The overall objectives of the proposed research are to gain insight into the mechanism by which cyclic ADP-ribose (cADPR) mobilizes intracellular calcium from intracellular stores and to elucidate the mechanisms by which cADPR metabolism is regulated, cADPR is a naturally occurring metabolite of beta-nicotinamide adenine dinucleotide (NAD) that has been shown to be involved in the release of intracellular calcium in a wide variety of cell types. Mechanistically, cADPR appears to mobilize calcium in a manner similar to caffeine. The specific aims of the proposal are: 1) to identify and characterize cADPR binding proteins, 2) to identify cell surface receptors that signal through the cADPR system, and 3) to characterize the effect of nicotine on cADPR metabolism, cADPR binding proteins will be characterized by photoaffinity labeling techniques using (32p)-8-azido-cADPR and conventional binding methods using (3zp)-3-deaza-cADPR, a high affinity, hydrolysis-resistant cADPR probe. The goal of specific aim 1 is to purify cADPR binding proteins to a state sufficient for obtaining sequence information by mass spectroscopy techniques. In the studies proposed for specific aim 2 the effects of 8-bromo-cADPR (8-Br-cADPR), a cell permeant cADPR antagonist on calcium signaling in cell lines known to possess cell surface receptors that signal through mobilization of intracellular calcium will be examined. We hypothesize that 8-Br-cADPR will attenuate the calcium responses to agonists that stimulate the cADPR pathway and this experimental paradigm will be useful in the identification of signaling systems that involve cADPR. Specific aim 3 will examine the effect of nicotine on the cADPR system. We have shown that nicotine, a pyridine containing alkaloid, is a substrate for the base-exchange reaction of ADP-ribosyl cyclase. As a consequence, the ability of ADP-ribosyl cyclase to generate cADPR is diminished. We hypothesize that nicotine will interfere with the cADPR signaling pathway in intact cells by participating in the base-exchange reaction and inhibiting cADPR production. The proposed studies should provide useful information on the role of cADPR in the regulation of calcium signaling.