CHLORIDE INFLUX IN HUMAN-LEUKOCYTES - A TRIPLE-ISOTOPE TECHNIQUE FOR THE ASSESSMENT OF CHLORIDE TRANSPORTERS

1. A novel triple-isotope method using (H2O)-H-3 [C-14]-sucrose and Cl-36- for measuring initial Cl- uptake rates in human leucocytes that had been preincubated in 10% (v/v) autologous serum is described. 2. There was a marked dependence of Cl- influx on intracellular pH, the flux at an intracellular pH of 7.20 being about 11.2% of that at an intracellular PH of 7.5 6. 3. This Cl- influx was inhibited by 4,4-di-isothiocyanatostilbene-2, 2-disulphonic acid in a dose-dependent manner. Half-maximal inhibition by 4,4-di-isothiocyanatostilbene-2, 2-disulphonic acid at an internal pH of 7.56 in the presence or absence of HCO3- was 0.30 or 0.35 mmol/l, respectively. 4. Depletion of intracellular Cl- resulted in a 70.7% decrease in Cl- influx, whereas depletion of cellular ATP led to a 3 7.7% decrease in Cl- influx. 5. The phorbol ester 12-O-tetradecanoyl phorbol acetate at a concentration of 0.1-mu-mol/l reduced Cl- influx, especially the 4,4-di-isothiocyanatostilbene-2,2-disulphonic acid-sensitive component. This effect was independent of intracellular pH changes or Na+/H+ anti-port activity.