Peçonha da serpente Micrurus lemniscatus lemniscatus (Roze, 1967), caracterização parcial das propriedades bioquímica e farmacológicas: neurotoxicidade e atividade pré sináptica de toxinas três-dígitos

Abstract

Accidents with snakes are a serious public health problem and were included as “neglected tropical diseases” by the World Health Organization (WHO). Micrurus sp is responsible for only 0.5% of the snake accidents. Clinical manifestations of envenoming by this snake consist primarily of the neurotoxic activity in the peripheral nervous system, mainly by the action of the three-finger toxins (3-Ftx) (6-8 KDa), present in the venom, that cause progressive blockade of nAChR, at the motor endplate. Previous studies showed that β-neurotoxins (phospholipase A2) isolated from elapidic venoms, including coral snakes, induce cortical neurons death and promote glutamatergic excitotoxicity, causing neuronal degeneration. L-glutamate, the main excitatory neurotransmitter in the central nervous system (CNS), when released in excess induces [Ca2+]i increase through the activation of ionotropic glutamate receptors, mainly postsynaptic NMDA receptors. Moreover, in pathological conditions as epilepsy and brain ischemia, there is a massive release of glutamate, leading to neurotoxicity. Although some symptoms associated to disorders in the CNS related with three-finger toxins have been described, little is known about their mechanisms of neurotoxicity. In the present PhD thesis, we proposed to evaluate the neurotoxic effects of M. lemniscatus crude venom and its fractions obtained through RPC-HPLC on neuroblastoma cells (Neuro2A), using MTT assays, after 24 hours of exposure. We observed that the crude venom (in a dose-dependent manner) and most of its fractions caused toxicity (40-80 %). In comparison, hippocampal neurons from newborn rats (P0-P5) were treated with the crude venom, at different concentrations (5x10-5 – 50x10-1 µg/mL) and at different times (1, 3 and 24 h). Neurotoxicity was observed after 3 hours of exposure. Fluorescence assays with calcein-AM and 1-homodimer ethidium confirmed this result, when neurons were exposed for 24 h. We investigated the possible pharmacological targets involved in neurotoxicity of the crude venom. Our results indicated that NMDA and nACh receptors were involved in the toxic effects. These effects were also observed for fraction 22 (F22). The venom, fraction F22, as well as two 3Ftxs (Ml7294_NTX and Ml7256_NTX) increased L-glutamate release in rat cortical brain synaptosomes. This release was time and concentration-dependent, in the case of the venom and F22. The contribution of N, L and P/Q calcium channels, ionotropic glutamatergic receptors (NMDA and AMPA/kainate), as well as the external and internal calcium concentration(evaluated using calcium chelators, EGTA and BAPTA-AM) was also evaluated. The results showed that calcium influx is involved in the L-glutamate release promoted by F22, and that this effect was abolished in the presence of glutamate ionotropic antagonists. These studies could lead to a better understanding of the neurotoxic effects of Micrurus lemniscatus venom on the CNS and showed for the first time the presynaptic activity of three-finger toxins.