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Suramin inhibits β-bungarotoxin-induced activation of N-methyl-d-aspartate receptors and cytotoxicity in primary neurons

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Abstract

We demonstrated that β-bungarotoxin (β-BuTX), a snake presynaptic neurotoxin, exhibited a potent cytotoxic effect on cultured cerebellar granule neurons. The mechanism of action of β-BuTX and the cytoprotective agents against β-BuTX were studied. The neuronal death of cerebellar granule neurons induced by β-BuTX was manifested with apoptosis and necrosis processes as revealed by neurite fragmentation, morphological alterations, and staining apoptotic bodies with the fluorescent dye Hoechst 33258. By means of microspectrofluorimetry and fura-2, we measured intracellular Ca2+ concentration, [Ca2+]i and found that [Ca2+]i was increased markedly prior to the morphological changes and cytotoxicity. The downstream pathway of the increased [Ca2+]i was investigated: there was increased production of free radicals, decreased mitochondrial membrane potential, and depleted cellular ATP content. MK801 and suramin effectively suppressed these detrimental effects of β-BuTX. Furthermore, the [3H]MK801 binding was reduced by unlabeled MK801, β-BuTX, and suramin. Thus, activation of N-methyl-d-aspartate (NMDA) receptors appeared to play a crucial role in the cytotoxic effects following βBuTX exposure. In conclusion, the novel finding of this study was that a polypeptide β-BuTX exerted a potent cytotoxic effect through sequential events, including activating NMDA receptors followed by increasing [Ca2+]i, ROS production, and impaired mitochondrial energy metabolism. Suramin, clinically used as a trypanocidal agent, was an effective antagonist against β-BuTX. Data suggest that suramin might have value to detect the possible pathway of certain neuropathological disorders.

Section snippets

Materials

β-Bungarotoxin was isolated from the venom of the Bungarus multicinctus by the method described by Lee et al. (1972). The homogeneity of the purified toxins as revealed in a single band was verified by disc gel electrophoresis (David, 1964). This toxin was prepared in 1 mg/ml and stored at −70°C. Poly-l-lysine, trypsin, soybean trypsin inhibitor, cytosine arabino-side, DNase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT), luciferin-luciferase, diltiazem, and carbonyl cyanide m

Effect of β-BuTX exposure on survival rate of cultured CGNs

The isolated CGNs were cultured for 7 days in vitro (DIV) to obtain maturation with a rich network of fasiculate fibers. We used seven DIV neurons throughout the whole experiment. The toxic effects of β-BuTX on survival rate of CGNs were concentration and time dependent (Fig. 1). After 24 h incubation, concentrations as low as 3 ng/ml of β-BuTX induced cytotoxicity. The EC50 estimated was 3 ng/ml (equivalent to 144 pM, Fig. 1A). However, exposure of astrocytes to 1 μg/ml β-BuTX did not result

Discussion

This study shows that β-BuTX is a potent toxicant to cultured CGNs. It was estimated that the toxic concentration of β-BuTX on cultured CGNs is nontoxic to mice in vivo in animals (Lin-Shiau and Lin, 1999). The selectivity of β-BuTX cytotoxicity to CGNs demonstrated that the cultured rat pheochromocytoma cell (PC12) and the human neuroblastoma cell (IMR32) were insensitive to β-BuTX (1 μg/ml, unpublished data). In this paper, we attempted to elucidate the possible molecular mechanism involved

Acknowledgments

This investigation was supported by a research grant (NSC90-2320-B-002-082) from the National Science Council, Taipei, Taiwan.

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