Activation of cloned BKCa channels in nitric oxide-induced apoptosis of HEK293 cells
Activation of cloned BKCa channels in nitric oxide-induced apoptosis of HEK293 cells
复制标题
一氧化氮诱导 HEK293 细胞凋亡中克隆 BKCa 通道的激活
DOI：
10.1007/s10495-009-0423-x
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发表时间：
2010-04-01
期刊：
影响因子：
7.2
通讯作者：
Xie, Man-Jiang
中科院分区：
文献类型：
--
作者：
Ma, Yu-Guang;Dong, Ling;Xie, Man-Jiang
大电导Ca（2+）激活的K(+) （BK(Ca)）通道在血管平滑肌细胞（VSMCs）中高度表达，并在多种生理功能的调节中发挥重要作用。除了其在血管舒张中的电生理功能外，BK（Ca）也被报道与一氧化氮（NO）诱导的VSMCs凋亡有关。然而，克隆通道的分子机制尚不清楚，尚未确定。本研究旨在阐明克隆BK（Ca）通道的激活是否参与no诱导的人胚胎肾293 （HEK293）细胞凋亡。将编码BK（Ca）通道α亚基的cDNA hSloalpha瞬时转染到HEK293细胞中。采用免疫细胞化学、琼脂糖凝胶电泳、MTT试验和流式细胞术检测HEK-hSloalpha细胞的凋亡。HEK-hSloalpha细胞的全细胞和单通道特征显示出与VSMCs中天然BK（Ca）通道相似的功能特征。暴露于s -亚硝基-n -乙酰青霉胺的HEK- hSloalpha细胞增加了全细胞和单通道的hSloalpha通道活性，并增加了细胞凋亡的百分比。然而，用1 mM四乙胺或100 nM iberiotoxin阻断hSloalpha通道可显著降低no诱导的细胞凋亡，而BK（Ca）的特异性激动剂NS1619可单独增加hSloalpha电流并诱导细胞凋亡。这些结果表明克隆的BK（Ca）通道的激活参与了no诱导的HEK293细胞凋亡。
The large conductance Ca(2+)-activated K(+) (BK(Ca)) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK(Ca) has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK(Ca) channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the alpha-subunit of BK(Ca) channel, hSloalpha, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloalpha cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloalpha cells exhibited functional features similar to native BK(Ca) channel in VSMCs. Exposuring of HEK- hSloalpha cells to S-nitroso-N-acetyl-penicillamine increased the hSloalpha channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloalpha channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 microM NS1619, the specific agonist of BK(Ca), independently increased hSloalpha currents and induced apoptosis. These results indicated that activation of cloned BK(Ca) channel was involved in NO-induced apoptosis of HEK293 cells.