Reader S, Moutardier V, Denizeau F
Université du Québec, Montréal, Département de Chimie, Canada.
Biochim Biophys Acta. 1999 Jan 11;1448(3):473-85. doi: 10.1016/s0167-4889(98)00166-9.
The purpose of the present study was to study the mechanisms involved in the induction of apoptosis and by tributyltin (TBT) in rainbow trout hepatocytes, and to examine the role of intracellular Ca2+, protein kinase C (PKC) and proteases in the apoptotic process. The intracellular Ca2+ chelator BAPTA-AM has a suppressive effect on TBT-mediated apoptosis. However, exposure to the ionophore A23187 is not sufficient to induce apoptosis in trout hepatocytes. The results obtained also show that TBT stimulates PKC gamma and delta translocation from cytosol to the plasma membrane in trout hepatocytes after 30 min of exposure. However, PKC gamma translocation is down-regulated after 90 min of treatment. The addition of protein kinase inhibitors (staurosporine and H-7) not only fails to inhibit apoptosis induced by TBT, but also leads to enhancement of DNA fragmentation. These inhibitors also afford a remarkable protection against the loss of plasma membrane integrity caused by TBT exposure. PMA, a direct activator of PKC, fails to stimulate DNA fragmentation. In addition, Z-VAD.FMK is an extremely potent inhibitor of TBT-induced apoptosis in trout hepatocytes, indicating that the activation of ICE-like proteases is a key event in this process. The cysteine protease inhibitor N-ethylmaleimide also prevented TBT-induced DNA fragmentation. Taken together, these data allow for the first time to suggest a mechanistic model of TBT-induced apoptosis. We propose that TBT could trigger apoptosis through a step involving Ca2+ efflux from the endoplasmic reticulum or other intracellular pools and by mechanisms involving cysteine proteases, such as calpains, as well as the phosphorylation status of apoptotic proteins such as Bcl-2 homologues.
本研究的目的是探讨三丁基锡（TBT）诱导虹鳟肝细胞凋亡的机制，并研究细胞内Ca2+、蛋白激酶C（PKC）和蛋白酶在凋亡过程中的作用。细胞内Ca2+螯合剂BAPTA-AM对TBT介导的凋亡具有抑制作用。然而，暴露于离子载体A23187不足以诱导鳟鱼肝细胞凋亡。所得结果还表明，TBT暴露30分钟后可刺激虹鳟肝细胞中PKCγ和δ从胞质溶胶向质膜转位。然而，处理90分钟后PKCγ转位下调。添加蛋白激酶抑制剂（星形孢菌素和H-7）不仅不能抑制TBT诱导的凋亡，反而导致DNA片段化增强。这些抑制剂还能显著保护细胞免受TBT暴露导致的质膜完整性丧失。PKC的直接激活剂PMA不能刺激DNA片段化。此外，Z-VAD.FMK是TBT诱导虹鳟肝细胞凋亡的极强抑制剂，表明ICE样蛋白酶的激活是该过程中的关键事件。半胱氨酸蛋白酶抑制剂N-乙基马来酰亚胺也可防止TBT诱导的DNA片段化。综上所述，这些数据首次提出了TBT诱导凋亡的机制模型。我们认为，TBT可能通过涉及内质网或其他细胞内池Ca2+外流的步骤以及涉及半胱氨酸蛋白酶（如钙蛋白酶）的机制，以及凋亡蛋白（如Bcl-2同源物）的磷酸化状态来触发凋亡。
