S. Shen 9631982 Changes in ionic activities, especially intracellular calcium activity ( Ca2+ i), have been linked to a wide variety of signal transduction pathways mediating the actions of growth factors, hormones and other extrinsic ligands. A model cell system for studying ionic events is the fertilization reaction in sea urchin eggs. Changes in ionic activities in the sea urchin egg during fertilization are necessary and sufficient for activation of the developmental program. Central to the ionic changes is a transient rise in Ca2+ i, which has been extensively characterized. The egg is similar to many somatic cells by having both conventional Ca2+ release mechanisms, that are sensitive to inositol 1,4,5-trisphosphate (IP3) and ryanodine. The egg also contains a recently discovered novel Ca2+ release that is induced by nicotinate adenine dinucleotide phosphate (NAADP). Despite the significance of the rise in Ca2+ i during fertilization, how the response is triggered by the sperm is still unknown. Based on pharmacological studies, the two conventional Ca2+ release mechanisms were considered to be redundant for triggering the fertilization response. But more recent pharmacological studies suggest that the IP3 receptor Ca2+ channel is the primary and required mechanism for Ca2+ release during fertilization, rather than equal redundant mechanisms. As with all pharmacological studies, the conclusion is limited by the specificity of the agents utilized. The objective of this proposal is to define the required mechanism(s) for the rise in Ca2+ i during fertilization. The experiments in this proposal are designed to establish the significance of inositol phospholipid hydrolysis during fertilization. Rather than block the activity of IP3 receptor Ca2+ channel, the requirement of IP3 production will be directly assessed by inhibiting inositol phospholipid hydrolysis. The significance of IP3 production is also evaluated indirectly by further characterization of the ryanodine and NAADP Ca 2+ release mechanisms.
