Pharmacological profile of inhibition of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein efflux in human HCT-8 intestinal epithelial cells

Abstract

The efflux of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from human HCT-8 intestinal epithelial cultured cells was time-dependent, and after 5 hr 76% of the fluorochrome was extracellular. The pharmacological profile for inhibition of this efflux has been investigated, focusing on agents which modulate anion transport. BCECF efflux was sensitive to inhibition by 0.5 mM indomethacin (50% inhibition at 20 microM) which reduced efflux to values observed after depletion of ATP with azide and 2-deoxy-D-glucose. Indomethacin inhibition of BCECF efflux was not reversed with prostaglandin. The stilbene derivatives 4-acetamido-4'-isothiocyano-2-2'-disulphonic stilbene and 4,4'-diisothiocyano-2,2'-disulphonic stilbene only resulted in partial inhibition of BCECF efflux, even at 1 mM. Furosemide, bumetamide, probenecid and 5-nitro-2-(3-phenylpropyl-amino)-benzoate only reduced BCECF efflux at 1 mM. The cationic agent vinblastine was as active as indomethacin as an inhibitor of BCECF efflux (50% inhibition) with 10 microM) while actinomycin D was also a good inhibitor (50% inhibition with 100 microM). Several other cationic agents, including nifedipine, amiloride and reserpine, were ineffective as inhibitors of BCECF efflux in concentrations up to 1 mM. Thus, the pharmacological profile for inhibition of BCECF efflux does not fully equate with any recognised transport system. Agents such as cytochalasin B and chloroquine did not fully equate with any recognised transport system. Agents such as cytochalasin B and chloroquine did not effect BCECF efflux suggesting accumulation and subsequent discharge from endosomes is not a pathway for secretion. BCECF may be a substrate for a cellular secretory detoxifying system in epithelial cells.