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Effect of autologous serum on human leucocyte Na+/H+ exchange and intracellular pH
D J Goldsmith1, L Poston, M Watson
1Department of Medicine, United Medical School of Guy's Hospital, London.
Insights
The study shows that 5-(N,N-hexamethylene) amiloride effectively inhibits sodium-hydrogen exchange in human leucocytes, particularly during acidosis. Autologous serum enhances this exchange, suggesting its importance in physiological and pathological conditions.
Area of Science:
- Cellular Physiology
- Biochemistry
- Immunology
Background:
- Leucocyte intracellular pH regulation is crucial for cellular function.
- Sodium-hydrogen (Na+/H+) exchange plays a significant role in maintaining pH balance.
- Understanding Na+/H+ exchange in leucocytes is vital for studying physiological and pathological states.
Purpose of the Study:
- To investigate the role of Na+/H+ exchange in human leucocytes.
- To assess the efficacy of 5-(N,N-hexamethylene) amiloride as an inhibitor of leucocyte Na+/H+ exchange.
- To determine the influence of autologous serum on leucocyte Na+/H+ exchange and intracellular pH.
Main Methods:
- Measurement of leucocyte intracellular pH and Na+ influx.
- Utilized ammonium chloride washout to induce intracellular acidosis.
- Employed 5-(N,N-hexamethylene) amiloride to inhibit Na+/H+ exchange.
- Incubation of leucocytes with varying concentrations of autologous serum.
Main Results:
- 5-(N,N-hexamethylene) amiloride effectively inhibited Na+/H+ exchange stimulated by intracellular acidosis.
- Recovery from acidosis was dependent on extracellular sodium and inhibited by 5-(N,N-hexamethylene) amiloride (Ki = 0.5 µmol/l).
- Autologous serum significantly increased Na+ influx and intracellular pH, with partial inhibition by 5-(N,N-hexamethylene) amiloride.
Conclusions:
- 5-(N,N-hexamethylene) amiloride is a potent inhibitor of human leucocyte Na+/H+ exchange.
- Autologous serum modulates leucocyte Na+/H+ exchange and intracellular pH.
- Studying leucocyte Na+/H+ exchange in the presence of autologous serum provides a more accurate reflection of in vivo conditions.
Abstract:
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 x 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 mumol/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.