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

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.

Characterization of the K+ (Na+)/H+ monovalent cation exchanger in the human red blood cell membrane: effects of transport inhibitors.

The (ouabain + bumetanide + EGTA)-insensitive K+ influx (defined as residual K+ influx) in the human erythrocyte was investigated with respect to the characterization of the recently identified K+(Na+)/H+ exchanger (Richter et al. 1997). In particular, the effects of selected ion transport inhibitors on this flux in physiological ionic strength (high ionic strength, HIS) as well as low ionic strength (LIS) solutions were qstudied. The stimulation of the K+ influx observed in LIS medium was further enhanced when DIDS, phloretin, eosin-5-maleimide, furosemide, DIOA, NPPB, or DCDPC was present at a concentration of 0.1 mmol/l. This paradoxical, inhibitor-induced increase of the K+ influx was more pronounced in LIS media where chloride (7.5 mmol/l) was replaced by nitrate. For DNDS, niflumic acid, and MK-196 (0.1 mmol/l) an enhanced K+ transport could only be observed in nitrate-containing LIS solution. Bumetanide and purine riboside, at a concentration of 0.1 mmol/l, did not cause significant changes of the K+ influx in either chloride- or nitrate-containing LIS media. Dipyridamole and ruthenium red (0.1 mmol/l), which are positively charged, significantly reduced the K+ influx in both chloride- and nitrate-containing LIS media. In nitrate-containing HIS solution only dipyridamole inhibited the K+ influx. The residual K+ influx in LIS solution was significantly increased by removing internal [Mg2+], and decreased by quinacrine (1 mmol/l). In HIS solution, no effect of altering intracellular Mg2+ occurred but a stimulation of the flux by quinacrine was observed. The results are discussed in terms of a more general surface charge effect of the used inhibitors on the K+(Na+)/H+ exchanger.

The monovalent cation "leak" transport in human erythrocytes: an electroneutral exchange process.

The mechanism of the "ground permeability" of the human erythrocyte membrane for K+ and Na+ was investigated with respect to a possible involvement of a previously unidentified specific transport pathway, because earlier studies showed that it cannot be explained on the basis of simple electrodiffusion. In particular, we analyzed and described the increase in the (ouabain+bumetanide+EGTA)-insensitive unidirectional K+ and Na+ influxes as well as effluxes (defined as "leak" fluxes) observed in erythrocytes suspended in low-ionic-strength media. Using a carrier-type model and taking into account the influence of the ionic strength on the outer surface potential according to the Gouy-Chapman theory (i.e., the ion concentration near the membrane surface), we are able to describe the altered "leak" fluxes as an electroneutral process. In addition, we can show indirectly that this electroneutral flux is due to an exchange of monovalent cations with protons. This pathway is different from the amiloride-sensitive Na+/H+ exchanger present in the human red blood cell membrane and can be characterized as a K+(Na+)/H+ exchanger.