Influence of lead ions on cation permeability in human red cell ghosts

Summary Intracellular Pb²⁺ ions can replace Ca²⁺ ions in stimulating the Ca-dependent K permeability of human red blood cells. In metabolically depleted resealed ghosts, the threshold for stimulation of⁸⁶Rb efflux by internal Pb²⁺ is around 5×10^–10 m, and stimulation is half-maximal at about 2×10^–9 m, and maximal at 10^–8 m Pb²⁺. There is no effect on²²Na efflux in this concentration range.⁸⁶Rb efflux is antagonized by internal Mg²⁺ ions, and by the channel-blocking drugs quinidine and diS-C₂(5), as observed for the Ca-dependent K permeability in red cells. In ghosts containing EDTA, which prevents any internal effects of Pb²⁺ ions, external Pb²⁺ increases both²²Na and⁸⁶Rb permeability when its concentration exceeds 6×10^–7 m. This effect is seemingly unrelated to the Ca-dependent K permeability. This work makes extensive use of Pb²⁺ ion buffers, and gives information about their preparation and properties.     

Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.     

Studies on the cation permeability of human red cell ghosts. Characterization and biological significance of two membrane sites with high affinities for Ca.

Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant ap pH 7.2 for Ca (K'p1) of 3-5 X 10(-7) M, for Sr of 7 X 10(-6) M. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes. K'D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK' values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mM. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1-140 mM) or K (0.1-6 mM) concentrations had little effect and did not change K'p1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K'p2) varied between 6 X 10(-7) and 4 X 10(-6) M at pH 7.2 Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca greater than Sr greater than Ba greater than Mg. K'p2 was independent on the pH value in the range between 6.0 and 8.0 Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill cofficients were affected neither by the ion composition nor by the Ph values of the intra-and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific "channel" has properties similar to the K channel in excitable tissues.     

The birefringence of the human red cell ghosts

The type of birefringence described by Mitchison, which extends some 0.5 µ in from the surface of the human red cell ghost in glycerol and which shows a maximum retardation of about 7 A, is only found in ghosts which are sufficiently well hemoglobinised to be seen with the ordinary microscope. Ghosts from which all hemoglobin has been lost are not visible with the ordinary microscope and are not birefringent, although they are clearly visible with phase contrast. About 90 per cent of the ghosts in glycerol preparations are of the latter type, the exact percentage being a function of time. Mitchison's measurements of birefringence, although reproducible, accordingly apply only to ghosts in which some hemoglobin still remains complexed with the lipoprotein layers of the red cell ultrastructure, and do not enable one to draw conclusions as to the thickness and orientation of the lipoprotein surface layers.     

Physiological characteristics of human red blood cell ghosts

The properties of ghosts prepared by hypotonic hemolysis at various ratios of cells (C) to hemolyzing solution (H) have been studied. At all ratios, hemoglobin (Hb) was found to be distributed equally between the ghost and supernatant compartments. Techniques employing Fe(59)-labelled Hb showed that during hemolysis all of the Hb is exchangeable and that following hemolysis the ghost is impermeable to Hb. Ghosts containing defined fractions of their original Hb were prepared by appropriately altering the ratio C/H. When washed and suspended in 0.17 M NaCl-PO(4)-buffered media, the ghosts returned to their initial volume, recovered normal shape, and behaved as osmometers. The rate of rehemolysis of these reconstituted ghosts was observed to be proportional to the concentration of Hb in the ghosts. The rate of rehemolysis was accelerated by the addition of n-butyl alcohol (BA). For a given concentration of BA, temperature, and Hb content the rate of rehemolysis was minimal around the isoelectric point of Hb. Rehemolysis by BA was inhibited by the addition of sucrose to the medium. K influx and outflux were measured and found to be increased by the addition of BA and not influenced by the presence of sucrose. These results on the rehemolytic characteristics of ghosts are consistent with and support the colloid-osmotic theory of hemolysis.     

Volume-sensitive K influx in human red cell ghosts

K influx into resealed human red cell ghosts increases when the ghosts are swollen. The influx demonstrates properties similar to volume-sensitive K fluxes present in other cells. The influx is, for the most part, insensitive to the nature of the major intracellular cation and therefore is not a K-K exchange. The influx is much greater when the major anion is Cl than when the major anion is NO3; Cl stimulates the flux and, at constant Cl, NO3 inhibits it. Increase in the influx rate is rapid when shrunken ghosts are swollen or when NO3 is replaced by Cl. The volume-sensitive K influx requires intracellular MgATP at low concentrations, and ATP cannot be replaced by nonhydrolyzable ATP analogues. The volume-sensitive influx is inhibited by Mg2+ and by high concentrations of vanadate, but is stimulated by low concentrations of vanadate. It is not modified by cAMP, the removal of Ca2+ by EGTA, substances that activate protein kinase C, or by inhibition of phosphatidylinositol kinase. The influx is inhibited by neomycin and by trifluoperazine.     

Improved stop-flow apparatus to measure permeability of human red cells and ghosts

An improved stop-flow apparatus has been designed and constructed to measure the permeability characteristics of human red cells, which can be inferred from the time course of red cell volume changes following a sudden change in cellular environment produced by a rapid mixing device. The improved apparatus is directly coupled to a computer which automates thesubtraction and averaging procedures that have been developed to minimize the noise generated in the system by the cessation of red cell forward motion when the flow is suddenly stopped. Real time data acquisition also makes it possible to increase the number of data points by an order of magnitude, thus improving accuracy significantly. The apparatus has been tested by measurements of the human red cel hydraulic permeability coefficient. Data are presented to validate the subtraction procedure. Experiments have also been carried out on red cell ghosts which indicate that the hydraulic conductivity of the ghost is similar to that of the undisturbed red cell.     

Transport of sugars into human red blood cell ghosts

It has been found that human red cell ghosts react differently in the presence of various sugars in the medium. The stability of spheric ghosts is preserved in solutions of sugars entering red cells by means of the common carrier. In media of other sugars the ghosts' shapes change to shrunken, crenated forms and between the microscope slides to discoid ones. Under the conditions employed it was further observed that the incubation of fructose- or rhamnose-containing ghosts in solutions of sugars sharing the carriers led to an equilibration of sugars between the medium and the ghosts. The impermeability of ghosts for sugars not sharing the carriers was supported by the finding that fructose could be washed out to a much less extent than glucose. These results suggest that sugars without affinity to the carriers may move in the erythrocyte membrane through nonspecific sites (pores, channels).     

The effect of ethanol on the passive Ca permeability of human red cell ghosts measured by means of arsenazo III

Ethanol in the range of 0.76-2.40 M caused an immediate increase in the Ca permeability of the plasma membrane of resealed human red blood cell ghosts in which intracellular free Ca could be continuously monitored by means of the Ca chromophore arsenazo III. At a given concentration of ethanol, the Ca permeability increased markedly a few minutes following the mixing of the ghosts and the ethanol, and continued to increase over at least the next 30 min. Preincubating the ghosts in ethanol for 15, 60 and 120 min before measuring the rate of free Ca accumulation, progressively increased the effect of a given concentration of ethanol. These results indicate that the effect of a given concentration of ethanol is a complex function of concentration and exposure time. The effects of ethanol in this concentration range were completely reversible. The resealed ghosts used in these experiments were depleted of ATP to avoid interference from the Ca pump and all experiments were carried out with 150 mM KCl on both sides of the membrane to minimize changes in either the volume or membrane potential associated with activation of the Ca-dependent K channel.     

Influence of preparative procedures on the membrane viscoelasticity of human red cell ghosts

The effects of systematic variations in the preparative procedures on the membrane viscoelastic properties of resealed human red blood cell ghosts have been investigated. Ghosts, prepared by hypotonic lysis at 0 degrees C and resealing at 37 degrees C, were subjected to: measurement of the time constant for extensional recovery (tc); measurement of the membrane shear elastic modulus (mu) via three separate techniques; determination of the membrane viscosity (eta m) via a cone-plate Rheoscope. Membrane viscosity was also determined as eta m = mu X tc. Compared to intact cells, ghosts had shorter tc, regardless of their residual hemoglobin concentration (up to 21.6 g/dl). However, prolonged exposure to hypotonic media did increase their recovery time toward the intact cell value. The shear elastic modulus, as judged by micropipette aspiration of membrane tongues (mu p), was similar for all ghosts and intact cells. This result, taken with the tc data, indicates that ghosts have reduced membrane viscosity. Rheoscopic analysis also showed that eta m was reduced for ghosts, with the degree of reduction (approx. 50%) agreeing well with that estimated by the product mu p X tc. However, flow channel and pipette elongation estimates indicated that the ghost membrane elastic modulus was somewhat elevated compared to intact cells. We conclude that: ghosts have reduced membrane viscosity; ghosts have membrane rigidities close to intact cells, except possibly when the membrane is subjected to very large strains; the reduction in eta m is not directly related to the loss of hemoglobin; prolonged exposure of ghosts to low-ionic strength media increases the membrane viscosity toward its initial cellular level. These data indicate that the mechanical characteristics of ghost membranes can be varied by changing the methods of preparation and thus have potential application to further studies of the structural determinants of red cell membrane viscoelasticity.     

Membrane transport of L-triiodthyronine by human red cell ghosts.

L-T₃ transport has been investigated in human red cell ghosts. Determination of initial T₃ uptake revealed two separate saturable uptake systems, one with a K_m of 1.6 × 10^?8M, the other with a K_m of 3.3 × 10^?6M. Binding experiments resulted in two dissociation constants, 1.4 × 10^?7M.and 2.6 × 10^?6M. Uptake was dependent on the ghost volume, indicating an intravesicular location of T₃. The T₃ was concentrated 6 times by the ghosts. Ouabain reduced the uptake by the low K_m system, but was without effect on the high K_m system. Thus evidence is provided both of binding of T₃ to the ghost membrane and of its uphill transport across the membrane.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

Calcium-calcium and calcium-strontium exchange across the membrane of human red cell ghosts

Summary Stationary and nonstationary state⁴⁵Ca fluxes as well as Sr–Ca exchange movements were studied in energy-depleted human erythrocyte ghosts at different intra-and extracellular Ca concentrations. Influx and efflux followed the kinetics of a closed two-compartment system. The influx and efflux rate constants (k _in andk _out, respectively, fractions of total extra- or intracellular⁴⁵Ca that move in one direction per unit time) were similar in magnitude. They decreased with increasing Ca concentration on the cisside and increased with increasing Ca concentration on the trans-side of the membrane. Hence, the fluxes in both directions were characterized by saturation kinetics and appeared to be partially caused by an exchange diffusion mechanism. In the presence of a moderate inward (up to 8mm) or outward (up to 2mm) Ca concentration gradient, k_in andk _out did not vary in the course of an experiment and did not differ significantly from rates which were measured under stationary state conditions. Extracellular Sr induced an outward transport of intracellular Ca against the concentration gradient (counter-transport). The resulting inward Ca concentration gradient (maximal inside-to-outside concentration ratio as 1 to 3) persisted since extra- and intracellular Sr did not equilibrate. Analogous results were obtained studying⁴⁵Ca–⁴⁰Ca countertransport. In net flow experiments Ca–Sr exchange proved to occur on a one-for-one basis. Ca–Sr exchange was additive to the noncoupled Ca and Sr net downhill movements. The experimental results suggest that a specific ATP-independent Ca transfer system exists in the erythrocyte membrane which acts symmetrically on the two sides of the membrane and is restricted to a tightly coupled one-for-one exchange diffusion.