Characterization of apical and basal thiol-disulfide redox regulation in human colonic epithelial cells.
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.
Mannery
Yanci O
YO
Dept. of Medicine, Emory Univ., Atlanta, GA 30322, USA.
Ziegler
Thomas R
TR
Hao
Li
L
Shyntum
Yvonne
Y
Jones
Dean P
DP
D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism*; D003106:Colon / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D003553:Cystine / Q000097:blood / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism; D045604:Extracellular Fluid / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008239:Lysine / Q000494:pharmacology; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D012964:Sodium / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013997:Time Factors
D004220:Disulfides; D013438:Sulfhydryl Compounds; D003553:Cystine; D012964:Sodium; D005978:Glutathione; D008239:Lysine; D003545:Cysteine
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