High Glucose Alters Apoptosis and Proliferation in HEK293 Cells by Inhibition of Cloned BKCa Channel
High Glucose Alters Apoptosis and Proliferation in HEK293 Cells by Inhibition of Cloned BKCa Channel
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高葡萄糖通过抑制克隆的 BKCa 通道改变 HEK293 细胞的凋亡和增殖
DOI：
10.1002/jcp.22497
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发表时间：
2011-06-01
影响因子：
5.6
通讯作者：
Xie, Man-Jiang
中科院分区：
文献类型：
--
作者：
Chang, Hui;Ma, Yu-Guang;Xie, Man-Jiang
糖尿病血管功能障碍与大电导钙激活钾通道（BKCa）功能受损有关。然而，目前尚不清楚受损的BKCa通道是否直接参与调节糖尿病血管重塑，改变细胞生长反应高血糖。在本研究中，我们研究了在高浓度葡萄糖（25 mM）下BKCa通道在控制细胞凋亡和增殖中的具体作用。将编码BKCa通道α + β 1亚基的cDNA hSlo α + β 1瞬时转染入人胚肾293（HEK 293）细胞。采用全细胞膜片钳技术和细胞贴附膜片钳技术记录克隆的BKCa电流。用免疫细胞化学和琼脂糖凝胶电泳分析DNA片段来评估细胞凋亡。流式细胞术、MTT法、免疫细胞化学法检测细胞增殖情况。此外，还检测了抗凋亡蛋白Bcl-2的表达、细胞内Ca 2+和线粒体膜电位（Delta psi m），以探讨可能的机制。我们的研究结果表明，抑制克隆BKCa通道可能是负责高血糖改变HEK-hSlo α + β 1细胞的凋亡和增殖。然而，在高血糖条件下，NS 1619或他莫昔芬激活BKCa通道显著诱导HEK-hSlo α + β 1细胞凋亡并抑制其增殖。当大鼠脑平滑肌细胞培养在高血糖，观察到类似的结果。此外，BKCa通道激活的可能机制与Bcl-2表达降低、细胞内Ca 2+升高以及HEK-hSlo α + β 1细胞中Δ psi m的伴随去极化有关。结论：克隆的BKCa通道直接调控高糖条件下HEK 293细胞的凋亡和增殖。J.细胞。226：1660-1675，2011。(C)2010 Wiley-Liss，Inc.
It has been reported that diabetic vascular dysfunction is associated with impaired function of large conductance Ca2+-activated K+ (BKCa) channels. However, it is unclear whether impaired BKCa channel directly participates in regulating diabetic vascular remodeling by altering cell growth in response to hyperglycemia. In the present study, we investigated the specific role of BKCa channel in controlling apoptosis and proliferation under high glucose concentration (25 mM). The cDNA encoding the alpha+beta 1 subunit of BKCa channel, hSlo alpha+beta 1, was transiently transfected into human embryonic kidney 293 (HEK293) cells. Cloned BKCa currents were recorded by both whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry and analysis of fragmented DNA by agarose gel electrophoresis. Cell proliferation was investigated by flow cytometry assays, MTT test, and immunocytochemistry. In addition, the expression of anti-apoptotic protein Bcl-2, intracellular Ca2+, and mitochondrial membrane potential (Delta psi m) were also examined to investigate the possible mechanisms. Our results indicate that inhibition of cloned BKCa channels might be responsible for hyperglycemia-altered apoptosis and proliferation in HEK-hSlo alpha+beta 1 cells. However, activation of BKCa channel by NS1619 or Tamoxifen significantly induced apoptosis and suppressed proliferation in HEK-hSlo alpha+beta 1 cells under hyperglycemia condition. When rat cerebral smooth muscle cells were cultured in hyperglycemia, similar findings were observed. Moreover, the possible mechanisms underlying the activation of BKCa channel were associated with decreased expression of Bcl-2, elevation of intracellular Ca2+, and a concomitant depolarization of Delta psi m in HEK-hSlo alpha+beta 1 cells. In conclusion, cloned BKCa channel directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemia condition. J. Cell. Physiol. 226: 1660-1675, 2011. (C) 2010 Wiley-Liss, Inc.