Dickson J L, Conner T D, Ecay T W
Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
Am J Physiol Cell Physiol. 2000 May;278(5):C865-72. doi: 10.1152/ajpcell.2000.278.5.C865.
Wortmannin is a potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and membrane trafficking in many cells. To test the hypothesis that cystic fibrosis transmembrane conductance regulator (CFTR) traffics into and out of the plasma membrane during cAMP-stimulated epithelial Cl(-) secretion, we have studied the effects of wortmannin on forskolin-stimulated Cl(-) secretion by the human colonic cell line T84. At the PI3K inhibitory concentration of 100 nM, wortmannin did not affect significantly forskolin-stimulated Cl(-) secretion measured as short-circuit current (I(SC)). However, 500 nM wortmannin significantly inhibited forskolin-stimulated I(SC). cAMP activation of apical membrane CFTR Cl(-) channels in alpha-toxin-permeabilized monolayers was not reduced by 500 nM wortmannin, suggesting that inhibition of other transporters accounts for the observed reduction in T84 Cl(-) secretion. Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP), but wortmannin did not alter forskolin inhibition of apical HRP endocytosis. In the absence of forskolin, wortmannin stimulated HRP endocytosis significantly. We conclude that, in T84 cells, apical fluid phase endocytosis is not dependent on PI3K activity and that CFTR does not recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.
渥曼青霉素是磷脂酰肌醇3激酶（PI3K）的强效抑制剂，在许多细胞中可抑制膜运输。为了验证在cAMP刺激的上皮细胞氯离子（Cl⁻）分泌过程中，囊性纤维化跨膜传导调节因子（CFTR）进出质膜这一假说，我们研究了渥曼青霉素对人结肠细胞系T84中毛喉素刺激的Cl⁻分泌的影响。在100 nM的PI3K抑制浓度下，渥曼青霉素对以短路电流（I(SC)）衡量的毛喉素刺激的Cl⁻分泌没有显著影响。然而，500 nM的渥曼青霉素显著抑制了毛喉素刺激的I(SC)。在α-毒素通透的单层细胞中，500 nM的渥曼青霉素并未降低cAMP对顶端膜CFTR Cl⁻通道的激活，这表明对其他转运体的抑制导致了观察到的T84细胞Cl⁻分泌减少。毛喉素抑制辣根过氧化物酶（HRP）的顶端内吞作用，但渥曼青霉素并未改变毛喉素对顶端HRP内吞作用的抑制。在没有毛喉素存在的情况下，渥曼青霉素显著刺激了HRP的内吞作用。我们得出结论，在T84细胞中，顶端液相内吞作用不依赖于PI3K活性，并且CFTR不会通过PI3K依赖且对渥曼青霉素敏感的膜区室进行循环。
