DETERMINATION OF THE NA PERMEABILITY OF THE TIGHT JUNCTIONS OF MDCK CELLS BY FLUORESCENCE MICROSCOPY

The kinetics of Na movement across the tight junctions of MDCK cells, grown on coverslips and perfused with HEPES or bicarbonate Ringer at 3 7 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 ce ll tight junctions, although cation-selective, were poorly permeable t o N-methyl-D-glucamine Cl (NMDG) but freely permeable to Li. In previo us experiments in which Na was replaced by NMDG, a very slow decrease in LIS Na concentration (time constant = 4.8 min) resulted. In the pre sent 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 con stant 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 t he transcellular component and equalized the time constants for Na inf lux and efflux. These results were incorporated into a mathematical mo del which enabled calculation of the transcellular and paracellular Na fluxes during fluid reabsorption. Regulation of the Na permeability o f individual tight junctions by protein kinase A (PKA) was evaluated b y treating the monolayers with the Sp-cAMPS, a cAMP substitute, or Rp- cAMPS, a specific inhibitor of PKA. Stimulation of PKA strikingly incr eased tight junctional permeability while PKA inhibition diminished ju nctional Na permeability.