Effect Phoneutria Nigriventer spider toxin, on oxide nitric synthase activity in endothelial cells.
Andrade, E(1); Messina, L(1); Andrade, P(1); Leite, K(1); Ítalo, C(1); Paranhos, M(1); Claro, J(1); Villanova, F(1); Srougi, M(1)
(1)Faculdade de Medicina da Universidade de São Paulo, Departamento de Urologia, Setor de Medicina Sexual.
Introduction and Objective: The activities of the toxin TX2-5 were associated with symptoms like: scratching, lacrimation, hipersalivation, priapism, sweating and agitation followed by spastic paralysis and death.Recently, we demonstrated that NOS inhibitors blocked partial or totally all the described symptoms, including the mortality. It allows to suggest that NO is a major mediator in pathological process induced by the toxin and that neural and endothelial mechanism were involved. The primary goal of this study was to investigate whether TX2-5 act in endothelial cells in culture.
Methods: Human endothelial cell line ECV304 was cultured in RPMI-1640 medium supplemented with 10% fetal 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. The lyophilized crude venom was obtained from Butantan Institute and the toxin was purified by gel filtration and HPLC. The analysis by MALDI-TOF and ES-MS yield a Mr 5116 Da and N-terminal amino acid analysis confirmed the identity of TX2-5. In this study two concentrations of toxin (0,1µM and 0,7µM) were tested in incubation with ECV304 cells for 30,60,90 and 120 minutes. NOS activities associated with the cells were determined following the convertion of 3[H]-arginine to 3[H]-citruline. Statistical comparisons were performed by one-way ANOVA followed by the Newman-Keuls test.
Results: The results showed a significant increase of NOS activity (p<0,05) in the cells that were treated with the toxin. The increase was dose dependent and the higher NOS activity occurred at 60 minutes incubation. Endothelial cells are nonexcitable but several reports have nevertheless clearly shown the incident of voltage-gated ion channels in both cultured and freshly isolated endothelial cells.
Conclusions: In conclusion the results demonstrated that TX2-5 also act directly on endothelial cells in culture.
