Efeito das neurotoxinas da aranha phoneutra nigriventer na isquemia cerebral in vitro

Abstract

The role of calcium channel blockers in ischemic conditions has been studied and described in literature. The PhTx3 fraction of the venom of Phoneutria nigriventer spider is a broad spectrum calcium channel blocker, which inhibits glutamate release and calcium uptake in synaptossomes. The PhTx3 fraction contains six isotoxins: Tx3-1 to Tx3-6. Some of these isotoxins block calcium channels type -P/Q, and other blocks the N-type calcium channels. With this action, these isotoxins inhibit calcium uptake and the release of neurotransmitters. The Tx3-3 and Tx3-4 toxins inhibit calcium channels type P/Q and the release of glutamate. It was also shown that the Tx3-4 toxin inhibit calcium-independent glutamate release via glutamate transporter. In our work we demonstrated that the PhTx3 (1µg/mL) was capable of providing neuroprotection (82±4.1%) in an experimental in vitro model using hippocampal slices of rats submitted to ischemic event. The PhTx3 fraction and the Tx3-3 and Tx3-4 toxins presented neuroprotector effect up to one hour after the ischemic event was induced. This effect was significantly reduced after one hour and thirty minutes after the ischemic event and no significant neuroprotection was observed after two hours. The role of intracellular calcium was also investigated in our work. It was observed that the BAPTA-AM and the 2-APB presented neuroprotection (55±2.9% and 45±2.4%), respectively. No neuroprotection was observed using dantrolene. Thus we may suggest that intracellular calcium released from intracellular stocks sensible to IP3 is involved in the process of cellular death in brain ischemia in vitro. Furthermore, we observed that the pharmacological tools used to reduce the amount of intracellular calcium, BAPTA-AM, and blockers of intracellular calcium 2-APB and dantrolene were able to reduce the caspase-3 activity of the ischemic tissue (115 ±- 4.32, 136± 5.6 e 157± 5.85 nmol/mg of proteina), respectively, compared to the hippocampal slices submitted to ischemia without the addition of test compounds (227±5.4 nmol/mg of protein). On the same experiments with the PhTx3 fraction and the Tx3-3 and Tx3-4 toxins no significant reduction on the caspase-3 activity (203 ± 4.7, 210 ±3.35 e 212± 3.37nmol/mg of protein), respectively, was observed when compared to the ischemia submitted slices without the addition of test compounds (223 +/- 4.7nmol/mg de proteína). Thus we concluded calcium channels are not involved in the caspase activity while blockers of intracellular calcium stores reduced the caspase-3 activity on brain ischemia. Based on our results, we can conclude that the toxins present on the Phoneutria nigriventer spider venom (PhTx3 fraction, Tx3-3 and Tx3-4) showed significant neuroprotection of brain ischemia and thus have potential as a new class of therapeutical agents to be used on brain ischemia.