BAPTA/AM, an intracellular calcium chelator, induces delayed necrosis by lipoxygenase-mediated free radicals in mouse cortical cultures

1. Disruption of calcium homeostasis during neurodegenerative diseases is k nown to trigger apoptotic or necrotic death in neuronal cells. Recently, th e authors reported that intracellular calcium restriction by NMDA receptor antagonists induces apoptosis in cortical cultures. To evaluate whether fur ther restriction of intracellular free calcium can induce apoptosis or necr osis, we examined the neurotoxic characterization of BAPTA/AM, a permeable free calcium chelator, in mouse cortical cultures. 2. Exposure of mixed (glia and neuron) cortical cultures (DIV 13-16) to 3-1 0 muM BAPTA/AM (non-toxic concentration for glial cells) for 24-48 hr resul ted in delayed and necrotic neuronal death. The necrotic findings included swelling and loss of mitochondria and endoplasmic reticulum (ER) with neuro nal membrane rupture 24 hr after treatment with BAPTA/AM. Simultaneously, w e observed a few TUNEL-positive cells in the neuronal subpopulation of the same cultures. 3. The neurotoxicity evoked by BAPTA/AM (10 muM) was significantly attenuat ed by the addition of 0.5 muM cycloheximide (a protein synthesis inhibitor) , 10 muM actinomycin D (an RNA transcription inhibitor), a high extracellul ar potassium concentration (total 15 mM KCI), 100 muM t-ACPD (a metabotroph ic agonist), 100 muM alpha -tocopherol (a free radical scavenger), 100 muM deferoxamine (a ferric ion chelator), 100 muM L-NAME (a nitric oxide syntha se (NOS) inhibitor), 50 muM DNQX (a non-NMDA receptor blocker), and 3-30 mu M esculetin (a lipoxygenase inhibitor). However, 0.3-3 MM ASA (a cyclooxyge nase inhibitor), 100 ng/ml nerve growth factor (NGF), 10 muM MK-801 (a NN D A receptor antagonist), 20 muM zVAD-fmk (caspase inhibitor) and 50 U/ml cat alase failed to inhibit the injury. 4. However, NGF and catalase blocked the neurotoxicity induced by BAPTA/AM in young neuronal cells (DIV 6). BAPTA/AM (10 muM) did not alter the expres sion of inducible nitric oxide synthase (NOS) on glial cells. 5. These results suggest that the feature of neuronal death induced by BAPT A/AM exhibits predominantly delayed necrosis mediated by lipoxygenase-depen dent free radicals.