Calcium Signaling by Cyclic ADP-ribose, NAADP, and Inositol Trisphosphate Are Involved in Distinct Functions in Ascidian Oocytes*

Abstract

ADP-ribosyl cyclase catalyzes the synthesis of two structurally and functionally different Ca²⁺releasing molecules, cyclic ADP-ribose (cADPR) from β-NAD and nicotinic acid-adenine dinucleotide phosphate (NAADP) from β-NADP. Their Ca²⁺-mobilizing effects in ascidian oocytes were characterized in connection with that induced by inositol 1,4,5-trisphosphate (InsP₃). Fertilization of the oocyte is accompanied by a decrease in the oocyte Ca²⁺ current and an increase in membrane capacitance due to the addition of membrane to the cell surface. Both of these electrical changes could be induced by perfusion, through a patch pipette, of nanomolar concentrations of cADPR or its precursor, β-NAD, into unfertilized oocytes. The changes induced by β-NAD showed a distinctive delay consistent with its enzymatic conversion to cADPR. The cADPR-induced changes were inhibited by preloading the oocytes with a Ca²⁺ chelator, indicating the effects were due to Ca²⁺ release induced by cADPR. Consistently, ryanodine (at high concentration) or 8-amino-cADPR, a specific antagonist of cADPR, but not heparin, inhibited the cADPR-induced changes. Both inhibitors likewise blocked the membrane insertion that normally occurred at fertilization consistent with it being mediated by a ryanodine receptor. The effects of NAADP were different from those of cADPR. Although NAADP induced a similar decrease in the Ca²⁺ current, no membrane insertion occurred. Moreover, pretreatment of the oocytes with NAADP inhibited the post-fertilization Ca²⁺ oscillation while cADPR did not. A similar Ca²⁺ oscillation could be artificially induced by perfusing into the oocytes a high concentration of InsP₃ and NAADP could likewise inhibit such an InsP₃-induced oscillation. This work shows that three independent Ca²⁺ signaling pathways are present in the oocytes and that each is involved in mediating distinct changes associated with fertilization. The results are consistent with a hierarchical organization of Ca²⁺ stores in the oocyte.