L-type calcium channels reduce ROS generation in cerebellar granule cells following kainate exposure.

Abstract

Cerebellar granule cells (CGC) deprived of serum or trophic factors develop sensitivity to kainate neurotoxicity that is mediated by the alpha-amino-3-hydroxy-5-methyl-isoxazole proprionic acid (AMPA) subtypes of glutamate receptors (GluR). The L-type voltage-gated calcium channel (L-type VGCC) blocker nifedipine increases the potency of kainate 50-fold. Thus, one goal of this laboratory is to determine the underlying protective mechanism triggered by calcium influx through this channel. The cell-permeable heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine effected complete protection against kainate treatment in the presence of nifedipine, as did the iron chelator deferoxamine. The chelatable heavy metal pool decreased approximately 70% immediately following treatment with kainate, but did not change following kainate/nifedipine treatment. Tetramethylrhhodamine ethyl ester (TMRE) fluorescence, an indicator of mitochondrial membrane potential, decreased approximately 70% following kainate treatment but displayed a more modest decrease ( approximately 15%) when CGC were treated with kainate/nifedipine. Reactive oxygen species (ROS) formation decreased in CGC immediately following kainate treatment but was slightly elevated following kainate/nifedipine treatment. Electron microscopic examinations of the CGC indicated severe swelling and distortion of mitochondria immediately following kainate/nifedipine treatment and the appearance of mitochondrial herniations, whorls, and bridges 2 h later, features that were rarely observed following kainate treatment. These results support the hypothesis that calcium entry through L-type VGCCs protects CGC during kainate treatment by lowering the chelatable heavy metal pool and the mitochondrial membrane potential, thereby mitigating the formation of ROS.