely understood. Since cADPR appears to be the endogenous modulator of calcium release from caffeine-sensitive stores, a systematic study of cADPR function and metabolism in neuronal cells should provide useful information on stimulation of the central nervous system.