Calcium-induced heterogeneous changes in membrane potential detected by flow cytofluorimetry

Ionophore-induced changes in the cell-associated fluorescence of samples of approx. 50000 individual murine L1210 leukemia cells which had been incubated with the voltage-sensitive dye 3,3'-dihexyloctacarbocyanine iodide (DiOC6(3] were monitored by flow cytometry. The K+ ionophore valinomycin (1 microM) produced homogeneous changes in the fluorescence of the entire population, the magnitude of which was dependent upon the concentration of extracellular K+. These changes allowed the estimation of the potassium equilibrium potential of the cells, by the null-point method, to be -11.9 mV. The Ca2+ ionophore A23187 (500 nM) produced heterogeneous changes in fluorescence, with populations of both hyperpolarised and depolarised cells. In addition, the depolarised population underwent an apparent size change, with a reduction in cell volume. This heterogeneity of response resulted in a minimal change in the median fluorescence value for the whole population, which suggests that it would not have been detectable by methods dependent upon net population-averaged changes in fluorescence. Removal of extracellular Na+ or preincubation of cells with amiloride (500 microM) effectively eliminated the depolarised population. Removal of extracellular K+ increased the hyperpolarised population. These findings provide evidence for the presence of Ca2+-induced Na+ exchange and Ca2+-induced K+ efflux mechanisms in these cells which may be expressed simultaneously in the cell population.