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

Abstract

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