Fig 5.
Effects on hypoxanthine plus XO on short-circuit current, electrical resistance, and Stx translocation in polarized T84 cell monolayers studied in the Ussing chamber. Short-circuit current (Isc) represents chloride secretion toward the apical (mucosal or lumenal) side of the tissue in this configuration. (A) Comparison of the short-circuit current triggered by 1 mM hypoxanthine plus XO (black tracing) with that triggered by 1 mM H2O2 (gray tracing), showing similar peak currents and similar offsets of secretion. (B) Hypoxanthine alone, without added XO, triggered a very small short-circuit current of ∼2 μA/cm2. (C) Dose-response relationship between the amount of hypoxanthine added and the short-circuit current; mean ± standard deviation (SD) of 3 tracings per concentration. (D) Effect of catalase on short-circuit current triggered by hypoxanthine plus XO. When 1,200 U/ml catalase was added simultaneously with hypoxanthine and XO (arrow 1, gray tracing), the secretory response was prevented. When hypoxanthine and XO were added first (black arrow) and a current was allowed to develop, subsequent addition of catalase (2nd black arrow) promptly reversed the secretion. (E) Effect of hypoxanthine (hypo) and XO on transepithelial electrical resistance 6 h after addition of hypoxanthine, XO, or both. *, significantly decreased compared to the control and to XO alone. (F) Effect of hypoxanthine (hypoxanth) plus XO on Stx translocation across T84 cell monolayers. T84 cells were grown to confluence in Transwell inserts, reaching a mean initial TER value of 1,648 Ω. Hypoxanthine and XO were added to the apical side of the monolayers, followed by Stx2 3 h later. The amount of Stx2 detectable in the lower basolateral chamber was measured at various times after the start of the experiment. *, significant compared to the control, DMSO vehicle, and XO alone.