Silva Carolina Nunes da, Lomeo Rosângela Silva, Torres Fernanda Silva, Borges Marcia Helena, Nascimento Marta Cordeiro, Rodrigues Mesquita-Britto Maria Helena, Rapôso Catarina, Pimenta Adriano Monteiro de Castro, da Cruz-Höfling Maria Alice, Gomes Dawidson Assis, de Lima Maria Elena
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG, Brazil.
Toxicon. 2018 Aug;150:280-288. doi: 10.1016/j.toxicon.2018.06.073. Epub 2018 Jun 15.
PhTx2 is the most toxic fraction from the venom of the spider Phoneutria nigriventer, being responsible to sodium entry into cortical synaptosomes, increasing the release of neurotransmitters, such as l-glutamate (L-Glu) and; acetylcholine. In this study, we investigated the action of a toxin purified from; PhTx2 fraction, called PnTx2-6 or δ-CNTX-Pn2a, on L-Glu release from rat; brain cortex synaptosomes, as well as its ability to induce blood-brain barrier permeability. PnTx2-6 increased L-Glu release from rat cortical brain synaptosomes in a time- and dose-dependent manner (EC50 = ∼20 nM; Tm = 16min), as measured by a fluorimetric method. The increase of L-Glu by PnTx2-6 was inhibited by tetrodotoxin. And partially inhibited by EGTA. Calcium channel blockers ω-conotoxin MVIIC (P/Q-types) and ω-conotoxin GVIA (N-type), were able to reduce the PnTx2-6-induced release of L-Glu, while nifedipine (L-type) did not show any inhibition. These findings suggest that thew release of L-Glu by PnTx2-6 is due its primary action on sodium channels, well-known to be target of this toxin. PnTx2-6 is able to potentiate penile erection and this effect may be related with the release of l-glutamate from the CNS, besides a local effect on corpus carvenosum, as previously shown by our group. If L-Glu release and penile erection potentiation are indeed correlated, then this toxin should be able to cross the blood brain barrier (BBB). Results by immunoblotting assays indicated a change in the expression of proteins associated with the paracellular and transcellular transport at the blood-brain barrier, suggesting a BBB dysfunction mediated by PnTx2-6. Therefore, PnTx2-6 may induce the release l-glutamate in the central nervous system, when injected peripherally.
PhTx2是黑腹捕鸟蛛毒液中毒性最强的组分，可促使钠离子进入皮层突触体，增加神经递质如L-谷氨酸（L-Glu）和乙酰胆碱的释放。在本研究中，我们研究了从PhTx2组分中纯化得到的一种毒素，即PnTx2-6或δ-CNTX-Pn2a，对大鼠脑皮层突触体释放L-Glu的作用，以及其诱导血脑屏障通透性的能力。通过荧光法测定，PnTx2-6以时间和剂量依赖性方式增加大鼠皮层脑突触体释放L-Glu（半数有效浓度[EC50]约为20 nM；峰值时间[Tm]为16分钟）。河豚毒素可抑制PnTx2-6引起的L-Glu释放增加，EGTA可部分抑制该作用。钙通道阻滞剂ω-芋螺毒素MVIIC（P/Q型）和ω-芋螺毒素GVIA（N型）能够减少PnTx2-6诱导的L-Glu释放，而硝苯地平（L型）未显示出任何抑制作用。这些发现表明，PnTx2-6引起的L-Glu释放是由于其对钠通道的主要作用，而钠通道是该毒素的已知靶点。PnTx2-6能够增强阴茎勃起，除了对海绵体的局部作用外，这种作用可能与中枢神经系统释放L-谷氨酸有关，正如我们小组之前所表明的那样。如果L-Glu释放与阴茎勃起增强确实相关，那么这种毒素应该能够穿过血脑屏障（BBB）。免疫印迹分析结果表明，血脑屏障处与细胞旁和跨细胞转运相关的蛋白质表达发生了变化，提示PnTx2-6介导了血脑屏障功能障碍。因此，外周注射PnTx2-6时，可能会诱导中枢神经系统释放L-谷氨酸。
