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.

Differential Effect of HOE642 on Two Separate Monovalent Cation Transporters in the Human Red Cell Membrane

“…However, as has been speculated elsewhere [15,16], it was assumed that the increase of the cation fluxes for RBCs suspended in LIS solutions is mediated by the K It is important to note that, after transferring cells from HIS to LIS solutions, a new steady-state is quickly established, associated with an increase of the internal pH from 7.2 to approximately 7.7 [9] which results in a depolarization from -10 mV to 18.5 mV. Although this calculation (which assumes a Nernst distribution of H + ) will be an underestimation given that in both extracellular and intracellular compartments there will be a significant buffer capacity (the extracellular solution was buffered in the investigations of the low ionic strength effect), the transmembrane potential calculated here for cells in LIS solution is below the level at which significant activation of the NSVDC channel will occur [4].…”

“…The first evidence for the involvement of two separate pathways in the enhancement of unidirectional Na Although it is now recognised that the first pathway is electrogenic [6,7] whereas the latter is electroneutral [8,9], a common pathway underlying these two transport modes cannot be entirely discounted [14]. However, as has been speculated elsewhere [15,16], it was assumed that the increase of the cation fluxes for RBCs suspended in LIS solutions is mediated by the K It is important to note that, after transferring cells from HIS to LIS solutions, a new steady-state is quickly established, associated with an increase of the internal pH from 7.2 to approximately 7.7 [9] which results in a depolarization from -10 mV to 18.5 mV.…”

“…At present, 10 different cation transport pathways and 5 Na + -dependent amino acid transporters have been characterised that can translocate Na + and/or K + across the membrane [4,5] [8,9]. In different studies, both pathways have been identified as the 602 principal pathway responsible for enhanced cation fluxes in LIS media.…”

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

“…Another indication for the stimulation of the KNHE in response to cooling is the fact that the temperature-sensitive K + influx in the patient's RBC was 6-fold higher when cold exposure studies were performed in low ionic strength medium (data not shown), a condition known to activate KNHE in erythrocytes from healthy humans. 33 Finally, HOE-642, an inhibitor that was shown to suppress KNHE in RBC from healthy donors, 15 blocked both the temperature-sensitive unidirectional K + influx (Table 1) and the K + leak from erythrocytes into plasma by about 50% when the patient's blood was exposed to cold ( Figure 2B). All these data indicate that an abnormally high activity of KNHE accounts, in part, for the high cation permeability of CHC RBC.…”

Influence of magnetic iron oxide nanoparticles on red blood cells and Caco-2 cells

“…For this reason 5 µM nigericin was added to RBCs suspended in the calibration buffer solutions containing 135 mM KCl, 10 mM NaCl, 10 mM glucose, 10 mM HEPES/NaOH [11,12]. The calibration curve was linear in the pH range 6.5-8.0.…”