Journal
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
Volume 299, Issue 2, Pages G523-G530Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpgi.00359.2009
Keywords
cell polarity; cysteine; oxidative stress; transport; amino acid
Categories
Funding
- National Institutes of Health [R01 ES009047, K24 RR023356, TL1 RR025010]
- Epithelial Pathobiology Core [R24DK064399]
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Mannery YO, Ziegler TR, Hao L, Shyntum Y, Jones DP. Characterization of apical and basal thiol-disulfide redox regulation in human colonic epithelial cells. Am J Physiol Gastrointest Liver Physiol 299: G523-G530, 2010. First published May 13, 2010; doi: 10.1152/ajpgi.00359.2009.-Control of extracellular thiol-disulfide redox potential (Eh) 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-acet-amido-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.
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