Franklin C C, Turner J T, Kim H D
Department of Pharmacology, School of Medicine, University of Missouri, Columbia 65212.
J Biol Chem. 1989 Apr 25;264(12):6667-73.
The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.
在培养的HT29人结肠腺癌细胞中研究了蛋白激酶C参与对Na+/K+/Cl-协同转运的调节作用。我们先前已证明HT29细胞中存在Na+/K+/Cl-协同转运途径（Kim, H.D., Tsai, Y-S., Franklin, C.C., 和Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397 - 404）。用佛波酯佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）和佛波醇12,13 - 二丁酸酯（PDBu）处理细胞会导致膜相关蛋白激酶C活性增加，同时伴随着胞质蛋白激酶C活性的相应降低。PMA还使协同转运在5分钟内迅速短暂增加至对照值的137%，随后逐渐降低，到2小时时降至对照值的19%。为了确定Na+/K+/Cl-协同转运减少的潜在机制，在使用[3H]布美他尼的放射性配体结合研究中测定了协同转运体数量和/或亲和力的变化。PMA和PDBu在特异性[3H]布美他尼结合上产生了基本相同的时间和剂量依赖性降低，这与观察到的协同转运降低相似。对饱和和竞争结合数据的分析表明，结合的降低是由于Bmax降低而亲和力没有变化。蛋白激酶抑制剂H7可阻止PMA引起的结合降低和协同转运变化。这些发现表明佛波酯导致HT29细胞中协同转运体数量减少，从而导致Na+/K+/Cl-协同转运活性降低。
