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Keywords:

  • Apoptosis;
  • Bcl-2;
  • Bax;
  • Caspase-3;
  • Inducible nitric oxide synthase;
  • Nitric oxide

Abstract

  1. Top of page
  2. Abstract

Abstract: To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1β with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1β was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by NG-monomethyl-l-arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of BCl-2 and up-regulation of Bax protein levels.

Abbreviations used: Ac-DEVD-CHO, acetyl-DEVD-aldehyde; AMC, 7-amino-4-methylcoumarin; H/R, hypoxia/reoxygenation; IFN-γ, interferon-γ; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; l-NMMA, NG-monomethyl-l-arginine; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; ODN, oligonucleotide; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; SNP, sodium nitroprusside; SSC, saline-sodium citrate.