Chloride influx in human leucocytes: a triple-isotope technique for the assessment of chloride transporters.

1. A novel triple-isotope method using 3H2O, [14C]-sucrose and 36Cl- 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 7.56. 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 37.7% decrease in Cl- influx. 5. The phorbol ester 12-O-tetradecanoyl phorbol acetate at a concentration of 0.1 mumol/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+ antiport activity.