Inhibition of calpain and caspase-3 prevented apoptosis and preserved electrophysiological properties of voltage-gated and ligand-gated ion channels in rat primary cortical neurons exposed to glutamate

• Published in: Neuroscience Vol. 139; no. 2; pp. 577 - 595
• Main Authors: Ray, S.K., Karmakar, S., Nowak, M.W., Banik, N.L.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01-01-2006; Elsevier
• Subjects: Amino Acid Chloromethyl Ketones - pharmacology; Animals; Animals, Newborn; ApopTag assay; Apoptosis - drug effects; Biological and medical sciences; Calpain - metabolism; calpeptin; Caspase 3; Caspase Inhibitors; Caspases - metabolism; Caspases - pharmacology; Cell Survival - drug effects; Cell Survival - physiology; Cells, Cultured; Cerebral Cortex - cytology; Chromatin - drug effects; Cysteine Proteinase Inhibitors - pharmacology; DNA Fragmentation - drug effects; Drug Interactions; Female; Fundamental and applied biological sciences. Psychology; Glutamic Acid - pharmacology; Ion Channel Gating - physiology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Membrane Potentials - radiation effects; Na + channels; Neurons - drug effects; Oligopeptides - pharmacology; Pregnancy; Rats; Rats, Sprague-Dawley; Vertebrates: nervous system and sense organs; whole-cell currents; z-DEVD-fmk; Ac-DEVD-pNA; DTT; APV; HRP; Na + channels; whole-cell currents; BSA; DNQX; FITC; TUNEL; ANOVA; OD; z-VAD-fmk; AMPA/KARs; TTX; MG; MUSC; EBSS; calpeptin; PBS; pNA; z-DEVD-fmk; DAB; TBS; TBST; NMDARs; CHAPS; S.E.M; ECL; SDS-PAGE; V REST; HEPES; PI; ApopTag assay; SBDP; Rat; Enzyme; Cysteine endopeptidases; Rodentia; Glutamatergic neuron; Caspase; Electrophysiology; Na+ channels; Calpain; Ionic channel; Peptidases; Vertebrata; Mammalia; Sodium; Cell death; Animal; Hydrolases; Apoptosis
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2005.12.057

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 μM 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 μM
N-benzyloxylcarbonyl-Leu-Nle-aldehyde (calpain-specific inhibitor) and 100 μM
N-benzyloxylcarbonyl-Asp(OCH
3)-Glu(OCH
3)-Val-Asp(OCH
3)-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.