The sodium concentration of the lateral intercellular spaces of MDCK cells: A microspectrofluorimetric study |
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Authors: | J. -Y. Chatton K. R. Spring |
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Affiliation: | (1) Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, 20892-1598 Bethesda, Maryland |
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Abstract: | 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 m of the membrane impermeant form of the dye. After dye loading, the monolayers were perfused at 37°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 × 10–4 m ouabain. Measurements of Na+ along the basolateral-to-apical axis of the LIS at 1.0 or 1.5 m 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, 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.We would like to thank P.H. Tran and C. Gibson for their technical and computational assistance as well as Dr. B.-E. Persson (University of Uppsala, Sweden) for his contribution in the early phases of the study. |
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Keywords: | Epithelia Fluid transport Fluorescence microscopy Tissue culture Na+ transport |
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