The sodium concentration of the lateral intercellular spaces of MDCK cells: a microspectrofluorimetric study.
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.
Chatton
J Y
JY
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1598, USA.
Spring
K R
KR
D000818:Animals; D002460:Cell Line; D004285:Dogs; D005110:Extracellular Space / Q000737:chemistry; D005456:Fluorescent Dyes; D007668:Kidney / Q000737:chemistry* / Q000166:cytology; D008832:Microchemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D010042:Ouabain / Q000494:pharmacology; D012964:Sodium / Q000032:analysis*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014741:Video Recording
D005456:Fluorescent Dyes; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase