Cecchini Alessandra L, Vasconcelos Flávio, Amara Susan G, Giglio José Roberto, Arantes Eliane Candiani
Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Av. do Café, s/n, 14040-903, Ribeirão Preto-SP, Brazil.
Toxicol Appl Pharmacol. 2006 Dec 1;217(2):196-203. doi: 10.1016/j.taap.2006.09.003. Epub 2006 Sep 12.
Scorpion neurotoxins targeting the Na(v) channel can be classified into two classes: alpha- and beta-neurotoxins and are reported as highly active in mammalian brain. In this work, we evaluate the effects of Tityus serrulatus venom (Ts venom) and its alpha-neurotoxin TsTX-V on gamma-aminobutyric acid (GABA), dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes. TsTX-V was isolated from Ts venom by ion exchange chromatography followed by reverse-phase (C18) high-performance liquid chromatography. Neither Ts venom nor TsTX-V was able to affect (3)H-Glu uptake. On the other hand, Ts venom (0.13 microg/mg) significantly inhibited both (3)H-GABA and (3)H-DA uptake ( approximately 50%). TsTX-V showed IC(50) values of 9.37 microM and 22.2 microM for the inhibition of (3)H-GABA and (3)H-DA uptake, respectively. These effects were abolished by pre-treatment with tetrodotoxin (TTX, 1 microM), indicating the involvement of voltage-gated Na(+) channels in this process. In the absence of Ca(2+), and at low Ts venom concentrations, the reduction of (3)H-GABA uptake was not as marked as in the presence of Ca(2+). TsTX-V did not reduce (3)H-GABA uptake in COS-7 cells expressing the GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced (3)H-GABA uptake by synaptosomes might be due to rapid cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes a reduction of (3)H-GABA and (3)H-DA uptake in a Ca(2+)-dependent manner, not directly affecting GABA transporters, but, in consequence of depolarization, involving voltage-gated Na(+) channels.
靶向钠通道（Na(v)）的蝎神经毒素可分为两类：α-和β-神经毒素，据报道在哺乳动物大脑中具有高活性。在本研究中，我们评估了锯齿脂鲤蝎毒液（Ts毒液）及其α-神经毒素TsTX-V对离体大鼠脑突触体中γ-氨基丁酸（GABA）、多巴胺（DA）和谷氨酸（Glu）摄取的影响。通过离子交换色谱法，随后进行反相（C18）高效液相色谱法从Ts毒液中分离出TsTX-V。Ts毒液和TsTX-V均不能影响³H-Glu的摄取。另一方面，Ts毒液（0.13微克/毫克）显著抑制³H-GABA和³H-DA的摄取（约50%）。TsTX-V对³H-GABA和³H-DA摄取抑制的半数抑制浓度（IC₅₀）值分别为9.37微摩尔和22.2微摩尔。用河豚毒素（TTX, 1微摩尔）预处理可消除这些效应，表明电压门控钠通道（Na⁺）参与了这一过程。在无Ca²⁺且Ts毒液浓度较低时，³H-GABA摄取的减少不如在有Ca²⁺时明显。TsTX-V在表达GABA转运体GAT-1和GAT-3的COS-7细胞中不降低³H-GABA摄取，表明该毒素间接降低转运。突触体对³H-GABA摄取的减少可能是由于C6胶质瘤细胞共聚焦显微镜显示的细胞快速去极化。因此，TsTX-V以Ca²⁺依赖的方式导致³H-GABA和³H-DA摄取减少，并非直接影响GABA转运体，而是由于去极化，涉及电压门控钠通道。
