1. Leucocyte Na+/H+ exchange and intracellular pH were investigated in physiological buffer containing bicarbonate.
2. The amiloride analogue 5-(N,N-hexamethylene) amiloride (1 × 10−5 mol/l), an inhibitor of Na+/H+ exchange, had no significant effect on resting leucocyte pH, Na+ influx or Na+ content.
3. Ammonium chloride washout induced a profound intracellular acidosis, stimulating Na+/H+ exchange. This led to a 236% increase in Na+ influx. Eighty-eight per cent of this increase was inhibited by 5-(N,N-hexamethylene) amiloride. This demonstrates that 5-(N,N-hexamethylene) amiloride is an effective inhibitor of Na+/H+ exchange in human leucocytes.
4. The recovery from intracellular acidosis was shown to be dependent entirely on the presence of extracellular Na+. The Ki for inhibition by 5-(N,N-hexamethylene) amiloride of the recovery was 0.5 μmol/l.
5. Incubation with serum increased Na+ influx and Na+ content: the maximal effect was reached at 20% dilution. Serum (10%, v/v) increased influx by 40%, and 20% (v/v) serum by 102%, over resting levels. Only 43% of the serum-induced increase in Na+ influx was inhibited by 5-(N,N-hexamethylene) amiloride. This represented one-fifth of total Na+ influx.
6. Leucocyte intracellular pH increased on incubation with serum. This alkalinization was inhibited using 5-(N,N-hexamethylene) amiloride.
7. Studies of Na+/H+ exchange in leucocytes in physiological and pathological states are more likely to reflect the state in vivo if carried out in the presence of autologous serum.