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目的探讨蛋白激酶p38,蛋白激酶C对几种哺乳动物细胞中水通道蛋白9磷酸化的调控作用,以及这些蛋白因子对细胞中砷摄入含量的影响。方法采用免疫印迹法和免疫共沉淀技术分别检测细胞株中p38,蛋白激酶C和水通道蛋白9表达水平及其磷酸化水平。电感藕合等离子体质谱法(ICP-MS)测定细胞内砷含量。结果在ECV-304,AsRE,Hep G2和L-02细胞株中,p38蛋白及其磷酸化表达水平在砷的刺激下随时间增加而有显著上升,相同条件下蛋白激酶C末见明显变化。当抑制细胞中p38活性,除了L-02细胞中水通道蛋白9的磷酸化水平受到抑制外,其余细胞中水通道蛋白9的蛋白含量及磷酸化水平都未见明显变化。砷含量也只在L-02细胞中看到有较显著的下降。结论水通道蛋白9磷酸化水平影响砷进入细胞的速度与含量,但不同的细胞中水通道蛋白9的磷酸化调控机制有所差异。p38蛋白激酶在正常肝细胞L-02中显示对水通道蛋白9磷酸化有一定的调控作用,蛋白激酶C则在本实验条件下未见入调控砷的入胞机制。
Objective To investigate the regulatory effect of protein kinase p38 and protein kinase C on aquaporin 9 phosphorylation in several mammalian cells and the effects of these protein factors on arsenic uptake in cells. Methods Western blotting and co-immunoprecipitation were used to detect the expression of p38, protein kinase C and aquaporin-9 and their phosphorylation levels in cell lines. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Determination of intracellular arsenic content. Results In the ECV-304, AsRE, Hep G2 and L-02 cell lines, the expression of p38 protein and its phosphorylation significantly increased with the increase of time under the stimulation of arsenic. The changes of protein kinase C terminal under the same conditions were observed. Inhibition of p38 activity in cells, in addition to L-02 cells in the aquaporin 9 phosphorylation was inhibited, the remaining cells in the water channel protein 9 protein content and phosphorylation levels were not significantly changed. Arsenic levels also showed a significant decrease only in L-02 cells. Conclusions Aquaporin 9 phosphorylation affects the rate and content of arsenic entering cells, but the phosphorylation mechanism of aquaporin 9 is different in different cells. p38 protein kinase showed a certain regulatory role on aquaporin 9 phosphorylation in normal liver cells L-02, and protein kinase C did not regulate the entry of arsenic in this experiment.