PLASMA MEMBRANE CALCIUM ATPASE IN DELAYED NEURONAL DEATH

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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.