Effect of Autologous Serum on Human Leucocyte Na⁺/H⁺ Exchange and Intracellular pH

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 K_i 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.