Calpain activation and Na+/Ca2+ exchanger degradation occur downstream of calcium deregulation in hippocampal neurons exposed to excitotoxic glutamate.
Delayed calcium deregulation (DCD) plays an essential role in glutamate excitotoxicity, a major detrimental factor in stroke, traumatic brain injury, and various neurodegenerations. In the present study, we examined the role of calpain activation and Na(+)/Ca(2+) exchanger (NCX) degradation in DCD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DCD accompanied by secondary mitochondrial depolarization. Activation of calpain was evidenced by detecting NCX isoform 3 (NCX3) degradation products. Degradation of NCX isoform 1 (NCX1) was below the detection limit of Western blotting. Degradation of NCX3 was detected only after 1 hr of incubation with glutamate, whereas DCD occurred on average within 15 min after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DCD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DCD and glutamate-induced neuronal death. On the other hand, MK801, an inhibitor of the NMDA subtype of glutamate receptors, added shortly after the initial glutamate-induced jump in cytosolic Ca(2+), completely prevented DCD and activation of calpain and strongly protected neurons against excitotoxicity. Taken together, our results suggest that, in glutamate-treated hippocampal neurons, the initial increase in cytosolic Ca(2+) that precedes DCD is insufficient for sustained calpain activation, which most likely occurs downstream of DCD.
Brustovetsky
Tatiana
T
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Bolshakov
Alexey
A
Brustovetsky
Nickolay
N
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism / Q000633:toxicity*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000378:metabolism / Q000633:toxicity; D051381:Rats; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D013997:Time Factors
D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D018691:Excitatory Amino Acid Antagonists; D009498:Neurotoxins; C491102:Slc8a3 protein, rat; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C071482:calpeptin; D018698:Glutamic Acid; D016291:Dizocilpine Maleate; D002154:Calpain; D002118:Calcium