Electron microprobe analysis of electrolytes in whole cultured epithelial cells

Microprobe analysis was used to determine electrolyte contents in whole epithelial sheets of A6 cells and to investigate the most critical points of this method. Analysis of dextran standard sections of different thickness revealed that low accelerating voltages of about 10 kV are best suited for whole freeze-dried cells on thick supports, since 5 microM thick sections are not penetrated by 10 kV electrons. Washing of A6 cells for 10 sec with distilled water led to cell swelling of about 40%, but the molar concentration ratios and the concentrations per dry weight (dw) were not altered. Washing for 60 sec with distilled water caused a further increase in cell volume (120%) and loss of cellular K and Cl (90 mmol/kg dw). Washing with isotonic NH4- acetate led to a loss of cell Cl already after 10 sec. To characterize the Na transport compartment, A6 cells cultured on permeable supports were washed for 5 sec with distilled water, freeze-dried, and analyzed. Inhibition of transepithelial Na transport by ouabain increased Na/P from 0.15 +/- 0.07 to 0.75 +/- 0.03 and Cl/P from 0.21 +/- 0.001 to 0.38 +/- 0.003 while K/P decreased from 0.83 +/- 0.08 to 0.32 +/- 0.03. The changes in cell Na and K contents can be explained by K/Na exchange; the increase in Cl content indicates some cell swelling. Since the ouabain-induced changes could be prevented by apical amiloride, the apical membrane provides the most important pathway for Na entry in A6 cells.