Xi D, Kurtz D T, Ramsdell J S
Marine Biotoxins Program of the U.S. Marine Fisheries Services, Charleston, SC 29412, USA
Biochem Pharmacol. 1996 Mar 22;51(6):759-69. doi: 10.1016/0006-2952(95)02392-5.
Maitotoxin includes an extracellular Ca2+-dependent membrane depolarization predominantly via activation of L-type voltage-dependent Ca2+ channels (L-VDCC) in GH4C1 rat pituitary cells. In contract to studies employing intracellular dyes, electrophysiological studies have indicated that maitotoxin activates voltage-independent conductances. In the present study, we used fura-2 calcium digital analysis to investigate the actions of very low concentrations of maitotoxin on cytosolic free calcium ([Ca2+]i) in GH4C1 cells in an effort to distinguish different calcium entry mechanisms. Maitotoxin at concentrations as low as 0.01 ng/mL elevated [Ca2+]i 35 +/- 3% and induced membrane depolarization. The concentration dependency for maitotoxin-elevated [Ca2+]i was biphasic with the first phase maximal at 0.05 to 0.5 ng/mL and the minimum EC50 of the second phase about 2.0 ng/mL. Nimodipine (100 nM), a dihydropyridine antagonist of L-VDCC, prevented the [Ca+2]i increase and depolarization induced by up to 0.1 ng/mL maitotoxin, but not at higher concentration (0.5 ng/mL) of maitotoxin. This indicates that lower concentrations (0.1 ng/mL) of maitotoxin require L-VDCC, whereas higher concentrations (>-0.5 ng/mL) of maitotoxin may require additional ionic mechanisms. Maitotoxin (0.5 ng/mL) induced 45Ca2+ uptake and depolarization in Ltk-cells which lack VDCC. Reducing extracellular Cl- from 123 to 5.8 microM increased the magnitude of membrane depolarization by maitotoxin (0.5 ng/mL), which suggests that a Cl- conductance participated in depolarization induced by higher maitotoxin concentrations. Taken together, our results indicate that maitotoxin activates at least two ionic mechanisms. At lower concentrations of maitotoxin, the primary ionic mechanism requires the activation of L-VDCC; however, at higher maitotoxin concentrations, additional ionic mechanisms are involve in the entry of extracellular Ca2+. This latter mechanism may represent the voltage-independent pathway evident under voltage clamp conditions.
刺尾鱼毒素可使细胞外钙离子依赖性膜去极化，主要是通过激活GH4C1大鼠垂体细胞中的L型电压依赖性钙通道（L-VDCC）。与使用细胞内染料的研究不同，电生理学研究表明刺尾鱼毒素可激活电压非依赖性电导。在本研究中，我们使用fura-2钙数字分析来研究极低浓度的刺尾鱼毒素对GH4C1细胞胞质游离钙（[Ca2+]i）的作用，以区分不同的钙内流机制。低至0.01 ng/mL的刺尾鱼毒素可使[Ca2+]i升高35±3%并诱导膜去极化。刺尾鱼毒素升高[Ca2+]i的浓度依赖性呈双相，第一相在0.05至0.5 ng/mL时最大，第二相的最小半数有效浓度（EC50）约为2.0 ng/mL。尼莫地平（100 nM）是一种L-VDCC的二氢吡啶拮抗剂，可阻止高达0.1 ng/mL的刺尾鱼毒素诱导的[Ca+2]i升高和去极化，但对更高浓度（0.5 ng/mL）的刺尾鱼毒素则无效。这表明较低浓度（0.1 ng/mL）的刺尾鱼毒素需要L-VDCC，而较高浓度（>0.5 ng/mL）的刺尾鱼毒素可能需要其他离子机制。刺尾鱼毒素（0.5 ng/mL）可诱导缺乏VDCC的Ltk细胞摄取45Ca2+并使其去极化。将细胞外氯离子浓度从123 μM降至5.8 μM可增加刺尾鱼毒素（0.5 ng/mL）引起的膜去极化幅度，这表明氯离子电导参与了较高浓度刺尾鱼毒素诱导的去极化过程。综上所述，我们的结果表明刺尾鱼毒素至少激活了两种离子机制。在较低浓度的刺尾鱼毒素作用下，主要的离子机制需要激活L-VDCC；然而，在较高浓度的刺尾鱼毒素作用下，其他离子机制参与了细胞外钙离子的内流。后一种机制可能代表了在电压钳制条件下明显的电压非依赖性途径。
