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.
Biophysics Section, Institute of Biology, Humboldt University Berlin, Invalidenstrasse 42, 10115 Berlin, Germany.
D
D
Kummerow
J
J
Hamann
J A
JA
Browning
R
R
Wilkins
J C
JC
Ellory
I
I
Bernhardt
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D000838:Anions / Q000097:blood; D002034:Bumetanide / Q000494:pharmacology; D002417:Cattle; D002712:Chlorides / Q000097:blood; D004533:Egtazic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000097:blood*; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009544:Niflumic Acid / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000097:blood*; D011796:Quinacrine / Q000494:pharmacology; D012964:Sodium / Q000097:blood*; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects; D013045:Species Specificity; D013267:Stilbenes / Q000494:pharmacology; D013552:Swine
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D002712:Chlorides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; D013267:Stilbenes; D002034:Bumetanide; D009544:Niflumic Acid; D004533:Egtazic Acid; D010042:Ouabain; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D000584:Amiloride; D006859:Hydrogen; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D011796:Quinacrine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D009550:Nigericin; D011188:Potassium