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 bifunctional enzymes with ADP-ribosyl cyclase and cADPR hydrolase activity. ADP-ribosyl cyclase and cADPR hydrolase activity have been reported in the brain, but the cellular localization of these activities has not been determined previously. In the present study, selective culturing techniques were employed to localize ADP-ribosyl cyclase activity and cADPR hydrolase activity to astrocytes or neurons in cultures derived from rat embryonic cerebral cortex. ADP-ribosyl cyclase activity was determined by incubating cultures with 1 mM NAD in the extracellular medium for 60 min at 37 degrees C and measuring formation of cADPR by bioassay and by HPLC. Astrocyte cultures and mixed cultures of astrocytes and 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-ribosyl cyclase activity was found in neuron-enriched/ astrocyte-poor cultures. cADPR hydrolase activity was detectable by incubating cultures with 300 microM cADPR for 60 min at 37 degrees C and assaying loss of cADPR or accumulation of ADPR. The demonstration of extracellular ADP-ribosyl cyclase and cADPR hydrolase activities associated with astrocytes may have important implications for the role of extracellular cADPR in signal transduction and in intercellular communication in the nervous system.
Pawlikowska
L
L
Department of Neurology, Children's Hospital, Boston, Massachusetts 02115, USA.
Cottrell
S E
SE
Harms
M B
MB
Li
Y
Y
Rosenberg
P A
PA
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000378:metabolism; D001253:Astrocytes / Q000378:metabolism*; D001681:Biological Assay; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005110:Extracellular Space / Q000378:metabolism*; D006868:Hydrolysis; D008562:Membrane Glycoproteins; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D051381:Rats / Q000196:embryology; D012617:Sea Urchins
D015703:Antigens, CD; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D009243:NAD; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498156:Cd38 protein, rat; D051997:ADP-ribosyl Cyclase 1; D002118:Calcium