2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein and Adenocarcinoma

The human pancreatic duct cell line, HPAF, has been shown previously to secrete Cl(-) in response to Ca(2+)-mobilizing stimuli. Our aim was to assess the capacity of HPAF cells to transport and secrete HCO3(-).. HPAF cells were grown as confluent monolayers on permeable supports. Short-circuit current was measured by voltage clamp. Intracellular pH (pHi) was measured by microfluorometry in cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).. In HCO3(-)-free solutions, ATP-evoked changes in short-circuit current were inhibited by bumetanide, and the recovery of pHi from acid loading was abolished by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). In the presence of HCO3(-), ATP-evoked secretion was no longer inhibited by bumetanide, and there was a strong EIPA-insensitive recovery from acid loading, which was inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS). ATP, but not forskolin, stimulated HCO3(-) efflux from the cells.. In the absence of HCO3(-), ATP-evoked Cl(-) secretion is driven by a basolateral Na(+)-K(+)-2Cl(-) cotransporter, and pH(i) is regulated by apical and basolateral Na(+)/H(+) exchangers. In the presence of HCO3(-), ATP-evoked secretion is sustained in the absence of Na(+)-K(+)-2Cl(-) cotransporter activity and is probably driven by basolateral Na(+)-HCO3(-) cotransport.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenocarcinoma; Adenosine Triphosphate; Amiloride; Bicarbonates; Biological Transport; Bumetanide; Cell Line, Tumor; Chlorides; Cytophotometry; Fluoresceins; Humans; Hydrogen-Ion Concentration; Intracellular Space; Ion Transport; Membrane Potentials; Pancreatic Ducts; Sodium Potassium Chloride Symporter Inhibitors; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2

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

The efflux of the intracellular pH fluorochrome 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was quantified in four cultured epithelial cell lines; HCT-8, T84, HGT-1 and MDCK. BCECF efflux was time-dependent, and after 5 h 45-91% of the initial BCECF loaded was extracellular, efflux being greatest in MDCK cells. Depletion of cellular ATP approximately halved BCECF efflux. BCECF efflux was inhibited by indomethacin, vinblastine and verapamil, but not by nifedipine or reserpine. Certain features of BCECF efflux resemble drug efflux in multidrug resistant cells, but inhibition of efflux displays a distinct pharmacological profile suggesting BCECF is a substrate for a novel ATP-dependent transport system.

Topics: Adenocarcinoma; Adenosine Triphosphate; Animals; Cell Line; Epithelium; Fluoresceins; Fluorescent Dyes; Humans; Indomethacin; Kinetics; Nifedipine; Reserpine; Verapamil; Vinblastine