烷化剂敏感的L1210小鼠白血病细胞中Na+/K+/Cl-共转运体的特性研究

Characterisation of a Na+/K+/Cl- cotransporter in alkylating agent-sensitive L1210 murine leukemia cells.

作者信息

Wilcock C, Hickman J A

机构信息

Cancer Research Campaign Experimental Chemotherapy Group, Aston University, Birmingham, U.K.

出版信息

Biochim Biophys Acta. 1988 Dec 22;946(2):359-67. doi: 10.1016/0005-2736(88)90411-7.

DOI:10.1016/0005-2736(88)90411-7

PMID:3207751

Abstract

The mode of influx of 86Rb+, a K+ congener, to exponentially proliferating L1210 murine leukemia cells, incubated in a Krebs-Ringer buffer, has been characterised. The influx was composed of a ouabain-sensitive fraction (approx. 40%), a loop diuretic-sensitive fraction (approx. 40%) and a fraction which was insensitive to both types of inhibitor (approx. 15%). The fraction of ouabain-insensitive 86Rb+ influx, which was fully inhibited by furosemide (1 mM) or bumetanide (100 microM), was completely inhibited when Cl- was completely substituted by nitrate or gluconate ions, but was slightly (29 +/- 12%) stimulated if the Cl- was substituted by Br-. The substitution of Na+ by Li+, choline or tetramethylammonium ions inhibited the loop diuretic-sensitive fraction of 86Rb+ uptake. These results suggested that a component of 86Rb+ influx to L1210 cells was mediated via a Na+/K+/Cl- cotransporter. 86Rb+ efflux from L1210 cells which had been equilibrated with 86Rb+ and incubated in the presence or absence of 1 mM ouabain, was insensitive to the loop diuretics. Additionally, efflux rates were found to be independent of the external concentration of K+, suggesting that efflux was not mediated by K+-K+ exchange. The initial rate of 86Rb+ influx to L1210 cells in the plateau phase of growth was reduced to 44% of that of exponentially dividing cells, the reduction being accounted for by significant decreases in both ouabain- and loop diuretic-sensitive influx; these cells were reduced in volume compared to cells in the exponential phase of cell growth. In cells which had been deprived of serum for 18 h, and which showed an increase of the proportion of cells in the G1 phase of the cell cycle, the addition of serum stimulated an immediate increase in the furosemide-sensitive component of 86Rb+ influx. Diuretic-sensitive 86Rb+ influx was not altered by the incubation of the cells with 100 microM dibutyryl cyclic AMP, but was inhibited by 10 microM of the cross-linking agent nitrogen mustard (bis(2-chloro-ethyl)methylamine, HN2).

摘要

已对在 Krebs-Ringer 缓冲液中培养的指数增殖的 L1210 小鼠白血病细胞摄取钾离子类似物 86Rb⁺的方式进行了表征。摄取由哇巴因敏感部分（约 40%）、襻利尿剂敏感部分（约 40%）和对两种抑制剂均不敏感的部分（约 15%）组成。哇巴因不敏感的 86Rb⁺摄取部分，可被呋塞米（1 mM）或布美他尼（100 μM）完全抑制，当 Cl⁻被硝酸根离子或葡萄糖酸根离子完全替代时，该部分摄取被完全抑制，但如果 Cl⁻被 Br⁻替代，则摄取略有增加（29±12%）。用 Li⁺、胆碱或四甲基铵离子替代 Na⁺可抑制 86Rb⁺摄取的襻利尿剂敏感部分。这些结果表明，L1210 细胞摄取 86Rb⁺的一个成分是通过 Na⁺/K⁺/Cl⁻共转运体介导的。在存在或不存在 1 mM 哇巴因的情况下培养并已与 86Rb⁺平衡的 L1210 细胞的 86Rb⁺外流对襻利尿剂不敏感。此外，发现外流速率与细胞外 K⁺浓度无关，这表明外流不是由 K⁺-K⁺交换介导的。生长平台期 L1210 细胞摄取 86Rb⁺的初始速率降至指数分裂细胞的 44%，这种降低是由于哇巴因和襻利尿剂敏感摄取均显著降低所致；与细胞生长指数期的细胞相比，这些细胞体积减小。在血清剥夺 18 小时且细胞周期 G1 期细胞比例增加的细胞中，添加血清可立即刺激 86Rb⁺摄取的呋塞米敏感成分增加。用 100 μM 二丁酰环磷腺苷孵育细胞不会改变利尿剂敏感的 86Rb⁺摄取，但会被 10 μM 的交联剂氮芥（双（2-氯乙基）甲胺，HN2）抑制。