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Fig. 8. Paracellular flux of neutral molecules. (A) Paracellular urea flux. Cells were grown on Transwell membranes until confluent. 50 mM urea was added to the basal solution. After incubation for 1 hour at 37°C, 300 µl aliquot was removed from the apical solution. Urea concentration was measured by an ACE clinical chemistry analyzer. Data are represented as means ± s.e.m. from three independent clones (n=12 for each experiment). Urea flux in claudin-7-overexpressing cells (2) was 120% higher (P<0.001) compared to that of the control cells (1). TER was measured in growth media before and after each paracellular urea flux experiment to confirm that the monolayer remained intact during the flux experiments. (B) Mannitol flux. Control or claudin-7-overexpressing cells were plated on Transwell plates until confluent. The basal culture medium was supplemented with 1 mM cold mannitol. The apical medium was the same as the basal medium except that it contained 1 µCi/ml [³H]mannitol. After 1 hour incubation at 37°C, 100 µl of basal medium were taken and replaced with 100 µl fresh medium containing 1 mM unlabeled mannitol. The radioactivity of the 100 µl samples was measured. Data are means ± s.e.m. from three independent clones (n=12 for each experiment). Mannitol flux in claudin-7-overexpressing cells (2) was significantly higher (P<0.001) compared to controls (1).

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