The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K+ efflux and Cl− loss. When human erythrocytes were suspended in a physiological NaCl solution (pH o = 7.4), the measured pH i was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH i to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl− content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H+ influx with the measured unidirectional K+ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H+ influx and a K+ efflux in LIS solution of 108.2 ± 20.4 mmol (l cells hr)−1 and 98.7 ± 19.3 mmol (l cells hr)−1, respectively. For bovine and porcine erythrocytes, in LIS media, H+ influx and K+ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K+(Na+)/H+ exchanger, inhibited the K+ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K+(Na+)/H+ exchanger in the human erythrocyte membrane.
The Journal of Membrane Biology – Springer Journals
Published: Aug 1, 2000
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