Plasma membrane potential of murine erythroleukemia cells: Approach to measurement and evidence for cell‐density dependence

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Abstract
The plasmamembrane potential (ΔΨp) of murine erythroleukemia (MEL) cells has been determined by measuring the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) across the plasmamembrane. TPP+ accumulation within the cells (experimental accumulation ratio, ARexp) was measured by adding 2 μM TPP+ directly to the culture flasks, avoiding any other perturbation of the experimental system. The mitochondrial contribution to ARexp, evaluated by adding carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP) or 2,4‐dinitrophenol (DNP), was apparently negligible in standard cultures, ARexp being substantially the same in either the absence or presence of these uncouplers. However, the addition of oligomycin produced a strong ARexp enhancement, which was abolished by FCCP, suggesting that MEL cell mitochondria are in state 3. The aspecific TPP+ binding was estimated by a new mathematical approach worked out to fit ARexp values measured in the presence of valinomycin at various extracellular K+ concentrations and plotted against the ratio of intracellular to extracellular K+ concentration ([K+]i/[K+]e). This binding was found to be close to zero in MEL cells. By applying the Nernst equation directly to ARexp values, ΔΨp of these cells was then measured; this potential varying from −65 mV to −16 mV (inside negative) is inversely related to the cell density on the culture surface on which the cells sediment (cells/cm2; CD). The dependence of ΔΨp on CD is practically eliminated by valinomycin and appears to be related to a cell interaction with the culture surface of the flasks, suggesting that in the immediate environment of MEL cells one or more factors are produced that modulate the K+ plasma membrane permeability (Pk).