Chloride permeability in human red cells: Influence of membrane protein rearrangement resulting from ATP depletion and calcium accumulation

Summary As 15% of band 3 protein, the assumed chloride channel, is associated with spectrin, the major peripheral protein of a lattice located at the red cell membrane-cytosol interface, the present study was undertaken to evaluate whether a rearrangement of the lattice modifies the functional property of band 3 protein. Such a rearrangement was modulated by depletion of cell ATP and/or by accumulation of Ca²⁺ ions within the cell.ATP depletion induces an inhibition of the electroneutral one-for-one chloride exchanges. Neither the modification of red cell morphology due to ATP depletion (discocyte-echinocyte transformation) nor a direct effect of the decrease in internal ATP level can account for this inhibition. On the other hand, it seems reasonable to consider that inhibition is related to the changes in membrane protein organization (formation of heteropolymers) induced by the decrease in ATP level. But it does not appear that the degree of inhibition is modified when this altered assembly of membrane protein is stabilized by disulfide linkages.Accumulation of Ca²⁺ ions in the cell at a relatively low concentration (10m range) inhibits chloride exchange without apparent modification of the assembly of membrane proteins. This effect of calcium on chloride exchanges is speculatively denoted as a direct effect of calcium.Calcium loading of fresh red cells at higher concentrations (500 to 1000 m) obtained by use of the ionophore A23187 induces a very strong inhibition of chloride exchanges. In this case, inhibition can be reasonably accounted for by two simultaneous effects of calcium: a direct effect which explains half of the inhibition and an indirect effect due to the formation of membrane protein complexes stabilized by covalent crosslinkages (activation by Ca²⁺ ions of a transglutaminase).It is interesting to note that intracellular calcium, whatever the level, inhibits electroneutral exchanges of chloride but increases net chloride movements.     

The red cell membrane and its cytoskeleton.

Gel-filtration (Sephadex G-75) analysis of hepatic cytosol reveals both qualitative and quantitative sex differences in oestrogen-binding proteins. The elution profile of [³H]oestradiol-labelled cytosol shows four species of oestrogen-binding proteins (peaks I, II, IV and V) common to both sexes. The amount of [³H]oestradiol binding in peak I is equivalent in both males and females and corresponds quantitatively to the specific oestrogen receptor. The amount of binding in the remaining three peaks is greater in males than females. In addition, an oestrogen-binding protein (peak III) is present that is unique to male cytosol. Proteinase-inhibition studies demonstrate that the observed multiplicity of oestrogen-binding proteins is not an artefact of proteolytic breakdown. Sex differences in oestrogen-binding proteins are absent in immature male and female animals; the oestrogen-binding protein profile in immature rats resembles that of an adult female. Gonadectomy of adult animals does not affect the oestrogen-binding-protein profile. In contrast, neonatal (day 1) castration results in partial feminization of the characteristic oestrogen-binding protein profile seen in the adult male; the appearance of Peak III is suppressed and marked decreases in the amount of oestradiol binding occurs in the remaining peaks. Hypophysectomy of adult animals results in near abolishment of the observed sex differences; the male oestrogen-binding protein profile is partially feminized and the female profile is partially masculinized, as characterized by the appearance of [³H]oestradiol binding in the region of peak III and increased amounts of binding in peaks IV and V. The present studies demonstrate a multiplicity of oestrogen-binding proteins in liver cytosol and raise the possibility that the presence of some of these proteins may be imprinted at birth through the hypothalamic–pituitary axis, by a mechanism requiring neonatal androgen exposure.     

Pyruvate prevents the ATP depletion caused by formaldehyde or calcium-chelator esters in the human red cell

Formaldehyde released during hydrolysis of calcium-chelator esters incorporated into cells blocks glycolysis in the human erythrocyte (Tiffert, T., García-Sancho, J. and Lew, V.L. (1984) Biochim. Biophys. Acta 773, 143-156). This blockade is due to the inhibition of glyceraldehyde-3-phosphate dehydrogenase by NAD+ depletion caused by enzymatic oxidation of formaldehyde coupled to NADH production. The addition of pyruvate to the incubation medium prevents or reverts ATP depletion.     

Further investigation on ATP metabolism and red cell membrane integrity

Among 15 enzymes PFK decreased most during preservation at 4 degrees C for 6-8 weeks, and this was prevented by the addition of adenine and inosine, but not by adenine or inosine only. PFK inactivation in hemolysate was also prevented by ATP. In order to maintain low fragility, isotonic sucrose solution was newly devised. Maltose and lactose followed and mannitol was also effective. The decrease in fragility accompanied the decrease in the cell volume and the increase of Na+, K+, H+ and Cl- in the medium. However, the filtrability of red cells was sometimes decreased in case of extremely lowered fragility. Therefore, appropriate ratios of isoosmotic sucrose (hardly permeable) and NaCl (relatively rapidly permeable) solutions must be selected for preservation of blood. Various properties in vitro of rabbit cells were well maintained and their posttransfusion viability was also prolonged when using this medium. Elimination of hypoxanthine could be achieved by hydron coated charcoal prior to transfusion.     

Copper-induced generation of superoxide in human red cell membrane.

The addition of CuSO₄ to erythrocyte membrane preparations causes the generation of superoxide radicals as measured by the superoxide dismutasesensitive oxidation of epinephrine. The formation of O₂^? is accompanied by the reduction of copper to the cuprous form. These events are inhibited by pCMB suggesting the involvement of membrane -SH groups in the reduction of copper, and a subsequent autoxidation of Cu⁺ to generate O₂^? and Cu⁺⁺. It is proposed that these mechanisms may be the basis for the cytotoxicity of copper in individuals exposed to copper either through a genetic deficiency of copper binding proteins or as a result of the acute ingestion of copper salts.     

Potassium influx in human neonatal red blood cells. Partition into its major components.

The potassium influx in human neonatal red blood cells (nRBC) shows an approximately 25% lower value compared to the total potassium influx in adult red blood cells (aRBC). The ouabain-sensitive potassium influx component represents approximately 70-75% of the total potassium influx for both types of cells but with an absolute value significantly lower in nRBC. In nRBC, the half maximum inhibitory effect for ouabain was obtained at a 10(-9) M concentration. The ouabain-insensitive nRBC potassium influx fractions showed two components: (i) a bumetanide-sensitive component, significantly lower than that of aRBC, (ii) a ouabain-bumetanide-insensitive (leak) component with a similar value in both cell types. The sum of the ouabain-sensitive and furosemide-sensitive components amounted in nRBC to a greater value than the total potassium influx. This behaviour could be interpreted as a superposition of the action of the inhibitors on the components affected.     

ATP regulation of the human red cell sugar transporter

Purified human red blood cell sugar transport protein intrinsic tryptophan fluorescence is quenched by D-glucose and 4,6-ethylidene glucose (sugars that bind to the transport), phloretin and cytochalasin B (transport inhibitors), and ATP. Cytochalasin B-induced quenching is a simple saturable phenomenon with Kd app of 0.15 microM and maximum capacity of 0.85 cytochalasin B binding sites per transporter. Sugar-induced quenching consists of two saturable components characterized by low and high Kd app binding parameters. These binding sites appear to correspond to influx and efflux transport sites, respectively, and coexist within the transporter molecule. ATP-induced quenching is also a simple saturable process with Kd app of 50 microM. Indirect estimates suggest that the ratio of ATP-binding sites per transporter is 0.87:1. ATP reduces the low Kd app and increases the high Kd app for sugar-induced fluorescence quenching. This effect is half-maximal at 45 microM ATP. ATP produces a 4-fold reduction in Km and 2.4-fold reduction in Vmax for cytochalasin B-inhibitable D-glucose efflux from inside-out red cell membrane vesicles (IOVs). This effect on transport is half-maximal at 45 microM ATP. AMP, ADP, alpha, beta-methyleneadenosine 5'-triphosphate, and beta, gamma-methyleneadenosine 5'-triphosphate at 1 mM are without effect on efflux of D-glucose from IOVs. ATP modulation of Km for D-glucose efflux from IOVs is immediate in onset and recovery. ATP inhibition of Vmax for D-glucose exit is complete within 5-15 min and is only partly reversed following 30-min incubation in ATP-free medium. These findings suggest that the human red cell sugar transport protein contains a nucleotide-binding site(s) through which ATP modifies the catalytic properties of the transporter.     

The association of human erythrocyte catalase with the cell membrane

A specific interaction of human erythrocyte catalase with the inner surface of the red cell membrane was demonstrated. The dependency of catalase affinity on pH and ionic strength implies that the interaction is dominated by electrostatic forces. Scatchard analysis of the binding at pH 6.0 and 5 mm Mes buffer reveals a single class of approximately 10⁶ binding sites/ghost with an association constant of 2.5 × 10⁷m^?1. The membrane-bound catalase retains its enzymatic activity. Competition binding studies of catalase and other proteins known to associate with the membrane inner surface were carried out. It was found that the binding of catalase is inhibited by aldolase, glyceraldehyde-3-phosphate dehydrogenase as well as by hemoglobin. The advantage of membrane-bound catalase in protection of the cell membrane against peroxidative damages is discussed.     

Ca binding to the human red cell membrane: characterization of membrane preparations and binding sites.

Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces...     

Hydrogen-peroxide-induced heme degradation in red blood cells: the protective roles of catalase and glutathione peroxidase

Catalase and glutathione peroxidase (GSHPX) react with red cell hydrogen peroxide. A number of recent studies indicate that catalase is the primary enzyme responsible for protecting the red cell from hydrogen peroxide. We have used flow cytometry in intact cells as a sensitive measure of the hydrogen-peroxide-induced formation of fluorescent heme degradation products. Using this method, we have been able to delineate a unique role for GSHPX in protecting the red cell from hydrogen peroxide. For extracellular hydrogen peroxide, catalase completely protected the cells, while the ability of GSHPX to protect the cells was limited by the availability of glutathione. The effect of endogenously generated hydrogen peroxide in conjunction with hemoglobin autoxidation was investigated by in vitro incubation studies. These studies indicate that fluorescent products are not formed during incubation unless the glutathione is reduced to at least 40% of its initial value as a result of incubation or by reacting the glutathione with iodoacetamide. Reactive catalase only slows down the depletion of glutathione, but does not directly prevent the formation of these fluorescent products. The unique role of GSHPX is attributed to its ability to react with hydrogen peroxide generated in close proximity to the red cell membrane in conjunction with the autoxidation of membrane-bound hemoglobin.     

Creep and stress relaxation of human red cell membrane

In contrast to most mechanical properties of the red cell, experimental information on stress relaxation (SR) of the membrane skeleton is scarce. On the other hand, many postulates or assumptions as to the value of the characteristic time of SR \((\tau _{\mathrm{SR}})\) can be found in the literature. Here, an experiment is presented that allows measurement of \(\tau _{\mathrm{SR}}\) up to values of about 10 h. The membrane skeleton was deformed passively by changing the spontaneous curvature of the bilayer thus transforming the natively biconcave red cells into echinocytes. This shape and the concomitant deformation of the skeleton were kept up to 4 h by incubation at 37 ℃. During this period, no plastic deformation (creep) was observed. After the incubation, the spontaneous curvature was returned to normal. The resulting shape was smooth showing no remnants of the echinocytic shape. Both observations indicate \(\tau _{\mathrm{SR}}\gtrapprox \) 10 h. This result is in gross disagreement to postulates or assumptions existing in the literature.     

Radiation resistance and the CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase activities of seven human cell lines

CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase form the primary enzymic defense against toxic oxygen reduction metabolites in cells. To test the importance of these protective enzymes in the cellular radiation response, the enzymic activities of seven different human cell lines were determined in parallel with their clonogenic survival characteristics. A positive correlation between the content of glutathione peroxidase in cell lines and their extrapolation numbers (n) and quasithreshold doses (Dq) was detected. Between the cellular contents of the other enzymes and D0, n, and Dq no positive correlations could be established. An interesting finding was a very high Mn superoxide dismutase content in a malignant mesothelioma cell line P7, which had an extremely high D0, 5.0 Gy.     

Molecular properties of the red cell calcium pump: II. Effects of calmodulin,proteolytic digestion and drugs on the calcium-induced membrane phosphorylation by ATP in inside-out red cell membrane vesicles

In inside-out human red cell membrane vesicles /IOV/, in the absence of Mg²⁺, the only calcium-induced labelling by γ³²P-ATP occurs in a 140–150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated intermediate /EP/ of the calcium pump. In the presence of Mg²⁺ calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accelerates EP formation both in the absence and presence of Mg²⁺.Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVs, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of EP. In trypsin-digested IOVs the molecular weight of the ³²P-labelled EP is shifted to lower values /110–120 000/ We suggest that trypsin digestion cleaves off a 20–40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.     

Water-soluble proteins of the human red cell membrane

Summary Procedures were developed for preparation of red cell membranes almost free of hemoglobin but with minimal loss of membrane proteins. Two water-soluble protein fractions are described, each constituting about 25% of the ghost protein. The first is ionically bonded and can be solubilized in water rapidly at pH 7.0 and more slowly at higher ionic strength solutions, with a minimal rate at 20mm. This fraction contains four major components with molecular weights ranging from 30,000 to 48,000. The second fraction can only be solubilized at an appreciable rate if Ca⁺⁺ is absent and at higher pH (9.0). It is predominantly a single molecular weight component (150,000). It tends to aggregate at higher ionic strength and in the presence of Ca⁺⁺. Evidence is presented suggesting that the water-soluble proteins are present at the inner face of the membrane. The lipids remain in a water-insoluble residue that contains four major protein components ranging in molecular weight from 30,000 to 100,000. The latter is the predominant component. Only the residue contains the Na⁺–K⁺-activated ATPase, the cholinesterase, antigenic activity and most of the sialic acid and carbohydrate. The first water-soluble fraction contains a Mg⁺⁺-activated ATPase. The extraction of the water-soluble proteins is accompanied by anatomical changes resulting finally in the formation of small membranous vesicles.     

Glucose transport in human red cell membranes. Dependence of age, ATP, and insulin

Glucose self-exchange flux (Jex) and net efflux (Jnet) in human red cells and ghosts were studied at 25 degrees C and pH 7.2 by means of the combined use of the Millipore-Swinnex filtering method and the continuous flow tube method to show the dependence of time of storage after aspiration, ATP and insulin. In fresh cells (RBC), ghosts (G), ghosts with 2 mM ATP (G +), and cells stored at 4 degrees C greater than 60 days (OC) both Jex and Jnet follow simple Michaelis-Menten kinetics where J = Jmax X Ci X (K1/2 + Ci)-1. Jmaxex and Jmaxnet (nmol X cm-2 X s-1), respectively, was: (RBC) 0.27 and 0.19, (G) 0.24 and 0.27, (G +) 0.23 and 0.24, (OC) 0.23 and 0.20. K1/2,ex and K1/2,net (mM), respectively, was: (RBC) 7.5 and 1.3, (G) 4.8 and 14.2, (G +) 11.6 and 6.8, (OC) 3.8 and 9.0. In ghosts, the ATP-dependent fraction of the permeability shows a hyperbolic dependence on glucose concentrations lower than 80 mM. Insulin up to 1 microM had effect on neither Jex nor Jnet in RBC. Based on reported values of cytochalasin B binding sites the turnover rate per site in RBC appears to be as high as in maximally insulin-stimulated fat cells. Our results suggest that the number of transport sites remains constant, independent of age, ATP and insulin.