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-diisothiocyanatostilbene-2,2-disulphonic acid at an internal pH of 7.56 in the presence or absence of HCO−3 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 μmol/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.