Variations of intracellular pH in human erythrocytes via K(+)(Na(+))/H(+) exchange under low ionic strength conditions.

Abstract

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 + or - 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 + or - 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 + or - 20.4 mmol (l(cells) hr)(-1) and 98.7 + or - 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.