Monitoring membrane potentials in Ehrlich ascites tumor cells by means of a fluorescent dye

Abstract

1. The fluorescent intensity of the dye 3,3'-dipropylthiodicarbocyanine iodide was measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potentials under a variety of different ionic and metabolic conditions. 2. In the presence of valinomycin, fluorescent intensity is dependent on log [K+]medium (the fluorescent intensity increased with increasing [K+]medium) where K+ replaced Na+ in the medium. Cellular K+ content also influenced fluorescent intensity in the presence of valinomycin. With lower cellular K+, fluorescent intensity in the presence of valinomycin for any given concentration was increased. 3. In the presence of gramicidin fluorescent intensity was highest in Krebs-Ringer and decreased with the substitution of choline+ for Na+. 4. The observations with ionophores are consistent with the hypothesis that the dye monitors membrane potential in these cells with an increase in fluorescence indicating membrane depolarization (internal becomes more positive). 5. The estimated membrane potentials were influenced by the way in which the cells were treated. Upon dilution of the cells from 1 in 20 to 1 in 300 the initial estimations were between -50 and -60 mV. With incubation at 1 in 300 dilution for 1 h at room temperature or a 37 degrees C, the membrane potentials ranged from -18 to -42 mV. 6. Estimations of membrane potential on the basis of chloride distribution (Cl-cell/Cl-medium) in equilibrated cells ranged from -13 to -32 mV. 7. Addition of glucose to cells equilibrated at 37 degrees C for 30 min in the presence of rotenone led to a decrease in fluorescent intensity indicating hyperpolarization. Addition of ouabain in turn led to a 70 to 100% reversal of fluorescent intensity. This hyperpolarization is therefore probably due to the electrogenic activity of the sodium pump. 8. The addition of amino acids known to require external Na+ for transport increased fluorescent intensity (depolarization) reaching a maximum at higher concentrations of amino acids. Plots of 1/deltafluorescence vs. 1/[glycine] were linear with an apparent Km of 2-3 mM. The increase in fluorescence with amino acids always required external Na+. Plots of 1/fluorescence vs. 1/[Na+]medium were also linear with an apparent Km of 29 mM. These apparent Km values compare favorably with those derived from amino acid transport studies using tracers. These data indicate that the Na+-dependent transport of amino acids in these cells is electrogenic.