2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein and Colonic-Neoplasms

Intracellular probes used for oxidative burst evaluation could be a substrate of the multidrug resistance proteins (MDR), which may cause misinterpretation of experimental data. We aimed to study the effect of the culture condition and of the MDR-interfering antioxidant quercetin on free radical measurement in serum depleted HCT-8 cells and chick embryo hepatocytes. Serum depletion and/or quercetin affected the traffic of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and calcein. The comparative results obtained with 2',7'-dichlorofluorescein (DCF) and with the plasma-membrane probe C11-BODIPY 581/591 [4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid], suggest caution in the use of intracellular probes to evaluate oxidative stress in vitro.

Topics: Amidines; Animals; Biological Transport; Boron Compounds; Cell Line, Tumor; Cells, Cultured; Chick Embryo; Chickens; Colonic Neoplasms; Culture Media, Serum-Free; Drug Resistance, Multiple; Fluoresceins; Fluorescent Dyes; Hepatocytes; Humans; Oxidative Stress; Quercetin; Reactive Oxygen Species; Spectrometry, Fluorescence

Glycylsarcosine (Gly-Sar) transport in human intestinal epithelial (Caco-2) cells has been investigated. Gly-Sar transport, from apical to basal surfaces (Ja-b), and intracellular accumulation are greatest when the apical medium is acidified (apical pH 6.0, basal pH 7.4). Both transport and accumulation are susceptible to saturation and competition. Similarly, Gly-Sar transport, from basal to apical surfaces (Jb-a), is increased with acidification of the basal medium (basal pH 6.0, apical pH 7.4). Apical addition of 20 mM Gly-Sar (pH 6.0) to Caco-2 cell monolayers loaded with the pH indicator BCECF (2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein) caused a marked cytosolic acidification. Basolateral application of 20 mM Gly-Sar (pH 6.0) also caused a fall in intracellular pH. These observations are consistent with the expression of H(+)-coupled dipeptide transporters at both membrane faces of the human intestinal epithelial Caco-2 cell line. We also provide direct evidence for dipeptide-stimulated H(+)-influx, across both apical and basolateral membranes, in this intact epithelial cell system.

Topics: Adenocarcinoma; Analysis of Variance; Biological Transport; Carrier Proteins; Cell Membrane; Colonic Neoplasms; Dipeptides; Epithelium; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Kinetics; Mannitol; Tumor Cells, Cultured