Characterization of apical and basal thiol-disulfide redox regulation in human colonic epithelial cells.

摘要

Control of extracellular thiol-disulfide redox potential (E(h)) is necessary to protect cell surface proteins from external oxidative and reductive stresses. Previous studies show that human colonic epithelial Caco-2 cells, which grow in cell culture with the apical surface exposed to the medium, regulate extracellular cysteine/cystine E(h) to physiological values (approximately -80 mV) observed in vivo. The present study tested whether extracellular E(h) regulation occurs on the basal surface of Caco-2 cells and investigated relevant mechanisms. Experiments were performed with confluent, differentiated cells grown on a permeable membrane surface. Cells were exposed to an oxidizing potential (0 mV) using a fixed cysteine-to-cystine ratio, and culture medium was sampled over time for change in E(h). Regulation of extracellular thiol-disulfide E(h) on the basal domain was faster, and the extent of change at 24 h was greater than on the apical surface. Mechanistic studies showed that redox regulation on the basal surface was partially sodium dependent and inhibited by extracellular lysine, a competitive inhibitor of cystine transport by the y(+)L system and by quisqualic acid, an inhibitor of the x(c)(-) system. Studies using the thiol-reactive alkylating agent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid and the glutathione synthesis inhibitor buthionine sulfoximine showed that extracellular redox regulation was not attributable to plasma membrane cysteine/cystine interconversion or intracellular glutathione, respectively. Thus the data show that redox regulation occurs at different rates on the apical and basal surfaces of the polarized Caco-2 epithelial cell line and that the y(+)L and x(c)(-) systems function in extracellular cysteine/cystine redox regulation on the basal surface.