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The kinetics of Na movement across the tight junctions of MDCK cells, grown on coverslips and perfused with HEPES or bicarbonate Ringer at 37 degrees C, were investigated after filling the lateral intercellular spaces (LIS) of the epithelium with SBFO, an Na-sensitive fluorescent dye. Dilution and bi-ionic potential measurements showed that MDCK cell tight junctions, although cation-selective, were poorly permeable to N-methyl-D-glucamine Cl (NMDG) but freely permeable to Li. In previous experiments in which Na was replaced by NMDG, a very slow decrease in LIS Na concentration (time constant = 4.8 min) resulted. In the present study, reduction of perfusate Na from 142 to 14 or 24 mM with Na replaced by Li caused LIS Na concentration to decrease with a time constant of 0.43 min. The time constant for Na increase of the LIS was 0.28 min, significantly shorter than that for Na decrease because of the additional component of transcellular Na influx. Ouabain eliminated the transcellular component and equalized the time constants for Na influx and efflux. These results were incorporated into a mathematical model which enabled calculation of the transcellular and paracellular Na fluxes during fluid reabsorption. Regulation of the Na permeability of individual tight junctions by protein kinase A (PKA) was evaluated by treating the monolayers with the Sp-cAMPS, a cAMP substitute, or Rp-cAMPS, a specific inhibitor of PKA. Stimulation of PKA strikingly increased tight junctional permeability while PKA inhibition diminished junctional Na permeability.

MDCK cell monolayers grown on glass coverslips were used to examine the Na+ concentration in individual lateral intercellular spaces (LIS) by video fluorescence microscopy. The LIS was filled with the Na(+)-sensitive fluorescent dye SBFO by incubation of the monolayers for 75-90 min with 250 microM of the membrane impermeant form of the dye. After dye loading, the monolayers were perfused at 37 degrees C with solutions buffered with HEPES or bicarbonate/CO2 containing 142 mM Na+. Ratios of the fluorescence images after sequential excitation with 340 nm and 380 nm light were performed and in situ calibration of LIS Na+ was accomplished after blocking the Na+ pump with 5 x 10(-4) M ouabain. Measurements of Na+ along the basolateral-to-apical axis of the LIS at 1.0 or 1.5 microns intervals did not reveal a Na+ gradient when the perfusate was either HEPES or bicarbonate/CO2 solutions. In bicarbonate solutions, the mean Na+ concentration (mM) was 157.2 +/- 2.3, approximately 15 mM higher than the bath Na+ concentration. In HEPES solutions, however, the Na+ concentration was not different from the bath concentration (142.7 +/- 3.1 mM). The time course of Na+ changes in LIS was investigated by rapidly switching the perfusate from 142 to 80 mM Na+ and measuring the Na+ changes at one focal plane.

The pH of the lateral intercellular space (LIS) of Madin-Darby canine kidney (MDCK) cell monolayers grown on permeable supports was investigated by microspectrofluorimetry using BCECF (2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein). The permeability of the support was selectively reduced by growing Zn-Al-silicate crystals inside its pores. The diffusion of BCECF across the filter was sufficiently retarded to allow measurements of fluorescence in the LIS. The LIS pH and intracellular pH of the cells surrounding them were determined in HEPES-buffered solutions. When the perfusate pH was 7.4, the LIS pH was more acidic (7.06 +/- 0.02) and equaled the cytoplasmic pH (7.08 +/- 0.05). When perfusate was changed to pH 7.0 or 7.8, the LIS changed linearly by about half the magnitude of the perfusate pH. Intracellular pH followed LIS pH variations between perfusate pH 7.0 and 7.4 but was significantly higher when perfusate pH was 7.8. Tight junctional H+ permeability was undetectably low. The low steady-state pH in the LIS was not altered by inhibitors of acid transport or low temperature. Rapid perturbations of pH in the LIS showed that protons were not immobilized in the LIS. The acidic microenvironment within the LIS may be the result of buffering by the cell surface proteins.