DOWN-REGULATION BY BUTYLATED HYDROXYTOLUENE OF THE NUMBER AND FUNCTION OF GAP-JUNCTIONS IN EPITHELIAL-CELL LINES DERIVED FROM MOUSE LUNG AND RAT-LIVER
DOWN-REGULATION BY BUTYLATED HYDROXYTOLUENE OF THE NUMBER AND FUNCTION OF GAP-JUNCTIONS IN EPITHELIAL-CELL LINES DERIVED FROM MOUSE LUNG AND RAT-LIVER
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DOI：
10.1093/carcin/16.10.2575
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发表时间：
1995-10-01
期刊：
影响因子：
4.7
通讯作者：
RUCH, RJ
中科院分区：
文献类型：
--
作者：
GUAN, X;HARDENBROOK, J;RUCH, RJ
在小鼠肺上皮细胞(C10)和大鼠肝上皮细胞(WB-F344)细胞间隙连接通讯(GJIC)中，荧光染料显微注射法检测到BHT以剂量和时间依赖的方式抑制这两种细胞的GJIC。当BHT浓度大于或等于62.5mU/M时，对WB-F344细胞和C10细胞均有抑制作用；当BHT浓度大于或等于150mU/M时，作用4h后，抑制作用在15-30min内发生，并可被从培养液中去除BHT所逆转实验还检测了毒性较大的BHT代谢产物6-叔丁基-2-(羟基-叔丁基)-4-甲基苯酚(BHTOH)和无毒的BHT代谢产物2，6-二叔丁基-4-羟甲基苯酚(BHTBzOH)，结果表明，BHTOH对GJIC的抑制作用强于BHT，而BHTBzOH对GJIC的抑制作用不明显，并探讨了BHT抑制GJIC的机制。在15~30min内观察到的快速抑制可能是由于缝隙连接通道的关闭或阻断，因为在2~4h内观察到缝隙连接数目、连接蛋白(Cx)43水平或Cx43磷酸化水平没有变化，但缝隙连接进入细胞质内，可检测到的质膜缝隙连接数量减少，磷酸化的Cx43-P2减少，12-O-十四烷酰佛波醇-13-乙酸酯(TPA)处理细胞24 h可阻止TPA对GJIC的抑制，但BHT不能。对TPA处理的WB-F344或C10细胞的Western印迹分析显示，Cx43(Cx43-P3)存在过度磷酸化，Cx43-P2中存在NO减少，与BHT处理的细胞相比，这些数据表明BHT和TPA通过不同的机制抑制肺和肝上皮细胞GJIC。
The mouse pneumotoxicant and lung and liver tumor promoter butylated hydroxytoluene (BHT) was examined for its effects on gap junctional intercellular communication (GJIC) in mouse lung epithelial (C10) and rat liver epithelial (WB-F344) cell lines, GJIC, as measured by fluorescent dye microinjection, was inhibited in both types of cells by BHT in dose- and time-dependent fashions, Inhibition was detected in WB-F344 cells at BHT concentrations greater than or equal to 62.5 mu M and in C10 cells at concentrations greater than or equal to 150 mu M after 4 h treatment, Inhibition occurred within 15-30 min and was reversed by removing BHT from the culture medium, The highly toxic BHT metabolite 6-t-butyl-2-(hydroxy-t-butyl)-4-methylphenol (BHTOH) and the non-toxic BHT metabolite, 2,6-di-t-butyl-4-hydroxymethylphenol (BHTBzOH) were also tested, In both cell lines BHTOH was a more potent inhibitor of GJIC than BHT, whereas BHTBzOH was ineffective, The mechanisms of inhibition of GJIC by BHT were also examined. The initial rapid inhibition detected within 15-30 min may have been due to gap junction channel closure or blockage, since no changes in gap junction number, connexin (Cx) 43 levels or Cx43 phosphorylation were observed, By 2-4 h, however, gap junctions were internalized into the cytoplasm, the number of immunodetectable plasma membrane gap junctions was reduced and phosphorylated Cx43-P2 was decreased, Treatment of the cells for 24 h with 12-O-tetradecanoylphorbol-13-acetate (TPA) prevented inhibition of GJIC by TPA, but not by BHT. Western blot analyses of TPA-treated WB-F344 or C10 cells revealed the presence of a hyperphosphorylated form of Cx43 (Cx43-P3) and no reduction in Cx43-P2, in contrast to BHT-treated cells, These data suggest that BHT and TPA inhibit lung and liver epithelial cell GJIC through distinct mechanisms.