The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3.radical.
The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3.radical.
复制标题
DOI：
10.1016/s0021-9258(19)38899-4
复制
发表时间：
1990-05
期刊：
影响因子：
--
通讯作者：
S. Srivastava;N. Chen;J. Holtzman
中科院分区：
文献类型：
--
作者：
S. Srivastava;N. Chen;J. Holtzman
四氯化碳的肝毒性是通过细胞色素P-450将其还原为四氯化碳自由基来介导的。这种自由基然后损害重要的代谢系统，如ATP依赖性微粒体Ca 2+泵。我们实验室以前对分离微粒体的研究表明，在没有毒性物质的情况下，NADPH会抑制这种泵。我们现在已经发现，在体外孵育中，CCl 4（0.5-2.5 mM）增强了NADPH依赖性的抑制Ca 2+的摄取，从28%，没有CCl 4的最大值为68%。这些浓度在致死中毒动物的肝脏和血液中发现的范围内（Dambrauskas，T.，和康沃尔语，H. H.（1970）Toxicol.应用药理学17，83-97; Long，R.M.，和摩尔，L.（1988）Toxicol.应用药理学92，295-306）并且对培养的肝细胞有毒（Long，R. M.，和摩尔，L.（1988）Toxicol.应用药理学92，295-306）。Ca ~（2+）摄取的抑制是由于Ca ~（2+）依赖性ATP酶活性降低和Ca ~（2+）从微粒体释放增加所致。NADPH依赖的CCl_4抑制作用在N_2下更大，并且完全被CO阻止。在与CCl_4孵育期间加入GSH（1-10 mM）阻止了抑制作用。当在氮气下进行孵育时，也可以看到这种保护作用。当样品与CCl 4预孵育时，CCl 4代谢停止，然后测定Ca 2+摄取; GSH逆转CCl 4对Ca 2+摄取的抑制。这种逆转表现出饱和动力学GSH与两个Km值为0.315和93 μ M时，预孵育和Ca 2+的吸收在空气中进行，和0.512和31 μ M时，都在氮气下进行。半胱氨酸不能阻止NADPH依赖的CCl 4抑制Ca ~（2+）摄取。CCl 4增加了空气中的脂质过氧化作用，但没有脂质过氧化作用的氮。脂质过氧化反应仅轻微逆转GSH。GSH没有去除与14 CCl 4预孵育的样品结合的14 C。尽管EDTA（100 μ M）降低了CCl 4的抑制作用，但金属络合剂去铁胺（100 μ M）和二乙基二硫代氨基甲酸盐（100 μ M）对泵的抑制作用没有影响。类似地，活性氧清除剂过氧化氢酶（65微克/毫升）、超氧化物歧化酶（15微克/毫升）、甘露醇（10 mM）和二甲亚砜（50 mM）也没有影响。我们的研究结果表明，初始毒性的CCl 4的Ca 2+泵的结果从CCl 4的代谢CCl 3。激进然后，这种自由基直接氧化Ca 2+泵，导致Ca 2+吸收减少。（400字处截断摘要）
The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3.radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals (Dambrauskas, T., and Cornish, H. H. (1970) Toxicol. Appl. Pharmacol. 17, 83-97; Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306) and are toxic to cultured hepatocytes (Long, R. M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306). The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4. Although EDTA (100 microM) decreased the CCl4 inhibition, the metal-complexing agents deferoxamine (100 microM) and diethyldithiocarbamate (100 microM) had no effect on the inhibition of the pump. Similarly, the reactive oxygen scavengers catalase (65 micrograms/ml), superoxide dismutase (15 micrograms/ml), mannitol (10 mM), and dimethyl sulfoxide (50 mM) also had no effect. Our results suggest that the initial toxicity of CCl4 for the Ca2+ pump results from the metabolism of CCl4 to the CCl3. radical. This radical then directly oxidizes the Ca2+ pump, leading to decreased Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)