Holm P K, Eker P, Sandvig K, van Deurs B
Department of Anatomy, Panum Institute, University of Copenhagen, Denmark.
Exp Cell Res. 1995 Mar;217(1):157-68. doi: 10.1006/excr.1995.1075.
We have examined the regulation of endocytosis in polarized Madin-Darby canine kidney (MDCK) cells. Using quantitative electron microscopy and biochemical measurements, we found that incubation with the tumor promoter phorbol 12-myristate 13-acetate (TPA) stimulated endocytosis of horseradish peroxidase (HRP) and ricin four- to fivefold at the apical side in MDCK cells, whereas the uptake at the basolateral membrane was unaffected. The use of several protein kinase inhibitors and TPA analogues indicated that the stimulation of apical endocytosis was mediated via protein kinase C independently of protein kinase A. This stimulation occurred even when the clathrin-dependent pathway was inhibited by acidification of the cytosol, suggesting that the TPA-stimulated uptake was associated with a clathrin-independent mechanism. Moreover, we found that TPA also stimulated recycling of ricin to the apical domain. Ultrastructural analysis of MDCK cells preincubated with TPA revealed that neither the morphology nor the size of the endosomes was altered compared to control cells. Using morphometry, no marked change in the apical plasma membrane area was detected after incubation with TPA. These data indicate that the TPA-stimulated endocytosis involved neither ruffling nor formation of macropinosomes in MDCK cells. Finally, we found that TPA also selectively stimulated apical endocytosis in the human colon adenocarcinoma cell line (Caco-2). The data suggest that protein kinase C is involved in a strong stimulation of apical endocytosis and might participate in the regulation of membrane trafficking between the apical plasma membrane and apical endosomes in polarized epithelial cells.
我们研究了极化的犬肾上皮细胞（MDCK细胞）中内吞作用的调控机制。通过定量电子显微镜和生化测量，我们发现用肿瘤促进剂佛波酯12-肉豆蔻酸酯13-乙酸酯（TPA）处理MDCK细胞后，顶侧辣根过氧化物酶（HRP）和蓖麻毒素的内吞作用增强了4至5倍，而基底外侧膜的摄取不受影响。使用几种蛋白激酶抑制剂和TPA类似物的实验表明，顶侧内吞作用的增强是由蛋白激酶C介导的，与蛋白激酶A无关。即使通过酸化细胞质抑制网格蛋白依赖性途径，这种刺激仍然发生，这表明TPA刺激的摄取与一种不依赖网格蛋白的机制有关。此外，我们发现TPA还刺激蓖麻毒素循环回到顶侧结构域。对预先用TPA孵育的MDCK细胞进行超微结构分析发现，与对照细胞相比，内体的形态和大小均未改变。通过形态计量学分析，在用TPA孵育后未检测到顶质膜面积有明显变化。这些数据表明，TPA刺激的内吞作用在MDCK细胞中既不涉及微绒毛的形成，也不涉及巨胞饮体的形成。最后，我们发现TPA也选择性地刺激了人结肠腺癌细胞系（Caco-2）的顶侧内吞作用。这些数据表明，蛋白激酶C参与了对顶侧内吞作用的强烈刺激，并且可能参与了极化上皮细胞顶质膜和顶侧内体之间膜运输的调控。
