Chloride gradient is the driving force for ammonia/ammonium influx in human red blood cells

Abstract

Ammonium/ammonia (AM) Rhesus transporters are expressed in tissues involved in AM generation, secretion and excretion, however, the physiological role of erythroid RhAG glycoprotein is not fully defined yet. AM concentration in red blood cells (RBCs) is approximately three times higher than in plasma and RBCs are the only blood cells that swell and lyse in isotonic ammonium (NH4+) buffer indicating that AM is transported inside the cell. Two main possible mechanisms of AM transport, including diffusion and active transport, are described, however, the real driving force for AM influx in RBCs is still under debate. RhAG and anion exchanger 1 (AE1, band 3, Cl-/HCO3--exchanger) form a structural complex in RBC membrane therefore we hypothesized that AE1 might be involved in AM influx in RBCs via the interplay with RhAG, and suggested that RhAG uses chloride (Cl-) gradient mediated by AE1 to import NH4+ against the gradient. Washed human RBCs were analyzed by flow cytometry, spectrophotometry and laser diffraction methods. For the evaluation of the interplay between RhAG and AE1 transporters HEPES buffer (in mM: NaCl, 140; KCl, 2; HEPES, 10, MgCl2, 2; Glucose, 5) and isotonic NH4+ buffer (in mM: NH4Cl, 140; KCl, 2; HEPES, 10; MgCl2, 2; Glucose, 5) were used. For the analysis of the role of Cl-gradient NaCl and NH4Cl were substituted by sodium glutamate and ammonium glutamate. Calcein-AM was used for cell vitality registration, hemoglobin (Hb) forms were analyzed spectrally, AM import was assessed by enzymatic and colorimetric AM assay kits, AE1 was inhibited by DIDS. In isotonic NH4+ buffer RBCs rapidly swelled and lysed within 3min at 37°C. The addition of AE1 substrate HCO3- (25mM) to isotonic NH4+ buffer increased the cell swelling rate 49±5 fold (n=11, Leven's test p>0.05, Tukey HSD post hoc, p<0.001). NH4+ import in media containing HCO3- increased by 74±11% (n=9, Leven's test p<0.05, Tamhane T2 post hoc, p<0.001). AE1 inhibition completely prevented hemolysis in isotonic NH4+ buffer and decreased NH4+ import in RBCs by 66±9% (n=9, Leven's test p<0.05, Tamhane T2 post hoc, p<0.001). The lack of Cl- gradient in ammonium glutamate and sodium glutamate isotonic buffers had no effects on cell viability (esterase activity) or Hb conformation. In isotonic ammonium glutamate buffer Cl- substitution prevented cell swelling and completely inhibited hemolysis in the same manner as AE1 inhibition by DIDS in NH4+buffer. AM import in conditions lacking Cl- gradient decreased by 62±11% (n=9, Leven's test p<0.05, Tamhane T2 post hoc, p<0.001) which is consistent with AE1 inhibition data. Our data clearly indicated that RhAG and AE1 are functionally connected, and AE1-mediatedCl-gradient drives RhAG-facilitated AM transport in RBCs. Therefore, we suggest that NH4+ is imported in human RBCs against its gradient by a secondary active transport mechanism, rather than via facilitated diffusion. Taken together our data revealed the new mechanism of AM transport against the gradient in RBCs and suggested that RBCs in addition to main functions are involved in the AM concentration regulation in plasma.