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 known to trigger apoptotic or necrotic death in neuronal cells. Recently, the authors reported that intracellular calcium restriction by NMDA receptor antagonists induces apoptosis in cortical cultures. To evaluate whether further restriction of intracellular free calcium can induce apoptosis or necrosis, 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 3-10 muM BAPTA/AM (non-toxic concentration for glial cells) for 24-48 hr resulted in delayed and necrotic neuronal death. The necrotic findings included swelling and loss of mitochondria and endoplasmic reticulum (ER) with neuronal membrane rupture 24 hr after treatment with BAPTA/AM. Simultaneously, we 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 attenuated by the addition of 0.5 muM cycloheximide (a protein synthesis inhibitor), 10 muM actinomycin D (an RNA transcription inhibitor), a high extracellular potassium concentration (total 15 mM KCI), 100 muM t-ACPD (a metabotrophic agonist), 100 muM alpha -tocopherol (a free radical scavenger), 100 muM deferoxamine (a ferric ion chelator), 100 muM L-NAME (a nitric oxide synthase (NOS) inhibitor), 50 muM DNQX (a non-NMDA receptor blocker), and 3-30 muM esculetin (a lipoxygenase inhibitor). However, 0.3-3 MM ASA (a cyclooxygenase inhibitor), 100 ng/ml nerve growth factor (NGF), 10 muM MK-801 (a NN DA receptor antagonist), 20 muM zVAD-fmk (caspase inhibitor) and 50 U/ml catalase 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 expression of inducible nitric oxide synthase (NOS) on glial cells. 5. These results suggest that the feature of neuronal death induced by BAPTA/AM exhibits predominantly delayed necrosis mediated by lipoxygenase-dependent free radicals.