Karhapää L, Titievsky A, Kaila K, Törnquist K
Department of Biosciences, University of Helsinki, Finland.
Cell Calcium. 1996 Dec;20(6):447-57. doi: 10.1016/s0143-4160(96)90086-x.
In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate intracellular free Ca2+ concentration ([Ca2+]i) in GH4C1 pituitary cells. In intact Fura-2 loaded cells, low concentrations of thimerosal potentiated the spike phase of the TRH-induced (thyrotropin-releasing hormone) rise in [Ca2+]i, whereas high thimerosal concentrations inhibited it. The effect of thimerosal on the plateau phase was always inhibitory. The effect of thimerosal on the IP3-induced calcium release (IICR) was studied in permeabilized cells using the Ca2+ indicator Fluo-3. A low concentration of thimerosal (10 microM) stimulated IICR: the Ca2+ release induced by 300 nM inositol-1,4,5-trisphosphate (IP3) was enhanced in cells treated with thimerosal for 1 or 6 min (67 +/- 11 nM and 34 +/- 5 nM, respectively) as compared to control cells (17 +/- 2 nM). On the other hand, a high concentration of thimerosal (100 microM) inhibited IICR: when IP3 (10 microM) was added after a 5 min preincubation with thimerosal, the IP3-induced rise in [Ca2+]i (46 +/- 14 nM) was 57% smaller as compared with that seen in control cells (106 +/- 10 nM). The effect of thimerosal on the voltage-operated Ca2+ channels (VOCCs) was studied by depolarizing intact Fura-2 loaded cells by addition of 20 mM K+ to the cuvette. The depolarization-evoked increase in [Ca2+]i was inhibited in a dose-dependent manner by thimerosal. Direct evidence for an inhibitory effect of thimerosal on VOCCs was obtained by using the whole-cell configuration of the patch-clamp technique: thimerosal (100 microM) potently inhibited the Ba2+ currents through VOCCs. In addition, our results indicated that thimerosal inhibited the caffeine-induced increase in [Ca2+]i, and activated a capacitative Ca2+ entry pathway. The actions of thimerosal were apparently due to its oxidizing activity because the effects were mostly reversed by the thiol-reducing agent dithiothreitol (DTT). We conclude that, in GH4C1 pituitary cells, the mobilization of intracellular calcium and the different Ca2+ entry pathways are sensitive to redox modulation.
在本研究中，我们研究了氧化型巯基试剂硫柳汞对调节GH4C1垂体细胞内游离钙离子浓度（[Ca2+]i）的不同机制的作用。在完整的、负载Fura-2的细胞中，低浓度的硫柳汞增强了促甲状腺激素释放激素（TRH）诱导的[Ca2+]i升高的峰值阶段，而高浓度的硫柳汞则抑制了该阶段。硫柳汞对平台期的作用始终是抑制性的。使用钙离子指示剂Fluo-3在透化细胞中研究了硫柳汞对肌醇-1,4,5-三磷酸（IP3）诱导的钙释放（IICR）的影响。低浓度的硫柳汞（10 microM）刺激IICR：与对照细胞（17 +/- 2 nM）相比，用硫柳汞处理1或6分钟的细胞中，由300 nM IP3诱导的钙释放增强（分别为67 +/- 11 nM和34 +/- 5 nM）。另一方面，高浓度的硫柳汞（100 microM）抑制IICR：在用硫柳汞预孵育5分钟后加入IP3（10 microM）时，IP3诱导的[Ca2+]i升高（46 +/- 14 nM）比对照细胞中观察到的升高（106 +/- 十 nM）小57%。通过向比色皿中加入20 mM K+使完整的、负载Fura-2的细胞去极化，研究了硫柳汞对电压门控性钙离子通道（VOCCs）的影响。硫柳汞以剂量依赖性方式抑制去极化诱发的[Ca2+]i升高。通过使用膜片钳技术的全细胞配置获得了硫柳汞对VOCCs具有抑制作用的直接证据：硫柳汞（100 microM）强烈抑制通过VOCCs的Ba2+电流。此外，我们的结果表明硫柳汞抑制咖啡因诱导的[Ca2+]i升高，并激活了一种容量性钙离子内流途径。硫柳汞的作用显然归因于其氧化活性，因为这些作用大多被巯基还原剂二硫苏糖醇（DTT）逆转。我们得出结论，在GH4C1垂体细胞中，细胞内钙的动员和不同的Ca2+内流途径对氧化还原调节敏感。
