Fig. 6.

Effect of GPI anchoring and catalytic activity of prostasin on transepithelial resistance, current, and permeability. Stably transfected M-1 cell lines (described in Results and Fig. 5) were grown on 0.4-μm polycarbonate filters. R_te and potential difference were measured with an epithelial voltohmeter, and equivalent short-circuit current (I_eq) was calculated by Ohm's law. Data are expressed as means ± SE from 5 independent experiments with triplicate wells. Data were analyzed when resistance and potential difference were stable and monolayers of all cell lines were impermeable to inulin (data in C). A: time course of R_te in stable transfected M-1 cells overexpressing prostasin variants (♦, mPR-ctrl; ■, mPR-wt; ▲, mPR-S238A; ×, mPR-Tpsg). ★P < 0.05 compared with mPR-ctrl; *P < 0.05 compared with mPR-Tpsg. B: time course of I_eq in stably transfected M-1 cell lines. ★P < 0.05 compared with mPR-ctrl; *P < 0.05 compared with mPR-Tpsg. C: paracellular permeability of M-1 cell lines at days 7 and 18 of culture was assayed by measuring apical-to-basal flux of FITC-inulin (20 μg/ml) over 24 h. Data are expressed as means ± SE relative fluorescence units (rfu) in basal medium at 0 (filled bars) and 24 (open bars) h. Mean apical and basal fluorescence at time 0 were ∼23,000 and ∼1,600 rfu, respectively. ★P < 0.05 for comparison between 0 and 24 h. D: effect of amiloride on I_eq (left) and R_te (right) in stably transfected M-1 cell lines. Ten micromolar amiloride was applied to the apical surface of cell monolayers on days 18–19 of culture. I_eq and R_te were measured 45–60 min after exposure to amiloride. Open and filled bars depict baseline and postamiloride measurements, respectively. Data are expressed as means ± SE from 5 independent experiments with triplicate wells. Exposure to amiloride abolished the differences in I_eq between cell lines and reduced I_eq to similarly low current (P > 0.05). There was no difference in baseline and postamiloride R_te in any of these cell lines (P > 0.05).