Induction by beta-bungarotoxin of apoptosis in cultured hippocampal neurons is mediated by Ca(2+)-dependent formation of reactive oxygen species.
The component of the venom of the Taiwanese banded krait Bungarus multicinctus, beta-bungarotoxin (beta-BuTx), acts as an extremely potent inducer of neuronal apoptosis when applied to rat hippocampal cultures. While induction of cell death is dependent on toxin binding to voltage-activated K+ channels and subsequent internalization, the pro-apoptotic signals triggered by picomolar concentrations of beta-BuTx are not understood. Following toxin binding, a dramatic increase in intracellular Ca2+ became detectable after 30 min, and in reactive oxygen species (ROS) after 3-4 h. Conversely, Ca2+ chelators, radical quenchers and antioxidants efficiently antagonized beta-BuTx induced apoptosis. As shown for the antioxidant 2,3-dihydroxybenzoic acid, analysis by matrix assisted laser desorbtion-time of flight (MALDI-TOF) mass spectrometry excluded the protective effects to be due to reductive cleavage of the toxic beta-BuTx dimer. Inhibitors of the intracellular antioxidant defence system enhanced neuronal susceptibility to beta-BuTx, supporting the essential role of ROS in beta-BuTx-initiated apoptosis. Cell damage was accompanied by an accumulation of markers of oxidative cell stress, phospholipid hydroxyperoxides and the lipid peroxidation product, malonyl dialdehyde. These observations indicate that beta-BuTx-induced cell death resulted from an intracellular signalling cascade involving subsequent stages of a dramatic rise in free Ca2+, the accumulation of ROS, membrane lipid peroxidation and, finally, apoptosis.
Shakhman
Olena
O
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Germany.
Herkert
Matthias
M
Rose
Christine
C
Humeny
Andreas
A
Becker
Cord-Michael
CM
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002038:Bungarotoxins / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000196:embryology; D015227:Lipid Peroxidation / Q000187:drug effects; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D009498:Neurotoxins / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D000975:Antioxidants; D002038:Bungarotoxins; D009498:Neurotoxins; D016877:Oxidants; D017382:Reactive Oxygen Species; D002118:Calcium