POLARIZED EFFLUX OF 2',7'-BIS(2-CARBOXYETHYL)-5(6)-CARBOXYFLUORESCEIN FROM CULTURED EPITHELIAL-CELL MONOLAYERS
POLARIZED EFFLUX OF 2',7'-BIS(2-CARBOXYETHYL)-5(6)-CARBOXYFLUORESCEIN FROM CULTURED EPITHELIAL-CELL MONOLAYERS
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DOI：
10.1016/0006-2952(92)90431-h
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发表时间：
1992-08-04
影响因子：
5.8
通讯作者：
HIRST, BH
中科院分区：
文献类型：
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作者：
COLLINGTON, GK;HUNTER, J;HIRST, BH
我们研究了生长在多孔膜上的Madin-Darby犬肾上皮细胞（MDCK，菌株I和II）和人肠上皮细胞（Caco-2，HCT-8和T84）的细胞内pH荧光染料2 '，7'-双（2-羧乙基）-5（6）-羧基荧光素（BCECF）流出的极性。在菌株I MDCK细胞中，吲哚美辛（100 μ M时抑制50%）和5-硝基-2-（3-苯丙基-氨基）-苯甲酸酯（NPPB; 10 μ M时抑制50%）可有效降低BCECF流出。用溴化物、碘化物或硝酸盐替代外部Cl-并没有改变BCECF流出量，而用甲磺酸盐替代导致了一个小但显着的减少。当在多孔膜上生长时，所有五种细胞系均形成汇合的上皮层。BCECF从菌株I MDCK上皮层流出到顶端溶液中的量约为流入基础溶液的3倍。顶侧溶液中加入吲哚美辛可减弱顶侧溶液的外排，但对基底侧溶液无效，而基底侧吲哚美辛可有效对抗基底侧外排。NPPB具有类似的作用特异性。肾上腺素，一种刺激电Cl-分泌，没有改变BCECF流出的模式。在MDCK菌株II和Caco-2上皮层中，BCECF外排也呈两极分化，顶端外排大于基底外排。相比之下，在HCT-8和T84细胞形成的层中，BCECF流出到基底和顶端培养基中是等同的。然而，吲哚美辛减少流出在所有五个上皮细胞系，虽然顶端和基底流出率吲哚美辛的相对敏感性变化，作为吲哚美辛的应用的偏侧的敏感性。在MDCK和HCT-8上皮层，P-糖蛋白介导的跨上皮长春碱分泌不受吲哚美辛的抑制。这些数据是一致的假设BCECF外排是一种新的ATP依赖性外源性分泌外排机制在肾脏和胃肠道上皮细胞的表现。调节BCECF流出极性的因素，包括吲哚美辛敏感和不敏感的成分，尚未阐明。
We have investigated the polarity of the efflux of the intracellular pH fluorochrome 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from layers of epithelial Madin-Darby canine kidney (MDCK, Strains I and II) and human intestinal (Caco-2, HCT-8 and T84) cells grown on porous membranes. In Strain I MDCK cells, BCECF efflux was effectively reduced by indomethacin (50% inhibition with 100-mu-M) and 5-nitro-2-(3-phenylpropyl-amino)-benzoate (NPPB; 50% inhibition with 10-mu-M). Replacement of external Cl- with bromide, iodide or nitrate did not alter BCECF efflux, while substitution with methanesulphonate resulted in a small but significant reduction. All five cell lines form confluent epithelial layers when grown on porous membranes. Efflux of BCECF from Strain I MDCK epithelial layers into the apical solution was approximately three times greater than into the basal solution. Addition of indomethacin to the apical solution attenuated efflux into the apical but not the basal solution, while basal indomethacin was effective against basal efflux. NPPB has a similar specificity of action. Adrenaline, a stimulant of electrogenic Cl- secretion, did not alter the pattern of BCECF efflux. BCECF efflux was also polarized, with apical efflux greater than basal efflux, in MDCK Strain II and Caco-2 epithelial layers. In contrast, BCECF efflux into the basal and apical media was equivalent in layers formed from HCT-8 and T84 cells. However, indomethacin reduced efflux in all five epithelial lines, although the relative sensitivities of the apical and basal efflux rates to indomethacin varied, as did the sensitivity to the sidedness of application of indomethacin. In MDCK and HCT-8 epithelial layers, transepithelial vinblastine secretion mediated by P-glycoprotein was not inhibited by indomethacin. The data are consistent with the hypothesis that BCECF efflux is a manifestation of a novel ATP-dependent xenobiotic secretory efflux mechanism in renal and gastrointestinal epithelia. The factors regulating the polarity of BCECF efflux, both the indomethacin-sensitive and -insensitive components, have yet to be elucidated.