Stoyanovsky D A, Cederbaum A I
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Biochemistry. 1996 Dec 10;35(49):15839-45. doi: 10.1021/bi961295p.
Elevation of cytosolic calcium levels has been shown to occur after exposure to hepatotoxins such as CCl4. This has been associated with inhibition of the Ca2+, Mg(2+)-ATPase which pumps calcium into the endoplasmic reticulum. Elevated cytosolic Ca2+ may also result from activation of calcium releasing channels. In the presence of NADPH, CCl4 produced a concentration-dependent release of calcium from liver microsomes after a lag period. The lag period was shorter with microsomes from pyrazole-treated rats in which CYP2E1 is induced, as compared to saline microsomes. The calcium releasing process appears to be very sensitive to activation by CCl4 as effective concentrations (e.g., 50 microM) did not affect the Ca2+, Mg(2+)-ATPase or produce lipid peroxidation. Inhibition of the CCl4-induced release of calcium by 4-methylpyrazole and by anti-CYP2E1 IgG, and the requirement for NADPH, indicates that CCl4 metabolism is required for the activation of calcium release. The lag period for CCl4-induced release of calcium was associated with the time required to deplete alpha-tocopherol from the microsomal membranes; however, lipid peroxidation was not observed at these levels of CCl4, and the lag period for CCl4-induced release of calcium was shorter under anaerobic than aerobic conditions, suggesting a possible role for CCl3 in the mechanism of activation. Production of CCl3 was observed by ESR spin-trapping experiments with PBN; PBN prevented the CCl4-induced calcium release, presumably by interacting with CCl3 and other reactive species. Calcium release was produced by thiol oxidants such as 2,2'-dithiodipyridine. Lipophilic thiols such as mercaptoethanol or cysteamine could partially reverse the CCl4-induced calcium release, whereas GSH was ineffective. While the IP3 receptor system is considered as the main regulator of calcium release, liver also contains ryanodine-sensitive calcium releasing stores. The CCl4-induced calcium release was blocked by ruthenium red, a specific inhibitor of the ryanodine receptor; ruthenium red did not block CCl4 metabolism to CCl3. CCl4 increased the binding of ryanodine, a specific ligand for the ryanodine-sensitive calcium channel. These results suggest that metabolism of CCl4 to reactive species by cytochrome P450 results in an activation of a ryanodine-sensitive calcium channel, perhaps due to oxidation of lipophilic thiols of the channel. Activation of calcium releasing channels may play a role in the elevated cytosolic calcium levels found in the liver after treatment with hepatotoxins.
已表明暴露于如四氯化碳等肝毒素后，胞质钙水平会升高。这与抑制将钙泵入内质网的Ca2 +、Mg(2 +)-ATP酶有关。胞质Ca2 +升高也可能源于钙释放通道的激活。在存在NADPH的情况下，四氯化碳在一段延迟期后从肝微粒体中产生浓度依赖性的钙释放。与盐水处理的微粒体相比，来自经吡唑处理的大鼠（其中CYP2E1被诱导）的微粒体的延迟期更短。钙释放过程似乎对四氯化碳的激活非常敏感，因为有效浓度（例如50 microM）不会影响Ca2 +、Mg(2 +)-ATP酶或产生脂质过氧化。4-甲基吡唑和抗CYP2E1 IgG对四氯化碳诱导的钙释放的抑制作用，以及对NADPH的需求，表明四氯化碳代谢是钙释放激活所必需的。四氯化碳诱导的钙释放的延迟期与从微粒体膜中耗尽α-生育酚所需的时间相关；然而，在这些四氯化碳水平下未观察到脂质过氧化，并且四氯化碳诱导的钙释放的延迟期在厌氧条件下比需氧条件下更短，这表明CCl3在激活机制中可能起作用。通过用PBN进行ESR自旋捕获实验观察到了CCl3的产生；PBN可能通过与CCl3和其他活性物质相互作用来阻止四氯化碳诱导的钙释放。硫醇氧化剂如2,2'-二硫代二吡啶可产生钙释放。亲脂性硫醇如巯基乙醇或半胱胺可部分逆转四氯化碳诱导的钙释放，而谷胱甘肽则无效。虽然IP3受体系统被认为是钙释放的主要调节因子，但肝脏中也含有对ryanodine敏感的钙释放储存。四氯化碳诱导的钙释放被ryanodine受体的特异性抑制剂钌红阻断；钌红不会阻断四氯化碳代谢为CCl3。四氯化碳增加了ryanodine（一种对ryanodine敏感的钙通道的特异性配体）的结合。这些结果表明，细胞色素P450将四氯化碳代谢为活性物质会导致对ryanodine敏感的钙通道的激活，这可能是由于通道亲脂性硫醇的氧化。钙释放通道的激活可能在肝毒素处理后肝脏中发现的胞质钙水平升高中起作用。
