1. A human lymphocyte preparation, obtained by Percoll gradient centrifugation and free of contaminating monocytes and granulocytes, was used to study the kinetics of the Na+-H+ exchanger through activation by nigericin-induced acidic loading. The fluorescent probe biscarboxyethylcarboxyfluorescein (acetoxymethyl ester) was used to determine cell pH and buffering capacity and to measure Na+-H+ exchange activity as external Na+-dependent H+ efflux.
2. At a cell pH of 6.2, H+ efflux was stimulated by external Na+ with a Km of 30 mmol/l (sem 6, n = 3) and a calculated Vmax of 0.73 mmol s−1 l−1 of cell water (sem 0.06, n = 6). External Na+-dependent H+ efflux was more than 98% inhibited and half-maximally inhibited by 200 μmol/l and 17 μmol/l amiloride, respectively. The external pH also inhibited Na+-H+ exchange, with a Ki of 93 nmol/l.
3. Na+-H+ exchange was sigmoidally activated by an internal pH lower than 7.0 with a Hill coefficient of 2.14 (sem 0.15, n = 6) and a pK of 6.57 (sem 0.03, n = 6). Cell buffering capacity was also measured as a function of cell pH and found to gradually increase from 14 to 26 mmol l−1 of cell water pH−1 when cell pH fell below 6.6. The maximal transport rate (cell pH 6.0–6.2) was 0.50 mmol (s 1 of cell water)−1 (sem 0.08, n = 12) and ranged between 0.25 and 1.10.
4. These results indicate that the human blood lymphocyte has a powerful Na+-H+ exchanger which is reproducibly activated by internal acidification with marked inter-individual variability in maximal rate and degree of co-operativity. This technique will be useful for future clinical, functional and genetic studies of the regulation of this antiporter.
