PLASMA MEMBRANE CALCIUM ATPASE IN DELAYED NEURONAL DEATH

拟议的实验将试图描绘出 质膜Ca~(2+)-ATPase(PMCA)在Ca~(2+)内稳态丧失中的作用 发生在癫痫发作期间。成年，雄性SD大鼠将成为 在腹膜内注射海人酸，允许抓住和 在不同的时间被牺牲。然后组织将被准备用于原位 杂交或蛋白质印迹分析。谷氨酸对细胞的影响 PMCA的功能将通过暴露差异化的 人NT2和小鼠P19畸胎瘤细胞系过度分化 谷氨酸，然后测量~(45)Ca~(+2)在微粒体的摄取 准备工作。一种潜在的神经生长保护机制 因子(NGF)将通过将PC12细胞暴露于NGF来确定 不同的时期。总的信使核糖核酸和蛋白质将被分离和分析 关于PMCA的表达的变化。功能界别 NGF诱导的变化的重要性将通过预先暴露来确定 PC12细胞暴露于亚毒性浓度的A23187和NGF 利用Fura-2测量细胞内钙离子浓度。PC12 细胞将被用来确定NGF是否诱导了 PMCA的表达可通过抑制Ca+2介导的细胞死亡 将PC12细胞与NGF预先孵育，暴露于有毒浓度的 A23187，然后用比色法测定细胞存活率。一位直接的 神经生长因子对细胞内钙离子丢失保护作用的研究 癫痫发作期间的动态平衡将通过以下方式解决 将分化的NT2和P19细胞与NGF预先孵育，暴露于这些细胞 细胞产生过量谷氨酸，45Ca+2测定PMCA活性 领悟。

The proposed experiments will attempt to delineate the role of the plasma membrane Ca+2 ATPase (PMCA) in the loss of Ca+2 homeostasis occurring during seizures. Adult, male Sprague-Dawley rats will be injected intraperitoneally with kainic acid, allowed to seize and sacrificed at various times. Tissue will then be prepared for in situ hybridization or western blot analysis. Glutamate effects on the functioning of the PMCAs will be determined by exposing differentiated human NT2 and mouse P19 teratocarcinoma derived cell lines to excessive glutamate and then measuring 45Ca+2 uptake into microsomal preparations. A potential neuroprotective mechanism for nerve growth factor (NGF) will be determined by exposing PC12 cells to NGF for various times. Total mRNA and protein will be isolated and analyzed for changes in the expression of the PMCAs. The functional significance of NGF-induced changes will be determined by preexposing PC12 cells to NGF, exposing to subtoxic concentration of A23187 and measuring intracellular Ca+2 concentrations utilizing fura-2. PC12 cells will be utilized to determine if NGF-induced changes in the expression of the PMCAs can prevent Ca+2 mediated cell death by preincubating PC12 cells with NGF, exposing to toxic concentrations of A23187 and then assessing cell survival colorimetrically. A direct assessment of NGF's ability to protect cells from the loss Ca+2 homeostasis occurring during seizures will be addressed by preincubating differentiated NT2 and P19 cells with NGF, exposing these cells to excessive glutamate and then assaying PMCA activity by 45Ca+2 uptake.