THE EFFECTS OF SICKLING ON ION TRANSPORT : II. THE EFFECT OF SICKLING ON SODIUM AND CESIUM TRANSPORT

When the red cells from patients with sickle cell anemia (S-S) were kept in the disk shape by incubation in O2, they maintained cell sodium in the steady state for at least 10 hours. The sodium flux in such cells at 37°C. was 6.0 ± 1.5 m.eq./ (liters RBC) x (hours). When S-S cells were sickled by incubation in N2, sodium outflux increased two- to threefold, while influx increased four- to fivefold and the cells gained net sodium. A small but undetermined fraction of the sodium in disk and sickle shaped S-S cells exchanges at one or more rates which are substantially slower than those calculated here from the initial rate of transfer of tracer from cells to the medium. The penetration of tracer Cs into normal and both disk and sickled S-S cells was markedly inhibited by increasing the K concentration in the medium, indicating that Cs and K compete for an entrance pathway in all three cell types. The ratio of the inward rate constant for tracer Cs to that for K42 in normal and disk-shaped S-S cells increased only slightly when the K concentration in the medium was increased, indicating that almost all the Cs entered such cells in competition with K. Sickling accelerated the entrance of tracer cesium into S-S cells. Furthermore, the rate constant ratio increased with increasing external K concentration in sickled cells, suggesting the simultaneous presence of a non-competitive route for cesium influx in this cell type. The results are interpreted to support the view that sickling (a) accelerates inward transport of K and Cs and outward transport of Na by a non-diffusion, assumed carrier, process and (b) opens pathways for the diffusion of all three ions

Ionic Permeability of Thin Lipid Membranes : Effects of n-alkyl alcohols, polyvalent cations, and a secondary amine

Ultrathin (black) lipid membranes were made from sheep red cell lipids dissolved in n-decane. The presence of aliphatic alcohols in the aqueous solutions bathing these membranes produced reversible changes in the ionic permeability, but not the osomotic permeability. Heptanol (8 mM), for example, caused the membrane resistance (Rm) to decrease from >108 to about 105 ohm-cm2 and caused a marked increase in the permeability to cations, especially potassium. In terms of ionic transference numbers, deduced from measurements of the membrane potential at zero current, Tcat/TCl increased from about 6 to 21 and TK/TNa increased from about 3 to 21. The addition of long-chain (C8ndash;C10) alcohols to the lipid solutions from which membranes were made produced similar effects on the ionic permeability. A plot of log Rm vs. log alcohol concentration was linear over the range of maximum change in Rm, and the slope was -3 to -5 for C2 through C7 alcohols, suggesting that a complex of several alcohol molecules is responsible for the increase in ionic permeability. Membrane permselectivity changed from cationic to anionic when thorium or ferric iron (10-4 M) was present in the aqueous phase or when a secondary amine (Amberlite LA-2) was added to the lipid solutions from which membranes were made. When membranes containing the secondary amine were exposed to heptanol, Rm became very low (103–104 ohm-cm2) and the membranes became perfectly anion-selective, developing chloride diffusion potentials up to 150 mv

Active Sodium and Potassium Transport in High Potassium and Low Potassium Sheep Red Cells

The kinetic characteristics of the ouabain-sensitive (Na + K) transport system (pump) of high potassium (HK) and low potassium (LK) sheep red cells have been investigated. In sodium medium, the curve relating pump rate to external K is sigmoid with half maximal stimulation (K1/2) occurring at 3 mM for both cell types, the maximum pump rate in HK cells being about four times that in LK cells. In sodium-free media, both HK and LK pumps are adequately described by the Michaelis-Menten equation, but the K1/2 for HK cells is 0.6 ± 0.1 mM K, while that for LK is 0.2 ± 0.05 mM K. When the internal Na and K content of the cells was varied by the PCMBS method, it was found that the pump rate of HK cells showed a gradual increase from zero at very low internal Na to a maximum when internal K was reduced to nearly zero (100% Na). In LK cells, on the other hand, no pump activity was detected if Na constituted less than 70% of the total (Na + K) in the cell. Increasing Na from 70 to nearly 100% of the internal cation composition, however, resulted in an exponential increase in pump rate in these cells to about ⅙ the maximum rate observed in HK cells. While changes in internal composition altered the pump rate at saturating concentrations of external K, it had no effect on the apparent affinity of the pumps for external K. These results lead us to conclude that the individual pump sites in the HK and LK sheep red cell membranes must be different. Moreover, we believe that these data contribute significantly to defining the types of mechanism which can account for the kinetic characteristics of (Na + K) transport in sheep red cells and perhaps in other systems

Separation of Adenosine Triphosphatase of HK and LK Sheep Red Cell Membranes by Density Gradient Centrifugation

Membrane fragments from high potassium (HK) and low potassium (LK) sheep red cells were separated by density gradient centrifugation. Three preparations were studied: (1) HK membranes sonicated for 20 minutes, (2) HK membranes sonicated for 3 minutes, and (3) LK membranes sonicated for 3 minutes. The adenosine triphosphatase (ATPase) activity in the maximally disrupted preparation (1) was not sensitive to Na + K and was recovered in relatively small but heavy (specific gravity 1.19) fragments which made up no more than 8 per cent of the total membrane. Both Na + K-sensitive (S) and Na + K-insensitive (I) ATPase activity were found in the more gently broken up preparations (2) and (3) but the ratio of S- to I-ATPase was much greater in HK than in LK membrane fragments. S-ATPase activity in preparation (2) was about 50 per cent that observed in HK membranes prior to sonication. S-ATPase activity was recovered from the density gradient in relatively large but light (specific gravity 1.10) fragments. As was the case with the maximally disrupted preparation (1), I-ATPase activity in both preparations (2) and (3) was recovered in small but heavy (specific gravity > 1.20) fragments. The possibility that sensitivity of sheep red cell membrane ATPase to Na + K depends on the association between units containing the enzyme(s) and large, light, phospholipid-containing components is discussed

Cytodifferentiation and Membrane Transport Properties in LK Sheep Red Cells

Young cells produced in LK sheep during rapid hematopoiesis after massive hemorrhage contain more K than the cells which are normally released into the circulation. The K content in these new cells falls to that characteristic of mature LK cells after a few days in the circulation. K transport properties in young and old cells before and after massive bleeding were studied. Young and old cells were separated by means of a density gradient centrifugation technique. Evidence showing that younger cells are found in the lower density fractions is presented. Active transport of K in the lightest fraction as measured by strophanthidin-sensitive influx was four to five times greater in red cells drawn 6 days after massive bleeding while the K leak as measured by strophanthidin-insensitive influx was only slightly larger. No change after bleeding was observed in older cells which had been present in the circulation prior to the hemorrhage. It is concluded that the high K content of young cells produced in LK sheep after bleeding is due to temporary retention of membrane K transport properties characteristic of HK cells. Thus, genetically determined modification of membrane transport properties has been shown to occur in nondividing circulating red cells

Effect of Peptide PV on the Ionic Permeability of Lipid Bilayer Membranes

This paper reports the effects of peptide PV (primary structure: cyclo-(D-val-L-pro-L-val-D-pro)δ) on the electrical properties of sheep red cell lipid bilayers. The membrane conductance (Gm) induced by PV in either Na+ or K+ medium is proportional to the concentration of PV in the aqueous phase. The PV concentration required to produce a comparable increase in Gm in K+ medium is about 104 times greater than for its analogue, valinomycin (val). Although the selectivity sequence for PV and val is similar, K+ ≳ Rb+ > Cs+ > NH4+ > TI+ > Na+ > Li+; the ratio of GGm in K+ to that in Na+ is about 10 for PV compared to > 103 for val. When equal concentrations of PV are added to both sides of a bilayer, the membrane current approaches a maximum value independent of voltage when the membrane potential exceeds 100 mV. When PV is added to only one side of a bilayer separating identical salt solutions of either Na+ or K+ salts, rectification occurs such that the positive current flows more easily away rather than toward the side containing the carrier. Under these conditions, a large, stable, zero-current potential (VVm) is also observed, with the side containing PV being negative. The magnitude of this VVm is about 90 mV and relatively independent of PV concentration when the latter is larger than 2 Times; 10–5 M. From a model which assumes that Vm equals the equilibrium potential for the PV-cation complexes (MS+) and that the reaction between PV and cations is at equilibrium on the two membrane surfaces, we compute the permeability of the membrane to free PV to be about 10–5 cm s–1, which is about 10–7 times the permeability of similar membranes to free val. This interpretation is supported by the fact that the observed values of Vm are in agreement with the calculated equilibrium potential for MS+ over a wide range of ratios of concentrations of total PV in the two bathing solutions, if the unstirred layers are taken into account in computing the MS+ concentrations at the membrane surfaces

The Permeability of Thin Lipid Membranes to Bromide and Bromine

Thin lipid (optically black) membranes were made from sheep red cell lipids dissolved in n-decane. The flux of Br across these membranes was measured by the use of tracer 82Br. The unidirectional flux of Br (in 50–100 mM NaBr) was 1–3 x 10-12 mole/cm2sec. This flux is more than 1000 times the flux predicted from the membrane electrical resistance (>108 ohm-cm2) and the transference number for Br- (0.2–0.3), which was estimated from measurements of the zero current potential difference. The Br flux was not affected by changes in the potential difference imposed across the membrane (±60 mv) or by the ionic strength of the bathing solutions. However, the addition of a reducing agent, sodium thiosulfate (10-3 M), to the NaBr solution bathing the membrane caused a 90% reduction in the Br flux. The inhibiting effect of S2O3= suggests that the Br flux is due chiefly to traces of Br2 in NaBr solutions. As expected, the addition of Br2 to the NaBr solutions greatly stimulated the Br flux. However, at constant Br2 concentration, the Br flux was also stimulated by increasing the Br- concentration, in spite of the fact that the membrane was virtually impermeable to Br-. Finally, the Br flux appeared to saturate at high Br2 concentrations, and the saturation value was roughly proportional to the Br- concentration. These results can be explained by a model which assumes that Br crosses the membrane only as Br2 but that rapid equilibration of Br between Br2 and Br- occurs in the unstirred layers of aqueous solution bathing the two sides of the membrane. A consequence of the model is that Br- "facilitates" the diffusion of Br across the unstirred layers

THE EFFECTS OF SICKLING ON ION TRANSPORT : I. EFFECT OF SICKLING ON POTASSIUM TRANSPORT

The conversion of red cells of patients with sickle cell anemia (S-S) from biconcave disk to sickle shape by removal of oxygen was found to increase the fraction of medium trapped in cells packed by centrifugation from 0.036 (S.E. 0.003) to 0.106 (S.E. 0.004). The fraction of water in the cells (corrected for trapped medium) was not affected by this shape transformation. Cation transport, however, was changed profoundly. S-S cells incubated in N2 rather than O2 showed net K loss with acceleration of both influx and outflux. That this change in K transport was due to the process of sickling was indicated by (1) the persistence of the effect in the absence of plasma, (2) the absence of the effect in hypoxic S-S cells in which sickling was inhibited by alkali or carbon monoxide, (3) the reversal of the effect when sickling was reversed by exposure to O2, and (4) the independence of the effect from such potentially important factors as age of the cell population. The acceleration of K transport by sickling is probably mediated by modification of the cell surface rather than the cell interior since concentrated sickle hemoglobin solutions in O2 or N2 did not show selective affinity for K. In molecular terms, the effect of sickling on K transport can be explained by presuming that the shape change (1) opens pathways for the free diffusion of K, and (2) accelerates K transport by a non-diffusion carrier process. The evidence for the former mechanism included (a) dependence of K influx into sickled cells on the concentration of K in the medium, and (b) increase in the total cation content of sickled cells with increasing pH. Observations suggestive of a carrier process included (a) the failure of sickled cell K concentration to become equal to external K concentration even after 48 hours, (b) the deviation of the flux ratio from that characteristic of diffusion, and (c) the dependence of K influx on glycolysis

Inhibition of Adenosine Triphosphatase in Sheep Red Cell Membranes by Oxidized Glutathione

This paper reports inhibition of Na+ + K+-stimulated, ouabain-inhibited adenosine triphosphatase (S-ATPase) in sheep red cell membranes by oxidized glutathione (GSSG). The results are consistent with the hypothesis that this inhibition depends upon the formation of a mixed disulfide between glutathione and -SH group(s) in the enzyme protein. Thus, inhibition of S-ATPase by GSSG proceeds more rapidly at alkaline than at neutral pH and is reversed by the addition of an excess of a compound containing reduced -SH groups (e.g. dithiothreitol). ATP protects S-ATPase against inhibition by GSSG and this protection depends on both the monovalent and divalent cation composition of the medium. Protection by ATP is more complete in the presence of K+ than in the presence of Na+