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 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.
Goldsmith
D J
DJ
Department of Medicine, United Medical School of Guy's Hospital, London.
Poston
L
L
Watson
M
M
Morris
J
J
Hilton
P J
PJ
Cragoe
E J
EJ
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D001769:Blood; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007474:Ion Exchange; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000097:blood*
D000643:Ammonium Chloride; C057360:5-(N,N-hexamethylene)amiloride; D000584:Amiloride; D012964:Sodium