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Polarized provision of cysteine affects redox homeostasis in intestinal Caco2‐BBE cells

Prior evidence for high first‐pass metabolism of enteral cysteine led to the hypothesis that the lack of luminal cysteine with parenteral feeding may perturb redox homeostasis in the intestinal epithelium. To explore possible underlying mechanisms, polarized Caco2‐BBE intestinal epithelial cells were seeded on cell culture inserts, grown to confluence determined by trans‐epithelial electronic resistance and subsequently provided cysteine in apical, basal, both, or neither compartments. HPLC analysis revealed a decrease (P < 0.05) in GSH concentrations in cells receiving cysteine from the basal compartment compared to apical only or both compartments determined 7d post‐confluence (differentiated cells). Similarly, redox potential as determined by the Nernst equation was significantly greater (P < 0.05) with apical (−205 mV) versus basal (−192 mV) provision of cysteine. These data indicate that differentiated intestinal epithelial cells may rely on apical provision of cysteine for the maintenance of redox homeostasis. This possibility was examined further by measuring 35S‐cysteine uptake and γ‐glutamylcysteine synthetase (γ‐GCS) RNA expression. Apical provision of cysteine spiked with 35S‐cysteine increased (P < 0.05) cellular radioactivity compared to basal supply indicating greater absorption from the apical surface. Quantitative RT‐PCR analysis demonstrated that steady‐state γ‐GCS RNA expression was also greater with apical versus basal cysteine provision. Together, these data indicate the importance of apical provision of cysteine for maintenance of redox homeostasis in Caco2‐BBE cells.