Amiloride-sensitive Na+ transport in human red cells: evidence for a Na/H exchange system

The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na+ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 microM) and methazolamide (400 microM). Red cells with different internal pH (pHi = 6.4, 7.0 and 7.8) were prepared and Na+ influx was measured at different external pH (pHo = 6.0, 7.0, 8.0). Na+ influx into acid-loaded cells (pHi = 6.4) markedly increased when pHo was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na+/H+ exchange systems blocked about 60% of the H+-induced Na+ entry, while showing small inhibitory effects in the absence of pH gradients. When pHo was kept at 8.0, the amiloride-sensitive Na+ entry was abolished as pHi was increased from 6.4 to 7.8. Moreover, measurements of H+ efflux into lightly buffered media indicated that the imposition of an inward Na+ gradient stimulated a net H+ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na+ (Nai+ = Nao+ = 15 mM, Em = +6.7 mV), an outward H+ gradient (pHi = 6.4, pHo = 8.0) promoted a net amiloride-sensitive Na+ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na+/H+ exchange system in human red cells.