Ionic and osmotic equilibria of human red blood cells treated with nystatin.

Abstract

Human red blood cells have been incubated in the presence of nystatin, which allows Na and K, as well as CI and pH to equilibrate rapidly when cell volume is set with external impermeant sucrose. The intracellular mean ionic activity coefficients, relative to values in the extraceUular solution, for KCI and NaCl are 1.01 4" 0.02 and 0.99 + 0.02 (SD, n = 10), respectively, and are independent of external pH, pH o, and of [sucrose]o. With nystatin the dependence of red cell volume on [sucrose]~ deviates from ideal osmotic behavior by as much as a factor of three. A virial equation for the osmotic coefficient, ~k, of human hemoglobin, Hb, accounts for the cell volumes, and is the same as that which describes Adair's measurements of ~#nb for Hb isolated from sheep and ox bloods. In the presence of nystatin the slope of the acid-base titration curve of the cells is independent of cell volume, implying that the charge on impermeant cellular solutes is independent of Hb concentration at constant pH.By modifying the Jacobs-Stewart equations (1947. J. Cell Comp. PhysioL 30:. 79-103) with the osmotic coefficients of Hb and of salts, a nonideal thermodynamic model has been devised which predicts equilibrium Donnan ratios and red cell volume from the composition of the extracellular solution and from certain parameters of the cells. In addition to accounting for the dependence of cell volume on osmotic pressure, the model also describes accurately the dependence of Donnan ratios and cell volumes on pHo either in the presence or absence of nystatin.

Volume-regulatory responses of Amphiuma red cells in anisotonic media. The effect of amiloride.

“…As discussed above and elsewhere (Siebens, 1985), these changes in Vm can be accounted for by changes in the relative contribution of the impermeant anions to the membrane potential . If this is true, it should be possible in future studies to use nystatin to make Amphiuma red cells permeable to cations and anions (Dalmark, 1975 ;Freedman and Hoffman, 1979) and to obtain values of rCl as a function of volume similar to those of the present study.…”

“…Because the osmotic coefficient of mammalian hemoglobin is known to change as a function of concentration (Adair, 1929;Dick and Lowenstein, 1958 ;McConaghey and Maizels, 1961 ;Freedman and Hoffman, 1979), the predicted value of aapp is actually expected to be slightly greater than the medium osmolality in the VRD and VRI responses because of changes in the osmotic coefficient of hemoglobin during volume recovery (Siebens, 1985). In the Amphiuma red cell, this slight effect predicts that a.pp will be^-6 and 13 mosmol hypertonic to the medium in the VRD and VRI responses, respectively .…”

On the mechanism of stomatocyte–echinocyte transformations of red blood cells: experiment and theoretical model

“…The latter assumption is not expect to lead to a considerable mistake, insofar as the modeling of the intracellular phase as a diluted electrolyte solution is found to give correct predictions about the concentrations of ions due to inorganic electrolytes, such as K + , Na + , Cl − , etc. [52].…”

My Passion and Passages with Red Blood Cells

“…Jeffrey Freedman designed and executed studies concerned with the conditions that account for ionic equilibrium of human red blood cells (86). Jeffrey showed that the charge of Hb was independent of its concentration and more importantly that evaluation of the ionic activity coefficients of Na + , K + , and Cl − and the osmotic coefficient of Hb, as a function of concentration, could quantitatively describe the dependence of the Donnan ratios and cell volumes on the ionic composition of the medium and its pH (86).…”

“…Jeffrey showed that the charge of Hb was independent of its concentration and more importantly that evaluation of the ionic activity coefficients of Na + , K + , and Cl − and the osmotic coefficient of Hb, as a function of concentration, could quantitatively describe the dependence of the Donnan ratios and cell volumes on the ionic composition of the medium and its pH (86). The analysis also corrected Ponder's "R" value (26) ++ on either the activation of the Gárdos channel or the inhibition of the Na + pump.…”