Sodium-phosphate cotransport in human red blood cells: Kinetics and role in membrane metabolism

Abstract

Orthophosphate (Pi) uptake was examined in human red blood cells at 37°C in media containing physiological concentrations of Pi (1.0-1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene-2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/P; cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]0 - 140 mM) and extracellular Na ([Pi]0 - 1-0 mM) to be 304 ± 24 µM and 139 ± 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a of 0.63 ± 0.05 mmol P; (kg Hb)-1(h)-1 at 140 mM Na0. Activation of Pi uptake by Na0 gave Hill coefficients that came close to a value of 1.0. The of the Na/P; cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 ± 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or P,. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane, however, substitution of extracellular sodium with jV-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate. These results demonstrate the existence of a Na/Pi cotransport system in the red cell membrane that is important in the delivery of extracellular phosphate to the membrane compartment of human red cells. © 1988, Rockefeller University Press., All rights reserved.