Intracellular Ca2+ Regulation in the Unfertilized Sea Urchin Egg and the Ca2+ Response During Fertilization

离子活性的变化，特别是细胞内钙离子活性（Ca2+ i）的变化，与多种信号转导途径有关，这些信号转导途径介导了生长因子、激素和其他外源性配体的作用。研究离子事件的一个模型细胞系统是海胆卵的受精反应。在受精过程中，海胆卵中离子活性的变化对于激活发育程序是必要和充分的。离子变化的中心是Ca2+ i的瞬态上升，这已被广泛表征。卵子与许多体细胞相似，具有传统的Ca2+释放机制，对肌醇1,4,5-三磷酸（IP3）和ryanodine敏感。卵还含有一种最近发现的由烟酸腺嘌呤二核苷酸磷酸（NAADP）诱导的新型Ca2+释放。尽管在受精过程中Ca2+ i的增加具有重要意义，但精子如何触发这种反应仍然未知。基于药理学研究，两种传统的Ca2+释放机制被认为是触发受精反应的冗余机制。但最近的药理学研究表明，IP3受体Ca2+通道是受精过程中Ca2+释放的主要和必需机制，而不是同等的冗余机制。与所有药理学研究一样，结论受到所用药物的特异性的限制。本提案的目的是确定所需的机制(s)在受精期间Ca2+ i的上升。本实验旨在确定肌醇磷脂水解在受精过程中的意义。而不是阻断IP3受体Ca2+通道的活性，IP3生产的需求将通过抑制肌醇磷脂水解直接评估。通过进一步表征ryanodine和NAADP ca2 +释放机制，间接评价了IP3产生的意义。

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.