Beta-bungarotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalization.
The neurotoxic phospholipase A(2), beta-bungarotoxin (beta-BuTx), is a component of the snake venom from the Taiwanese banded krait Bungarus multicinctus. beta-BuTx affects presynaptic nerve terminal function of the neuromuscular junction and induces widespread neuronal cell death throughout the mammalian and avian CNS. To analyse the initial events of beta-BuTx-mediated cell death, the toxin was applied to cultured rat hippocampal neurons where it induced neuronal cell death in a concentration-dependent manner (EC(50) approximately equal to 5 x 10(-13) M) within 24 h. Fluorescence labelled beta-BuTx was completely incorporated by neurons within < 10 min. Binding and uptake of beta-BuTx, as well as induction of cell death, were efficiently antagonized by preincubation with dendrotoxin I, a blocker of voltage-gated potassium channels devoid of phospholipase activity. Binding of beta-BuTx was selective for neurofilament-positive cells. As evident from intense annexin-V and TUNEL stainings, application of beta-BuTx induced apoptotic cell death exclusively in neurons, leaving astrocytes unaffected. No evidence was obtained for any contribution of either caspases or calpains to beta-BuTx-induced apoptosis, consistent with the inability of the inhibitors Z-Asp-DCB and calpeptin, respectively, to protect neurons from beta-BuTx-induced cell death. These observations indicate that induction of cell death by beta-BuTx comprises several successive phases: (i) binding to neuronal potassium channels is the initial event, followed by (ii) internalization and (iii) induction of apoptotic cell death via a caspase-independent pathway.
Herkert
M
M
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany.
Shakhman
O
O
Schweins
E
E
Becker
C M
CM
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects*; D002038:Bungarotoxins / Q000493:pharmacokinetics*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004546:Elapid Venoms / Q000494:pharmacology; D005260:Female; D006624:Hippocampus / Q000166:cytology; D020287:In Situ Nick-End Labeling; D009474:Neurons / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D009498:Neurotoxins / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D017304:Annexin A5; D002038:Bungarotoxins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D004546:Elapid Venoms; D009498:Neurotoxins; D011956:Receptors, Cell Surface; C108582:dendrotoxin I; C071482:calpeptin; D010741:Phospholipases A; D002154:Calpain; D020169:Caspases