THE CHLORIDE CONCENTRATION IN THE LATERAL INTERCELLULAR SPACES OF MDCK CELL MONOLAYERS

We measured the Cl concentration of the lateral intercellular spaces ( LIS) of MDCK cell monolayers, grown on glass coverslips, by video fluo rescence microscopy. Monolayers were perfused at 37 degrees C either w ith HEPES-buffered solutions containing 137 mM Cl or bicarbonate/CO2-b uffered solutions containing 127 mM Cl. A mixture of two fluorescent d yes conjugated to dextrans (MW 10,000) was microinjected into domes an d allowed to diffuse into the nearby LIS. The Cl-sensitive dye, ABQ-de xtran, was selected because of its responsiveness at high Cl concentra tions; a Cl-insensitive dye, Cl-NERF-dextran, was used as a reference. Both dyes were excited at 325 nm, and ratios of the fluorescence inte nsity at spectrally distinct emission wavelengths were obtained from t wo intensified CCD cameras, one for ABQ-dextran the other for Cl-NERF- dextran. LIS Cl concentration was calibrated in situ by treating the m onolayer with digitonin or ouabain and varying the perfusate Cl betwee n 0 and 137 mM (HEPES buffer) or between 0 and 127 mM (bicarbonate/CO2 buffer). LIS Cl in HEPES-buffered solutions averaged 176 +/- 19 mM (n = 12), calibrated with digitonin, and 170 +/- 9 mM (n = 12), calibrat ed with ouabain. LIS Cl in bicarbonate/CO2-buffered solutions averaged 174 +/- 10 mM (n = 7) using the ouabain calibration. The Cl concentra tion of MDCK cell domes, measured with Cl-sensitive microelectrodes an d by microspectrofluorimetry, did not differ significantly, Images of the LIS at 3 focal planes, near the tight junction, midway and basal, failed to reveal any gradients in Cl concentration along the LIS. LIS Cl changed rapidly in response to perfusate Cl with characteristic tim es of 0.8 +/- 0.1 min (n = 21) for Cl decrease and 0.3 +/- 0.04 min (n = 21) for CI increase. In conclusion, (i) Cl concentration is higher in the LIS than in the bathing medium, (ii) no gradients of Cl along t he depth of LIS are detectable, (iii) junctional Cl permeability is hi gh.