Fura-2 handling in a polarized epithelial barrier: the toad urinary bladder

Toad bladders sacs were placed inside quartz cuvettes. When fura-2 AM was added to the mucosal compartment, low temperature (4 degrees C) almost completely blocked the transepithelial transfer of fluorescence observed at 20 degrees C (20 degrees C = 371 +/- 56, 4 degrees C = 29 +/- 29 fluorescence intensity in arbitrary units (FIAU), excitation at 340 nm, emission at 510 nm). Simultaneously, fluorescence accumulation inside the tissue was significantly higher (20 degrees C = 25 +/- 5, 4 degrees C = 91 +/- 24% increase on basal levels (%IBL)). When fura-2 AM was added to the serosal side, low temperature also reduced the serosal to mucosal transfer (20 degrees C = 149 +/- 36, 4 degrees C = 61 +/- 35 FIAU). Nevertheless, in this situation tissue accumulation, that was significantly higher that the one observed when fura-2 AM was added to the mucosal side, was reduced at low temperature (20 degrees C = 300 +/- 30, 4 degrees C = 48 +/- 7 %IBL). Spectral analysis of the mucosal and serosal compartments indicated that free fura-2 was transferred from the intracellular to the serosal compartment, but not to the mucosal one. These results indicate that fura-2 appears as a useful tool to evaluate the cellular distribution and traffic of polycyclic charged and non-charged molecules.