Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly.
Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly.
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DOI：
10.1083/jcb.127.6.1895
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发表时间：
1994-12
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影响因子：
--
通讯作者：
Lampe PD
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作者：
Lampe PD
研究了12-O-十四烷基佛波醇-13-乙酸酯（TPA）对Novikoff肝癌细胞之间间隙连接组装的影响。用EDTA将细胞解离成单细胞，然后重新聚集以形成新的连接。当TPA（25 nM）在60分钟再聚集开始时加入到细胞中时，仅在0.6%的细胞-细胞界面处检测到染料转移，相比之下，未处理的对照为72%，4-α TPA为74%，4-α TPA是TPA的无活性异构体。冷冻断裂电子显微镜的重新聚集的控制细胞显示接口包含平均超过600个聚集的膜内间隙连接颗粒，而TPA处理的细胞没有间隙连接。然而，荧光黄染料之间的非解离细胞通过间隙连接转移不受TPA治疗60分钟。因此，TPA显着抑制间隙连接组装，但不改变通道门控，也不提高拆卸预先存在的间隙连接结构。短期TPA处理（< 30分钟）增加分子量为43，000的差距连接蛋白（Cx43）的磷酸化，但不改变Cx43的细胞水平。细胞表面生物素化实验表明，TPA并没有实质上降低Cx43的质膜浓度。因此，Cx43在质膜中的简单存在不足以进行间隙连接组装，蛋白激酶C可能在质膜水平上对间隙连接组装产生影响。
The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level.