Calpain Activation and Na+/Ca2+ Exchanger Degradation Occur Downstream of Calcium Deregulation in Hippocampal Neurons Exposed to Excitotoxic Glutamate

Delayed calcium deregulation (DOD) 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+/Ca2+ exchanger (NCX) degradation in DOD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DOD 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 DOD occurred on average within 15 min after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DOD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DOD 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 Ca2+, completely prevented DOD 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 Ca2+ that precedes DOD is insufficient for sustained calpain activation, which most likely occurs downstream of DOD. (C) 2009 Wiley-Liss, Inc.