Ouabain-resistant (Na+,K+)-ATPase enzyme activity in chemically induced ouabain-resistant C3H/10T1/2 cells.

Molecular toxicology Pub Date : 1989-04-01
M L Shibuya, T Miura, J R Lillehaug, R A Farley, J R Landolph
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Abstract

To further understand the molecular nature of changes leading to chemically induced ouabain resistance in C3H/10T1/2 Cl 8 (10T1/2) cells, we isolated plasma membranes from wild-type and ouabain-resistant (Ouar) 10T1/2 cells and characterized (Na,K)-ATPase activity in the plasma membrane fraction. (Na+,K+)-ATPase enzyme activity in membrane fractions extracted from wild-type 10T1/2 cells was inhibited in a concentration-dependent manner by ouabain and was completely inhibited by 2.4 mM ouabain. Lineweaver-Burke and Eisenthal/Cornish-Bowden analysis indicated that the inhibition was uncompetitive. Ten to 45% of (Na+,K+)-ATPase enzyme activity extracted from three Ouar 10T1/2 cell lines cultured in 1 mM ouabain was resistant to 2.4 mM ouabain, depending on the cell line. Resistance of (Na+,K+)-ATPase activity in the plasma membrane fraction of Ouar cells to inhibition by ouabain and resistance of cultured Ouar cells to the cytotoxicity of ouabain occurred over similar concentrations of ouabain (0.1-3mM). Two ouabain-resistant cell lines, Ouar MNNG Cl 2 and Ouar MCA Cl 16-7, demonstrated the same total (Na+,K+)-ATPase specific activity as 10T1/2 cells, but the fraction of Ouar enzyme activity increased (from 18 to 40% in MNNG Cl 2 cells and from 10 to 25% in Sp Ouar Cl 16 cells) when the cells were cultured in ouabain. Thermal denaturation profiles and pH dependence profiles of (Na+,K+)-ATPase activity in plasma membranes from wild-type and Ouar 10T1/2 cells were identical. A 3.9-kb (Na,K)-ATPase alpha subunit mRNA transcript was found in 10T1/2 cells, and in the Ouar MNNG Cl 2 cell line cultured in the presence or absence of ouabain. There was no amplification of the gene coding for the alpha subunit of (Na+,K+)-ATPase in the chemically induced Ouar MNNG Cl 2 cell line, whether this cell line was cultured in the presence or absence of ouabain. These studies provide further evidence that the Ouar phenotype of chemically induced and spontaneous Ouar 10T1/2 cell lines derives from Ouar (Na+,K+)-ATPase activity, and that this Ouar (Na+,K+)-ATPase activity increases further in some cell lines cultured in the presence of ouabain.

化学诱导的抗瓦阿因C3H/10T1/2细胞耐(Na+,K+)- atp酶活性
为了进一步了解化学诱导C3H/10T1/2 Cl 8 (10T1/2)细胞对瓦阿因耐药变化的分子性质,我们分离了野生型和抗瓦阿因(Ouar) 10T1/2细胞的质膜,并对质膜部分(Na,K)- atp酶活性进行了表征。(Na+,K+)-ATPase酶活性在野生型10T1/2细胞的膜组分中被瓦阿因呈浓度依赖性抑制,2.4 mM瓦阿因完全抑制。Lineweaver-Burke和Eisenthal/Cornish-Bowden分析表明抑制是非竞争性的。从3株在1mm瓦巴因中培养的Ouar 10T1/2细胞系中提取的(Na+,K+)- atp酶活性的10% ~ 45%对2.4 mM瓦巴因具有抗性,这取决于细胞系。在相同浓度(0.1 ~ 3mm)的瓦阿因中,Ouar细胞的质膜部分(Na+,K+)- atp酶活性对瓦阿因的抑制产生抗性,培养的Ouar细胞对瓦阿因的细胞毒性产生抗性。两个抗瓦阿因细胞系,即Ouar MNNG Cl 2和Ouar MCA Cl 16-7,表现出与10T1/2细胞相同的总(Na+,K+)- atp酶特异性活性,但当细胞在瓦阿因中培养时,Ouar酶活性的比例增加(MNNG Cl 2细胞从18%增加到40%,Sp Ouar Cl 16细胞从10%增加到25%)。野生型和Ouar 10T1/2细胞质膜(Na+,K+)- atp酶活性的热变性谱和pH依赖性谱相同。在10T1/2细胞和存在或不存在瓦巴因培养的Ouar MNNG Cl 2细胞系中发现了3.9 kb (Na,K)- atp酶α亚基mRNA转录物。化学诱导的(Na+,K+)- atp酶α亚基编码基因在化学诱导的Ouar MNNG Cl 2细胞系中均未扩增,无论该细胞系在有或无瓦巴因的情况下培养。这些研究进一步证明,化学诱导的和自发的Ouar 10T1/2细胞系的Ouar表型来源于Ouar (Na+,K+)- atp酶活性,并且在一些存在瓦巴因培养的细胞系中,这种Ouar (Na+,K+)- atp酶活性进一步增加。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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