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Evidence of amiloride-sensitive fluid absorption in rat descending colonic crypts from fluorescence recovery of FITC-labelled dextran after photobleaching

Jay R. Thiagarajah, Kevin C. Pedley and Richard J. Naftalin

1. Fluorescence recovery after photobleaching (FRAP) of fluorescein isothiocyanate (FITC)-labelled 10 and 250 kDa dextran (FITC dextran) in isolated rat descending colonic crypts was measured at 35 °C using laser scanning confocal microscopy.
2. FRAP of either 10 or 250 kDa FITC dextran in crypt lumens was almost complete within 2-3 min.
3. In the presence of amiloride (0.1 mM), or in the absence of Na⁺, the rate of FITC dextran uptake into the crypt lumens was reduced by 70-80 %.
4. The rate of fluid uptake into the crypt lumen, as estimated from the rate of total FITC dextran uptake into the crypt lumen and its adjacent pericryptal region after FRAP, was between 1.3 10^-3 and 1.7 10^-3 cm s^-1.
5. Convective flow during FRAP was also determined from the initial rate of FITC dextran advance along the crypt lumen. This effect was almost completely blocked by amiloride (0.1 mM).
6. The permeability of 10 kDa FITC dextran across the descending colonic crypt wall was found to be higher than that of 250 kDa FITC dextran (3.7 (± 0.6) 10^-5 and 1.8 (± 0.3) 10^-6 cm s^-1, respectively; n = 3 for both, P < 0.01). The permeability of the caecal crypt wall to 10 kDa dextran was higher than that of the descending crypt wall (2.03 (± 0.21) 10^-5 cm s^-1; n = 3, P < 0.025).
7. Simulation of the flow of Na⁺, water and FITC dextran into the crypt lumen and across the crypt wall and pericryptal sheath corroborates the observed parameters of water and Na⁺ flows.

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