EXTRACELLULAR SYNTHESIS OF CADP-RIBOSE FROM NICOTINAMIDE-ADENINE DINUCLEOTIDE BY RAT CORTICAL ASTROCYTES IN CULTURE

cADPR is an endogenous calcium-mobilizing agent that in vertebrates is synthesized from nicotinamide-adenine dinucleotide (NAD) by bifunctio nal enzymes with ADP-ribosyl cyclase and cADPR hydrolase activity. ADP -ribosyl cyclase and cADPR hydrolase activity have been reported in th e brain, but the cellular localization of these activities has not bee n determined previously. in the present study, selective culturing tec hniques were employed to localize ADP-ribosyl cyclase activity and cAD PR hydrolase activity to astrocytes or neurons in cultures derived fro m rat embryonic cerebral cortex. ADP-ribosyl cyclase activity was dete rmined by incubating cultures with 1 mM NAD in the extracellular mediu m for 60 min at 37 degrees C and measuring formation of cADPR by bioas say and by HPLC. Astrocyte cultures and mixed cultures of astrocytes a nd neurons had mean specific activities of 0.84+/-0.06 and 0.9+/-0.18 nmol cADPR produced/mg protein/hr, respectively. No detectable ADP-rib osyl cyclase activity was found in neuron-enriched/astrocyte-poor cult ures. cADPR hydrolase activity was detectable by incubating cultures w ith 300 mu M cADPR for 60 min at 37 degrees C and assaying loss of cAD PR or accumulation of ADPR. The demonstration of extracellular ADP-rib osyl cyclase and cADPR hydrolase activities associated with astrocytes may have important implications for the role of extracellular cADPR i n signal transduction and in intercellular communication in the nervou s system.