Poster 073 - Effect of TX2-5 Phoneutria nigriventer spider toxin on nitric oxide synthase activity

Poster 73. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.432.

Effect of TX2-5 Phoneutria nigriventer Spider Toxin on Nitric Oxide Synthase Activity in Endothelial Cells in Culture

^{1Yonamine, C.M.; ²Kawamoto, E.M.; ¹Higa, O.Z.; ³Troncone, L.R.P. & ¹Camillo MAP.}

1- Molecular Biology Center, IPEN, Av.Prof.Lineu Prestes 2242; SP; CEP.05508-900; Brazil; 2- Department of Pharmacology, ICB-1, USP. Av. Prof. Lineu Prestes 1524; SP; CEP.05508-900; Brazil; 3- Laboratory of Pharmacology; Instituto Butantan; Av.Vital Brasil 1500; SP; CEP.05503-900; Brazil.

Electrophysiological studies on frog skeletal muscle showed impairment of the fast inactivation of sodium channels by the toxin Tx2-5 and Tx2-6. In a previous study we demonstrated that inhibition of nNOS by L-NAME or 7-Nitroindazole abolished all the symptoms of intoxication by this toxin, suggesting that NO is an important mediator in this process. The primary goal of this study was to investigate whether TX2-5 is capable of activating NO synthesis in endothelial cells in culture. Tx2-5 was purified from crude desiccated venom by standard gel filtration and RP-HPLC. Identity of the toxin was confirmed by ES-MS (Mr=5116 Da) and N-terminal amino acid sequencing. Human endothelial cell line ECV304 was cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum and antibiotics. For the experiments, cells were plated in 50 mm dishes and cultured in 2 ml of medium in the presence or absence of the toxin. Cells were then exposed to two concentrations of toxin (0,1µM and 0,7µM) for 30, 60, 90 and 120 minutes. NOS activity in the cells was determined by the conversion of ³[H]-arginine to ³[H]-citruline. Statistical comparisons were performed by one-way ANOVA followed by the Newman-Keuls test. Results showed a significant increase of NOS activity (p<0,05) in cells treated with the toxin. The increase was dose dependent and the higher NOS activity occurred after 60 minutes of incubation. Although endothelial cells do not represent a typical excitable cell, several reports have clearly shown the presence of voltage-gated ion channels in both cultured and freshly isolated cells. In conclusion our results demonstrate that TX2-5 can directly activate NOS in endothelial cells in culture.