CALCIUM SIGNALING BY CYCLIC ADP-RIBOSE, NAADP, AND INOSITOL TRISPHOSPHATE ARE INVOLVED IN DISTINCT FUNCTIONS IN ASCIDIAN OOCYTES

ADP-ribosyl cyclase catalyzes the synthesis of two structurally and fu nctionally different Ca2+ releasing molecules, cyclic ADP-ribose (cADP R) from beta-NAD and nicotinic acid-adenine dinucleotide phosphate (NA ADP) from beta-NADP, Their Ca2+-mobilizing effects in ascidian oocytes were characterized ire connection with that induced by inositol 1,4,5 -trisphosphate (InsP(3)). Fertilization of the oocyte is accompanied b y a decrease in the oocyte Ca2+ current and an increase in membrane ca pacitance due to the addition of membrane to the cell surface, Both of these electrical changes could be induced by perfusion, through a pat ch pipette, of nanomolar concentrations of cADPR or its precursor, bet a-NAD, into unfertilized oocytes. The changes induced by beta-NAD show ed a distinctive delay consistent with its enzymatic conversion to cAD PR. 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, inhibi ted the cADPR induced changes. Both inhibitors likewise blocked the me mbrane insertion that normally occurred at fertilization consistent wi th it being mediated by a ryanodine receptor. The effects of NAADP wer e different from those of cADPR, Although NAADP induced a similar decr ease in the Ca2+ current, no membrane insertion occurred. Moreover, pr etreatment 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 concentr ation of InsP(3) and NAADP could likewise inhibit such an InsP(3)-indu ced oscillation. This work shows that three independent Ca2+ signaling pathways are present in the oocytes and that each is involved in medi ating distinct changes associated with fertilization. The results are consistent with a hierarchical organization of Ca2+ stores in the oocy te.