期刊
NEUROSCIENCE
卷 139, 期 2, 页码 577-595出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.neuroscience.2005.12.057
关键词
ApopTag assay; calpeptin; z-DEVD-fmk; Na+ channels; whole-cell currents
资金
- NCI NIH HHS [R01 CA-91460] Funding Source: Medline
- NINDS NIH HHS [R01 NS-38146, R01 NS-31622, R01 NS-41088] Funding Source: Medline
- NATIONAL CANCER INSTITUTE [R01CA091460] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R01NS041088, R01NS038146, R01NS031622] Funding Source: NIH RePORTER
Glutamate toxicity in traumatic brain injury, ischemia, and Huntington's disease causes cortical neuron death and dysfunction. We tested the efficacy of calpain and caspase-3 inhibitors alone and in combination to prevent neuronal death and preserve electrophysiological functions in rat primary cortical neurons following glutamate exposure. Cortical neurons exposed to 0.5 mu M glutamate for 24 h committed mostly apoptotic death as determined by Wright staining and ApopTag assay. Levels of expression, formation of active forms, and activities of calpain and caspase-3 were increased following glutamate exposure. Also, in situ double labeling identified conformationally active caspase-3-p20 fragment and chromatin condensation in apoptotic neurons. Pretreatment of cortical neurons with 0.2 mu M N-benzyloxyl-carbonyl-Leu-Nle-aldehyde (calpain-specific inhibitor) and 100 mu M N-benzyloxylcarbonyl-Asp(OCH3)-Glu(OCH3)-ValAsp(OCH3)-fluoromethyl ketone (caspase-3-specific inhibitor) provided strong neuroprotection. Standard patch-clamp techniques were used to measure the whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors. The lack of a change in capacitance indicated that neurons treated with inhibitor(s) plus glutamate did not undergo apoptotic shrinkage and maintained the same size as the control neurons. Whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors were similar in amplitude and activation/inactivation kinetics for cells untreated and treated with inhibitor(s) and glutamate. Spontaneous synaptic activity as observed by miniature end-plate currents was also similar. Prevention of glutamate-induced apoptosis by calpain and caspase-3 inhibitors preserved normal activities of crucial ion channels such as Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors in neurons. Our studies strongly imply that calpain and caspase-3 inhibitors may also provide functional neuroprotection in the animal models of traumatic brain injury and neurodegenerative diseases. (C) 2005 Published by Elsevier Ltd on behalf of IBRO.
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