Na-K-2Cl cotransporter inhibition impairs human lung cellular proliferation
Na-K-2Cl cotransporter inhibition impairs human lung cellular proliferation
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DOI：
10.1152/ajplung.00021.2004
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发表时间：
2004-09-01
影响因子：
4.9
通讯作者：
Wada, RK
中科院分区：
文献类型：
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作者：
Iwamoto, LM;Fujiwara, N;Wada, RK
Na-K-2Cl（NKCC）协同转运蛋白的广泛存在表明长期给予抑制剂可能导致不良反应。认为袢利尿剂对NKCC协同转运蛋白的抑制是该类药物利尿和肺平滑肌松弛作用的基础。然而，这种协同转运蛋白对盐和水运动的基本调节表明，它也可能介导细胞周期进程中发生的细胞体积变化。因此，我们假设髓袢利尿剂抑制NKCC协同转运蛋白会降低细胞增殖。与对照组相比，正常人支气管平滑肌细胞（BSMC）暴露于布美他尼（n = 5 - 10）和呋塞米（n = 6 - 16）7天后，细胞计数显示出显著的浓度依赖性降低。在正常人肺成纤维细胞（n = 5 - 9）中增殖也受到类似的抑制。为了确定这是否是由于细胞损失，我们对BSMC进行了细胞凋亡测定。在暴露于10 μ M布美他尼的BSMC中，膜联蛋白V-碘化丙啶染色（n = 5 - 10）和单细胞凝胶电泳测定（n = 4）均为坏死和凋亡阴性。随后通过流式细胞术分析细胞周期显示，与对照组相比，布美他尼暴露的BSMC在G（1）期延迟（n = 4 - 8）。这是袢利尿剂抑制气道平滑肌细胞增殖的第一个证据。NKCC协同转运蛋白抑制阻碍了G（1）-S相转变，但不促进细胞死亡。因此，尽管袢利尿剂的抑制作用使气道平滑肌松弛，但NKCC协同转运蛋白在细胞增殖调节中可能具有更重要的作用。
The widespread presence of the Na-K-2Cl (NKCC) cotransporter protein suggests that chronic administration of inhibitors may result in adverse effects. Inhibition of the NKCC cotransporter by loop diuretics is felt to underlie the diuretic and the pulmonary smooth muscle relaxant effects of this drug class. However, the fundamental regulation of salt and water movement by this cotransporter suggests that it may also mediate cell volume changes occurring during cell cycle progression. Thus we hypothesized that NKCC cotransporter inhibition by loop diuretics would decrease cellular proliferation. Normal human bronchial smooth muscle cells (BSMC) showed a significant concentration-dependent decrease in cell counts after 7 days of exposure to both bumetanide (n = 5 - 10) and furosemide ( n = 6 - 16) compared with controls. Proliferation was similarly inhibited in normal human lung fibroblasts ( n = 5 - 9). To determine whether this was due to loss of cells, we performed apoptosis assays on BSMC. Both annexin V-propidium iodide staining ( n = 5 - 10) and single cell gel electrophoresis assays ( n = 4) were negative for necrosis and apoptosis in BSMC exposed to 10 muM bumetanide. Subsequent analysis of the cell cycle by flow cytometry showed that bumetanide-exposed BSMC were delayed in G(1) phase compared with controls ( n = 4 - 8). This is the first evidence for loop diuretic inhibition of airway smooth muscle cell proliferation. NKCC cotransporter inhibition impeded G(1)-S phase transition without facilitating cell death. Thus although inhibition by loop diuretics relaxes airway smooth muscle, the NKCC cotransporter may have a more important role in cell proliferation regulation.