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

1. Fluorescence recovery after photobleaching (FRAP) of fluorescein isothio cyanate (FITC)-labelled 10 and 250 kDa dextran (FITC! dextran) in isolated rat descending colonic crypts was measured at 35 degreesC using laser scann ing confocal microscopy. 2. FRAP of either 10 or 250 kDa FITC dextran in crypt lumens was almost com plete within 2-3 min. 3. In the presence of amiloride (0.1 nM), or in the absence of Na+, the rat e 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 rat e of total FITC dextran uptake into the crypt lumen and its adjacent pericr yptal region after FRAP, was between 1.3 x 10(-3) and 1.7 x 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 comple tely blocked by amiloride (0.1 mM). 6. The permeability of 10 kDa FITC dextran across the descending colonic cr ypt wall was found to be higher than that of 250 kDa FITC dextran (3.7 (+/- 0.6) x 10(-5) and 1.8 (+/- 0.3) X 10(-6) cm s(-1), respectively; n = 3 for both, P < 0.01). The permeability of the caecal crypt wall to 10 kDa dextr an was higher than that of the descending crypt wall (2.03 (+/- 0.21) x 10( -5) cm s(-1); n = 3, P < 0.025). 7. Simulation of the flow of Na+, water and FITC dextran into the crypt lum en and across the crypt wall and pericryptal sheath corroborates the observ ed parameters of water and Na+ flows.