Synergistic Calcium Release in the Sea Urchin Egg by Ryanodine and Cyclic ADP Ribose

Abstract

A transient rise in cytoplasmic Ca2+ activity in the sea urchin egg occurs during fertilization due to release from an intracellular store. Two intracellular receptor Ca2+ channels for inositol 1,4,5-trisphosphate (IP3) and ryanodine have been identified by physiological and immunological techniques. While IP3 is the endogenous messenger for the IP3 receptor, a corresponding physiological messenger for the ryanodine receptor is unknown. A variety of recent experimental evidences suggest that cyclic ADP ribose (cADPR) may be a possible candidate. In this study using both egg homogenates and intact eggs, we show that subthreshold concentrations of cADPR and ryanodine can act synergistically to potentiate Ca2+ release. Addition of 10-20 nM cADPR, which causes little net increase in Ca2+, generally enhances the action of subthreshold concentrations of ryanodine. Similarly the addition of 60-80 μM ryanodine causes a slight transient increase but potentiates maximal Ca2+ increase by a subsequent subthreshold addition of cADPR. While the target of Ca2+ release by ryanodine and cADPR may be the ryanodine receptor, their actions appear to be different and more complex than simply opening the release mechanism. There are significant differences in the kinetics of release by the two agonists. In addition we used a poorly metabolized analog of IP3 and an inhibitor of endoplasmic reticulum Ca2+ ATPase activity, to show that the unfertilized egg contains a rapidly filled Ca2+ store, which is commonly released by both IP3-mediated and ryanodine-mediated release mechanisms.