Phospholipase A₂ activity of β‐bungarotoxin is essential for induction of cytotoxicity on cerebellar granule neurons

Abstract

The aim of this study was to investigate the mechanism of the cytotoxic effect of beta-bungarotoxin (beta-BuTX), a presynaptic neurotoxin, on rat cerebellar granule neurons (CGNs). The maturation of CGNs is characterized by the prominent dense neurite networks that became fragmented after treatment with beta-BuTX, and this cytotoxic effect of beta-BuTX on CGNs was in a dose- and time-dependant manner. The cytotoxic effect of beta-BuTX was found to be more potent than other toxins, such as alpha-BuTX, cardiotoxin, melittin, and Naja naja atra venom phospholipase A(2). Meanwhile, undifferentiated neuroblastoma neuronal cell lines, IMR-32 and SK-N-MC, and astrocytes were found to be resistant to beta-BuTX. These results indicated that only the mature CGNs were sensitive to beta-BuTX insults. None of the following chemicals: antioxidants, K(+)-channel activator, K(+)-channel antagonists, intracellular Ca(2+) chelator, Ca(2+)-channel blockers, NMDA receptor antagonists, and nitric oxide synthase inhibitor tested, were able to reduce beta-BuTX-induced cytotoxicity. However, secretory type phospholipase A(2) inhibitors (glycyrrhizin and aristolochic acid) and a free radical scavenger (5,5-dimethyl pyrroline N-oxide, DMPO) could attenuate not only beta-BuTX-induced cytotoxicity but also ROS production and caspase-3 activation. These data suggest that phospholipase A(2) activity of beta-BuTX may be responsible for free radical generation and caspase-3 activation that accounts for the observed cytotoxic effect. It is proposed that the CGNs can be a useful tool for studying interactions of the molecules on neuronal plasma membrane with beta-BuTX that mediates the specific cytotoxicity.

A liquid crystal-based sensor for real-time and label-free identification of phospholipase-like toxins and their inhibitors

“…Many phospholipase-like toxins are considered among the most potent toxins. Studies have reported that betabungarotoxin can induce more potent cytotoxic effects than other toxins such as alpha-bungarotoxin, cardiotoxin and melittin (Chen, 2005).…”

Modulation of TTX-sensitive voltage-dependent Na+ channels by β-bungarotoxin in rat cerebellar neurons

“…In summary, β-BuTX effects would lead to an increase of excitability of cerebellar neurons. In general, the increase in excitability would correspond to a toxic action of β-BuTX by inducing neurotoxicity which is based on over-excitation of the neurons involving NMDA receptors [14,16,20,22,27,32]. In addition to that, by its phospolipase activity β-BuTX leads to apoptosis.…”

“…In addition to that, by its phospolipase activity β-BuTX leads to apoptosis. Here, as a second effect, the products of phospholipase activity directly promote the signaling cascade leading to apoptosis [14-16,19,22,32]. Then the modulation of Na + channels to a higher activity would be a synergistic effect enhancing neurotoxicity.…”

“…In order to study mechanisms leading to apoptosis, animal toxins were often used. In this respect, the application of β-bungarotoxin (β-BuTX) is one of the most established models [14-21]. β-BuTX is a component of the venom of the Taiwanese banded krait Bungarus muticinctus .…”

“…β-BuTX is a component of the venom of the Taiwanese banded krait Bungarus muticinctus . The toxic effect of β-BuTX is related to its intrinsic phospholipase-A2 activity [14,15]. The mechanisms triggering neuronal cell death via β-BuTX include the increase in intracellular free Ca 2+ , increased Ca 2+ influx through NMDA-receptors, caspase-3 activation, production of reactive oxygen species and NO production, as well as K + channel inhibition [16-20,22-27].…”