PLASMA MEMBRANE CALCIUM ATPASE IN DELAYED NEURONAL DEATH

质膜钙ATP酶在延迟性神经元死亡中的作用

• 批准号: 2891520
• 负责人: MICHAEL L GARCIA
• 金额: $ 3.02万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2891520
• 关键词: calcium flux; calcium transporting ATPase; cell death; epilepsy; glutamates; in situ hybridization; kainate; laboratory rat; molecular pathology; neurons; neuroprotectants; neurotrophic factors; tissue /cell culture; western blottings

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.

拟议的实验将试图描绘的作用， 质膜Ca+2 ATP酶（PMCA）在Ca+2稳态丧失中的作用 发生在癫痫发作期间。 将成年雄性Sprague-Dawley大鼠 腹腔注射红藻氨酸，允许癫痫发作， 在不同的时间牺牲。 然后将准备组织用于原位 杂交或蛋白质印迹分析。 谷氨酸对 PMCAs的功能将通过暴露不同的 人NT 2和小鼠P19畸胎瘤衍生细胞系过度 谷氨酸盐，然后测量微粒体对45 Ca +2的吸收 准备工作。 神经生长的潜在神经保护机制 将通过将PC 12细胞暴露于NGF来测定细胞因子（NGF）。 不同的时间。 将分离并分析总mRNA和蛋白质 PMCAs表达的变化。 功能 通过预暴露来确定NGF诱导的变化的显著性 将PC 12细胞暴露于亚毒性浓度的A23187和 利用Fura-2测量细胞内Ca+2浓度。 PC12 将利用细胞来确定神经生长因子是否诱导了细胞的变化。 PMCA的表达可以通过以下方式阻止Ca+2介导的细胞死亡 用NGF预孵育PC 12细胞，暴露于毒性浓度的 A23187，然后用比色法评估细胞存活。 直接 评估NGF保护细胞免受Ca+2损失的能力 癫痫发作期间发生的内稳态将通过以下方式解决： 用NGF预孵育分化的NT 2和P19细胞，暴露这些细胞， 用~（45）Ca +2测定PMCA活性 摄取。