Regulation of phosphate metabolism in human red cells following prolonged incubation to steady state in vitro.

Human red cells were incubated aseptically in vitro for 24 or 48 h to allow the cellular concentrations of orthophosphate (Pi) and organic phosphates to attain steady state. In plasma at pH 7.0-8.0, the transmembrane Pi concentration ratio R (cellular Pi/plasma Pi) decreased with increasing pH, with a slope which was 2.7-times greater than that predicted if Pi simply distributed passively across the cell membrane. The concentration of 2,3-bisphosphoglycerate (2,3-BPG), the most abundant cytosolic organic phosphate, decreased at acidic pH and increased at alkaline pH, but stabilised at these values after 24 h. Therefore, while net generation or consumption of Pi by 2,3-BPG may initially have contributed to the steep dependence of R on pH, some other factor must have maintained this anomaly after 24 h. In plasma in which the Pi concentration was increased from 1 to 2.5 mM, the cellular Pi concentration increased from 0.6 to only 1.0 mmol/l cells, and 2,3-BPG increased by less than 20%. Thus, cellular Pi and 2,3-BPG concentrations seemed to be buffered or regulated in the face of changes in extracellular Pi. However, this regulation failed in a Pi-free balanced salt solution, as the 2,3-BPG concentration declined to half that observed in freshly drawn blood, although cell Pi remained at about 0.3 mM. Incubation in Pi-free solution with ouabain for 24 h to decrease the transmembrane sodium gradient, or incubation for 2 h in the absence of sodium, decreased this residual cellular Pi by about 20%, but did not abolish it. In Pi-free solution, but not with 1 mM Pi, cellular Pi increased when passive transmembrane Pi leakage was inhibited with 4-acetamido-4'-iso-thiocyanatostilbene-2,2'-disulphonate (SITS). We conclude that red cell Pi concentration cannot be explained fully by passive transmembrane distribution of Pi, nor by changes in 2,3-BPG, and that part of the anomaly may arise from sodium-linked active Pi transport.