Anion transport heterogeneity detected by flow cytometric measurement of NBD-taurine efflux kinetics.

NBD-taurine [N-(7-nitrobenzofuran-4-yl) taurine], a fluorescent substrate for the human erythrocyte anion exchange system, has been used to test the feasibility of making flow cytometric measurements of anion transport in K562 erythroleukemic cells. Cells were preloaded by incubation with 20 microM-2mM NBD-taurine, then diluted 10-30-fold, and efflux was monitored by measuring fluorescence intensity (FL) as a function of time using excitation at 488 nm. The observed rate of decrease in fluorescence was sensitive to temperature and also to phloretin, a compound known to inhibit anion transport and other carrier-mediated transport processes. The coefficient of variation (CV) of the fluorescence distribution increased markedly over the efflux period, suggesting heterogeneity of the K562 population with respect to the rate constant for NBD-taurine efflux. This heterogeneity was also reflected in the upward curvature of a first order plot of log (FLt - FL infinity) versus time. Half-times calculated from initial linear portions of the first-order plots were found to decrease as the loading concentration of NBD-taurine was decreased, as predicted for a saturable transport system. NBD-taurine is not an ideal anion transport substrate for flow cytometric studies. It appears to bind to high-affinity sites within the cells with consequent fluorescence quenching, complicating interpretation of kinetic curves at low concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)