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.

Net fluxes of orthophosphate across the plasma membrane in human red cells following alteration of pH and extracellular Pi concentration.

Even though net fluxes of Pi (orthophosphate) across the cell membrane may be important in clinical disorders involving the abnormal extracellular Pi concentration, in acid-base disturbances, and in the responses of some cells to hormones, relatively few studies have been made of these fluxes, owing to the complexities of interpretation. Here we have studied net fluxes in response to changes in extracellular pH and Pi concentration in the simple case of the human red cell. The permeability of the cell membrane to net Pi fluxes was described in terms of a first-order rate constant, epsilon. By means of a mathematical model, it was possible to discriminate between transmembrane Pi movement, net intracellular generation or consumption of Pi by organic phosphates, and extracellular generation of Pi from the cells lysing during the experiment. We show that net Pi influx into the cell during experimental alkalosis was probably driven by net consumption of Pi by organic phosphates, and that this was reversed during acidosis. Inhibition of net Pi influx by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate (SITS) suggests that, like Pi self-exchange, net influx is at least partly mediated by the band 3 transport protein. Unexpectedly, epsilon increased from 2 h-1 at extracellular pH 7.4 to approx. 7 h-1 at pH 7.8. From the value of epsilon at pH 7.4, we conclude that the apparent buffering or regulation of steady-state Pi concentrations, previously reported in red cells in vitro, was not an artifact of intracellular generation of Pi from organic phosphates.

32P-labelling anomalies in human erythrocytes. Is there more than one pool of cellular Pi?

1. Human erythrocytes were incubated in autologous plasma containing [32P]Pi, and sampled by a method which avoids washing the cells. 2. In experiments of up to 3 h duration, the specific radioactivity of cellular Pi stabilized at a value below that of extracellular Pi. This can be explained on the basis of a single cellular Pi pool exchanging with a large unlabelled pool of cellular organic phosphates. 3. However, a rapid initial phase of labelling, occurring within 30 s, was inconsistent with the situation described in point 2. A possible explanation is that about 1/4 of cellular Pi occurs in a separate, fast-labelling pool. 4. When the extracellular Pi concentration was doubled, most of the corresponding increase in the steady-state cellular Pi concentration was accounted for by the apparent fast-labelling Pi pool, which also doubled. 5. The observed initial rate of labelling of cellular organic phosphates [which probably occurs through the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12)] was considerably lower than that predicted from the flux through the Embden-Meyerhof pathway. This implies that the enzyme is exposed to Pi whose specific radioactivity is lower than the mean specific radioactivity of cellular Pi, and fails to support earlier suggestions that this enzyme uses extracellular Pi. 6. In 3 h incubations, the rate of organic phosphate labelling was roughly constant throughout, even though the specific radioactivity of cellular Pi had risen slowly to a plateau. Viewed in conjunction with point 5, this again suggests some inhomogeneity in cellular Pi. 7. Cellular Pi and extracellular Pi only reached isotopic steady state after 2 days. At this stage some organic phosphates were probably still incompletely labelled. 8. We conclude that, whatever their physical or technical reasons, such labelling inhomogeneities and slow attainment of isotopic steady state may cause serious misinterpretation of results if ignored during 32P-labelling of intact cells.