Membrane-bound form of ADP-ribosyl cyclase in rat cortical astrocytes culture

ADP-ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [ H-3]cyclic ADP-ribose production converted from [H-3]NAD(+) by ADP-ribosyl cyclase (2.015 +/- 0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained similar to 50% of the tota l cyclase activity in astrocytes. The formation rate of [H-3]ADP-ribose fro m cyclic ADP-ribose by cyclic ADP-ribose hydrolase and/or from NAD(+) by NA D glycohydrolase was low and enriched in the cytosolic fraction. Although N AD(+) in the extracellular medium was metabolized to cyclic ADP-ribose by i ncubating cultures of intact astrocytes,:the:presence of Triton X-100 in th e medium for permeabilizing cells increased cyclic ADP-ribose production th ree times as much. Isoproterenol and GTP increased [H-3]cyclic ADP-ribose f ormation in crude membrane-associated cyclase activity. This isoproterenol- induced stimulation of membrane-associated ADP-ribosyl cyclase activity was confirmed by cyclic GDP-ribose formation fluorometrically. This stimulator y action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin, These results suggest that ADP-ribosyl cyclase in as trocytes has both extracellular and intracellular actions and that signals of beta-adrenergic stimulation are transduced to membrane-bound ADP-ribosyl cyclase via G proteins within cell surface membranes of astrocytes.