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: 2398244
• 负责人: MICHAEL L GARCIA
• 金额: $ 2.5万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2398244
• 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述 Ca+2 稳态丧失中的质膜 Ca+2 ATP 酶 (PMCA) 发生在癫痫发作期间。 成年雄性 Sprague-Dawley 大鼠将 腹腔注射红藻氨酸，让其抓住并 在不同时期牺牲。 然后将在原位准备组织 杂交或蛋白质印迹分析。 谷氨酸对的影响 PMCA 的运作将通过暴露差异化来确定 人 NT2 和小鼠 P19 畸胎瘤细胞系过度 谷氨酸，然后测量微粒体摄取 45Ca+2 准备工作。 神经生长的潜在神经保护机制 通过将 PC12 细胞暴露于 NGF 来测定因子 (NGF) 不同的时间。 总 mRNA 和蛋白质将被分离并分析 PMCA 表达的变化。 功能性的 NGF 诱导的变化的重要性将通过预暴露来确定 PC12 细胞对 NGF 的作用，暴露于亚毒性浓度的 A23187 和 利用 fura-2 测量细胞内 Ca+2 浓度。 电脑12 细胞将被用来确定 NGF 是否诱导了 PMCA 的表达可以通过以下方式阻止 Ca+2 介导的细胞死亡 将 PC12 细胞与 NGF 预孵育，暴露于有毒浓度的 A23187，然后通过比色法评估细胞存活率。 直接一个 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稳态将通过 将分化的 NT2 和 P19 细胞与 NGF 预孵育，暴露这些细胞 细胞产生过量的谷氨酸，然后通过 45Ca+2 测定 PMCA 活性 吸收。