Establishment of a quantitative method for determining cyclic ADP-ribose concentration

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Recently, cyclic ADP-ribose, a metabolite from NAD^+, has been shown to be a second messenger for intracellular Ca^<2+> mobilization and to play an essential role in insulin secretion from pancreatic beta-cells. Thus, a quantitative method for determining cyclic ADP-ribose concentration has been required for studying of physiological functions of cyclic ADP-ribose in mammalian cells. The aim of this project was to establish a sensitive method for determining cyclic ADP-ribose concentration.In this project,(1)We have purified ADP-ribosyl cyclase, which converts NAD^+ to cyclic ADP-ribose, from Aplysia kurodai.(2)We synthesized cyclic ADP-ribose from NAD^+ using the purified Aplysia ADP-ribosyl cyclase and purified cyclic ADP-ribose by HPLC.(3)Using cyclic ADP-ribose synthesized by Aplysia ADP-ribosyl cyclase as a standard, we have developed a quantitative method for determining cyclic ADP-ribose concentration by monitoring the release of Ca^<2+> from mammalian cell (such as rat cerebell … More um or islet cells) microsomes in a cell free system.(4)We found that the addition of calmodulin to microsomes sensitized and activated the cyclic ADP-ribose-mediated Ca^<2+> release.(5)By addition of calmodulin to the cell free cyclic ADP-ribose assay system, we have established a sensitive method for determining cyclic ADP-ribose concentration.(6)We have also developed a method for preparing the cell extract, in which the cyclic ADP-ribose concentration is determined.Using above cyclic ADP-ribose assay system,(7)We found that cyclic ADP-ribose is generated in pancreatic islets by glucose stimulation.(8)We showed that CD38, which has been reported to be a human leukocyte antigen, has both ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities.(9)Moreover, we found that ATP,generated in the process of glucose metabolism, inhibits the cyclic ADP-ribose hydrolase activity, resulting in the increased formation of cyclic ADP-ribose.(10)We produced transgenic mice overexpressing CD38 in islet beta-cells and obtained the first evidance that CD38 has a regulatory role in insulin secretion by glucose in pancreatic beta-cells. Less