摘要
Voltage-gated Na+ channel (Nav channel) scorpion toxins are classified as α- and β-neurotoxins. Ts5 (α-neurotoxin) and Ts1 (β-neurotoxin) from Tityus serrulatus venom (TsV) interact with Nav channels, increasing Na+ influx and activating voltage-dependent Ca2+ channels. This study aimed to investigate the effect of TsV, Ts1 and Ts5 on the cytosolic Ca2+ concentration ([Ca2+]C) in rat aortic smooth muscle cells. Toxins were isolated by ion exchange chromatography (Ts1) followed by RP-HPLC (Ts5). The rat aortic smooth muscle cells were isolated in Hanks buffer pH 7.4 and loaded with 5 μmol/L of Fura-2AM (45 minutes at 37℃), in order to measure [Ca2+]C by fluorescence of Fura-2/AM (ratio 340/380 nm). The fluorescence was measured in one single cell (excitation: 340 and 380 nm;emission: 510 nm). TsV (100 and 500 mg/mL) and its toxins Ts1 and Ts5 (50 and 100 mg/mL each) led to a concentration-dependent increase in [Ca2+]C. Tetrodotoxin (1 mmol/L), a Nav channel blocker, and verapamil (1 mmol/L), a voltage-operated Ca2+ channel blocker, inhibited the increase in [Ca2+]C induced by TsV (500 mg/mL). In conclusion, TsV and its toxins induce a concentration-dependent increase in [Ca2+]C that probably occurs through interaction with Nav channels, thus inducing depolarization and consequent Ca2+ influx. This assumption is based on the fact that this effect is inhibited by tetrodotoxin and verapamil, showing a direct action of TsV toxins on aorta smooth muscle cells.
Voltage-gated Na+ channel (Nav channel) scorpion toxins are classified as α- and β-neurotoxins. Ts5 (α-neurotoxin) and Ts1 (β-neurotoxin) from Tityus serrulatus venom (TsV) interact with Nav channels, increasing Na+ influx and activating voltage-dependent Ca2+ channels. This study aimed to investigate the effect of TsV, Ts1 and Ts5 on the cytosolic Ca2+ concentration ([Ca2+]C) in rat aortic smooth muscle cells. Toxins were isolated by ion exchange chromatography (Ts1) followed by RP-HPLC (Ts5). The rat aortic smooth muscle cells were isolated in Hanks buffer pH 7.4 and loaded with 5 μmol/L of Fura-2AM (45 minutes at 37℃), in order to measure [Ca2+]C by fluorescence of Fura-2/AM (ratio 340/380 nm). The fluorescence was measured in one single cell (excitation: 340 and 380 nm;emission: 510 nm). TsV (100 and 500 mg/mL) and its toxins Ts1 and Ts5 (50 and 100 mg/mL each) led to a concentration-dependent increase in [Ca2+]C. Tetrodotoxin (1 mmol/L), a Nav channel blocker, and verapamil (1 mmol/L), a voltage-operated Ca2+ channel blocker, inhibited the increase in [Ca2+]C induced by TsV (500 mg/mL). In conclusion, TsV and its toxins induce a concentration-dependent increase in [Ca2+]C that probably occurs through interaction with Nav channels, thus inducing depolarization and consequent Ca2+ influx. This assumption is based on the fact that this effect is inhibited by tetrodotoxin and verapamil, showing a direct action of TsV toxins on aorta smooth muscle cells.
基金
supported by grants from Fundacao de Amparoa Pesquisa do Estado de Sao Paulo(FAPESP)
Conselho Nacional de Desenvolvimento Cientifico e Tecnologico(CNPq).
