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~ in media containing physiological concentrations of Pt (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 i cotransport pathway determined the KI/~ for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na ([Pi]o = 1.0 mM) to be 304 • 24/zM and 139 • 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a V~ of 0.63 _+ 0.05 mmol Pi (kg Hb)-l(h) -1 at 140 mM Na o. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The V=~, of the Na/P i cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/P i 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 S~P i 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 Pi. 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 N-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 Address reprint requests to Dr.

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2

“…The PiT1 K mPi and PiT2 K 0.5Pi values we obtained in oocytes are in good agreement with former studies on endogenously expressed human PiT proteins, which revealed K mPi values of 454 Ϯ 61 M in human osteoblastic SaOS2 cells (23), 280 Ϯ 30 M in human primary erythrocytes (35), 360 M in human erythroid precursor-like K562 cells (39), 110 Ϯ 20 M in human cervical epithelial-like HeLa cells (27), and 147 Ϯ 14 M in human embryonic kidney HEK-293 cells (10). Although Fernandes et al (10) assigned their K mPi values in HEK-293 cells to PiT1, their cells most likely also expressed PiT2 mRNA, because another study (40) showed its presence in HEK-293 cells and kidney.…”

Mice lacking the sodium-dependent phosphate import protein, PiT1 (SLC20A1), have a severe defect in terminal erythroid differentiation and early B cell development

“…In mature (enucleated) red blood cells, 75% of phosphate uptake occurs via the anion transporter, band 3; 20% occurs via a sodium-dependent transport mechanism, and 5% of uptake is mediated by a pathway linearly dependent on extracellular phosphate concentration [36]. In primary erythroid precursors, sodium-dependent phosphate uptake and the protein expression of sodium-dependent phosphate importers has not been reported.…”

Reductions in Red Blood Cell 2,3-Diphosphoglycerate Concentration during Continuous Renal Replacment Therapy

“…Extracellular phosphate and RBC intracellular phosphate concentrations are linked through at least three phosphate transporters in RBC membranes: band 3, Na/phos cotransporter, and sodium-independent transport (15). Activation of phosphate influx by these transport mechanisms depends on extracellular phosphate (15).…”

“…Extracellular phosphate and RBC intracellular phosphate concentrations are linked through at least three phosphate transporters in RBC membranes: band 3, Na/phos cotransporter, and sodium-independent transport (15). Activation of phosphate influx by these transport mechanisms depends on extracellular phosphate (15). During CRRT, phosphate is removed from the extracellular compartment and also from other compartments, as the entire extracellular mass of phosphate can be removed in 24 or 48 hours of CRRT using typical prescriptions (10).…”