Na+ for H+ exchange in rabbit erythrocytes

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Abstract
The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 μM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pHO = 8.0, in the presence and absence of 60 μM 5,N,N‐dimethyl‐amiloride (DMA). The H+ efflux rate in a K+‐containing medium was 116.38 ± 4.5 mmol/l cell × hr. Substitution of NaO+ for KO+ strongly stimulated H+ efflux to 177.89 ± 7.9 mmol/l cell × hr. The transtimulation of H+ efflux by NaO+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 × 10−7 M. The sequence of substrate selectivities for the external transport site were Na ⋙ Li > choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3−. The DMA sensitive H+ efflux was a saturable function of [Na+]O, with an apparent Km and Vmax of about 14.75 ± 1.99 mM and 85.37 ± 7.68 mmol/l cell × hr, respectively. However, the NaO+‐dependent and DMA‐sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 ± 3.0 mmol/l cell × hr) which was abolished when pHO was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.