Novel characteristics of glutamate-induced cell death in primary septohippocampal cultures: relationship to calpain and caspase-3 protease activation.
Studies examined the phenotypic characteristics of glutamate-induced cell death and their relationship to calpain and caspase-3 activation. Cell viability was assessed by fluorescein diacetate and propidium iodide staining and lactate dehydrogenase release. Calpain and caspase-3 activity was inferred from signature proteolytic fragmentation of alpha-spectrin. Characterization of cell death phenotypes was assessed by Hoechst 33258 and DNA fragmentation assays. Exposure of septohippocampal cultures to 1.0, 2.0, and 4.0 mmol/L glutamate induced a dose-dependent cell death with an LD50 of 2.0 mmol/L glutamate after 24 hours of incubation. Glutamate treatment induced cell death in neurons and astroglia and produced morphological alterations that differed from necrotic or apoptotic changes observed after maitotoxin or staurosporine exposure, respectively. After glutamate treatment, cell nuclei were enlarged and eccentrically shaped, and aggregated chromatin appeared in a diffusely speckled pattern. Furthermore, no dose of glutamate produced evidence of internucleosomal DNA fragmentation. Incubation with varying doses of glutamate produced calpain and caspase-3 activation. Calpain inhibitor II (N-acetyl-Leu-Leu-methionyl) provided protection only with a narrow dose range, whereas carbobenzoxy-Asp-CH2-OC(O)-2,6-dichlorobenzene (Z-D-DCB; pan-caspase inhibitor) and MK-801 (N-methyl-D-aspartate receptor antagonist) were potently effective across a wider dose range. Cycloheximide did not reduce cell death or protease activation.
Zhao
X
X
Vivian L. Smith Center for Neurologic Research, Department of Neurosurgery, University of Texas-Houston Health Science Center, USA.
Newcomb
J K
JK
Pike
B R
BR
Wang
K K
KK
d'Avella
D
D
Hayes
R L
RL
D000818:Animals; D002154:Calpain / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D018920:Coculture Techniques; D004789:Enzyme Activation; D005333:Fetus; D018698:Glutamic Acid / Q000494:pharmacology*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D009457:Neuroglia / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology; D012688:Septum Pellucidum / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D013049:Spectrin / Q000378:metabolism
D016194:Receptors, N-Methyl-D-Aspartate; D013049:Spectrin; D018698:Glutamic Acid; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases