Cyclooxygenase-2 contributes to N-methyl-D-aspartate-mediated neuronal cell death in primary cortical cell culture

Cyclooxygenase isozymes (COX-1 and COX-2) are found to be constitutively ex pressed in brain, with neuronal expression of COX-2 being rapidly induced a fter numerous insults, including cerebral ischemia. Because overactivation of N-methyl-D-aspartate (NMDA) receptors has been implicated in the cell lo ss associated with ischemia, we characterized the expression of the COX iso zymes in murine mixed cortical cell cultures and used isozyme-selective inh ibitors to determine their relative contribution to NMDA receptor-stimulate d prostaglandin (PG) production and excitotoxic neuronal cell death. Immuno cytochemical analysis of mixed cortical cell cultures revealed that COX-2 e xpression was restricted to neurons, whereas COX-1 was expressed in both ne urons and astrocytes. Brief exposure to NMDA (5 min; 100 mu M) elicited a t ime-dependent accumulation of PGs in the culture medium that preceded neuro nal cell death and correlated with the induction of COX-2 mRNA. COX-1 expre ssion remained unchanged. Flurbiprofen, a nonselective COX-1/COX-2 inhibito r, blocked NMDA-stimulated PG production and attenuated neuronal death in a concentration-dependent manner. Similar results were obtained with the spe cific COX-2 inhibitor NS-398 (10-30 mu M) but not with the selective COX-1 inhibitor valeryl salicylate (10-300 mu M). Inhibition of total constitutiv e COX activity with aspirin (100 mM, 1.5 h) before NMDA exposure did not pr event subsequent NMDA-mediated neuronal cell death. However, neuronal injur y in aspirin-pretreated cultures was attenuated by flurbiprofen administrat ion after NMDA exposure. Finally, the protection afforded by COX-2 inhibiti on was specific for NMDA because neither flurbiprofen nor NS-398 protected neurons against kainate-mediated neurotoxicity. Together, these results sup port the conclusion that newly synthesized COX-2 protein contributes to NMD A-induced neuronal injury.