ATP-depleted Red Cells Research Articles

Cell membrane fluctuations are regulated by medium macroviscosity: evidence for a metabolic driving force.

Extracellular fluid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body fluids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical fluctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase fluid viscosity. The parameters of cell membrane fluctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane fluctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATP-depleted red blood cells elevation of EMF did not affect cell membrane fluctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane fluctuations are driven by a metabolic driving force in addition to the thermal one.

Ca 2+ transients and Mn 2+ entry in human neutrophils induced by thapsigargin

Human neutrophils, preloaded with the fluorescent probe, Fura-2, were exposed to Ca 2+-releasing agents. The monitored traces of fluorescence were transformed by computer to cytosolic Ca 2+ concentration ([Ca 2+] i). Due to quenching of Fura-2, the addition of Mn 2+ enabled us to compute the cytosolic concentration of total manganese ([Mn] i). The agents used were the novel Ca 2+-mobilizing agent, thapsigargin (Tg), the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), and the divalent cation lonophore, A23187. The agents caused translent rises of [Ca 2+] i and monotonous rises of [Mn] i, suggesting influx but no efflux of Mn 2+. The rise time of [Ca 2+] i and the time constants and magnitude of the apparent Mn 2+ influx were strongly dependent on the sequence of addition of the agonist and Ca 2+. Contrary to FMLP, Tg needed several minutes to exert its full effect on the rise of [Ca 2+] i and on the influx of Mn 2+, the latter being dependent on two phases, activation and partial inactivation. Pretreatment with phorbol 12-myristate 13-acetate (PMA) inhibited the responses of Tg, FMLP and A23187. For comparison, human red blood cells were tested. Contrary to A23187, Tg did not Induced Ca 2+ uptake in ATP-depleted red cells but increased the Ca 2+ pump flux in intact red cells by 10%. The experimental data and computer simulations of the granulocyte data suggest that time-dependent changes of both passive Ca 2+ flux into the cytosol and Ca 2+ flux of the plasma membrane pump are involved in the translent [Ca 2+] i response.

Modulation of red cell vesiculation by protease inhibitors

Release of vesicles from human red cell membranes was induced either by ATP-depletion or by incubation of the cells in presence of sonicated dimyristoylphosphatidylcholine (DMPC) vesicles. Vesicles released from ATP-depleted red cells but not the DMPC-induced vesicles contained degradation products of band 3 protein. Furthermore, in ATP-depleted erythrocytes proteolytic breakdown products could be demonstrated that were not detected in cells incubated with DMPC. Proteolysis was neither significantly affected by the protease inhibitor N-α- tosyl- l-lysine chloromethyl ketone (TLCK) nor by other protease inhibitors tested in this study (diisopropylfluorophosphate, N-ethylmaleimide and phenylmethylsulfonyl fluoride). Both vesiculation processes were inhibited in a concentration dependent way by TLCK while other protease inhibitors did not significantly influence membrane vesiculation. Phase contrast microscopy showed that TLCK diminished the DMPC-induced formation of echinocytes which is known to precede vesicle release. These results suggest that the influence of TLCK on membrane vesiculation is not primarily due to inhibition of proteolysis but to a direct interaction of the inhibitor with the intrinsic domain of the erythrocyte membrane.

An increased calcium accumulation in ATP-depleted red cells of the patients with paroxysmal nocturnal hemoglobinuria.

A marked increase in calcium (Ca) uptake (85.5 +/- 39.0 nmoles/ml RBC/2 hr) was observed in ATP-depleted red cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) as compared to ATP-depleted normal red cells (8.0 +/- 2.0). The extent of increased Ca uptake in the PNH red cells was related to the extent of increased sensitivity to complement, as judged by the sugar water test (p less than 0.001) and the acidified serum test (p less than 0.001), and inversely correlated to the decreased activity of red cell acetylcholinesterase (p less than 0.01). The results may indicate that increased calcium uptake in PNH red cells is a possible intrinsic defect of the red cell membranes in the disorder.

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity.

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1–0.5 mM CdCl 2 in 10 mM Tris-HCl (pH 7.4) at 37°C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd 2+ concentration. The maximum release occurred at 37°C in the presence of 0.2 mM Cd 2+. The protein composition of Cd 2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1–0.2 mM Cd 2+, more than 90% of the Cd 2+ added to the incubation buffer was recovered in ghosts and 15–20% of the ghost Cd 2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd 2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd 2+ added to the incubation buffer and saturated at 0.2 mM Cd 2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd 2+. HgCl 2, PbCl 2 and ZnCl 2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl 2 and MgCl 2 did not.

A marked increase of calcium uptake in the ATP-depleted red cells of patients with iron deficiency.

Calcium (Ca) uptake was markedly increased in ATP-depleted red cells of patients with iron deficiency anemia (IDA) compared to ATP-depleted normal red cells. The extent of increased Ca uptake was related to the severity of iron deficiency as judged by decreased mean cell volume. Moreover, the increased Ca uptake returned to normal levels after oral iron supplementation therapy. The net calcium content of fresh red cells from iron-deficient individuals was the same as in red cells from normal subjects. Sodium influx and ferric ion uptake appeared to be virtually unaffected in the iron deficient red cells.

Surface charge topography in ATP-depleted red blood cells

Surface charge topography in ATP-depleted red blood cells

Increased calcium uptake in the red cells of unsplenectomized patients with hereditary spherocytosis: significant contribution of reticulocytosis

The presence of abnormal calcium (Ca) metabolism in the red cells of hereditary spherocytosis (HS) patients has long been in dispute. Thus, to clarify the discrepancies of experimental results published previously, Ca metabolism in the red cells was investigated in 12 unsplenectomized and 6 splenectomized HS patients. A marked increase of Ca uptake was observed in the ATP-depleted red cells of the unsplenectomized HS patients. The increased red cell Ca uptake was normalized after splenectomy in the same HS patients. The extent of increased Ca uptake was well correlated to the degree of reticulocytosis ( p < 0.001). The observation was confirmed by the fractionation studies of the HS red cells. Ca uptake was not increased in the spherocyte-rich bottom fraction, if reticulocytosis was not present. No significant difference was observed in red cell calcium content between normal and HS patients, neither unsplenectomized nor splenectomized.

Binding of autologous IgG to human red blood cells before and after ATP-depletion. Selective exposure of binding sites (autoantigens) on spectrin-free vesicles

Binding of autologous IgG to fresh, ATP-depleted red blood cells as well as to spectrin-free vesicles was studied by a non-equilibrium binding assay using 125I-iodinated protein A from Staphylococcus aureus. IgG binding was 14-times higher to spectrin-free vesicles than to ATP-maintaining red blood cells and 4-times higher than to ATP-depleted erythrocytes from which these vesicles were released. Protein A binding to vesicles that were released from washed and nutrient-deprived erythrocytes, was dependent on added autologous IgG. However, spectrin-free vesicles that were spontaneously released from erythrocytes conserved in whole blood, bound similar amounts of protein A with or without added autologous IgG (0.45–0.55 ng/μg band 3 protein). These findings demonstrate that opsonization of spectrin-free vesicles by autologous IgG occurs not only in the test tube, but also under blood blank conditions. The binding characteristics of IgG to spectrin-free vesicles are indicative of a natural autoantibody rather than an unspecific binding of autologous IgG. The preferential binding of IgG to spectrin-free vesicles implies a selective exposure of corresponding autoantigens in membrane regions that have lost cytoskeletal anchorage and bud off.

Analysis of calcium handling in erythrocyte membranes of genetically hypertensive rats.

Calcium handling by erythrocyte membranes was compared in genetically hypertensive (SHR) and normotensive (WKR) rats by direct measurement of calcium binding, passive influx, and adenosine triphosphate (ATP)-dependent extrusion. The SHR erythrocyte membranes exhibited the following abnormalities: 1) the binding capacity of the high affinity Ca2+-binding sites located on the inner side of the membrane was 0.84 +/- 0.07 nmole/mg protein compared with 1.17 +/- 0.08 nmole/mg protein in WKR, 2) ATP-dependent Ca2+ extrusion, measured as the Ca2+ influx into inside-out vesicles, was also lower than the WKR, as was the La3+ -sensitive, Ca2+ -dependent hydrolysis, indicating reduced activity of the calcium pump; 3) the passive calcium influx into ATP-depleted red blood cells was slightly accelerated. these abnormalities in Ca2+ binding and transport probably enhanced intracellular Ca2+ concentration, and were observed under both prehypertensive an hypertensive conditions, in 3-week-old and adult SHR respectively. Similar membrane defects in excitable cells may help to explain the pathogenesis of hypertension, since they may increase vascular tone and/or catecholamine release.

Metabolic Dependence of Protein Arrangement in Human Erythrocyte Membranes. II. Crosslinking of Major Proteins in Ghosts From Fresh and ATP-Depleted Red Cells

AbstractWe have investigated changes in membrane protein organization of ATP-depleted red cells by employing intermolecular crosslinking of the neighboring membrane proteins with glutaraldehyde or catalytic oxidation. After oxidative cross-linking, the complexes of crosslinked proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) and treated with a reducing agent, dithiothreitol (DTT), which subsequently cleaved the intermolecular disulfide bonds. The individual protein components of the crosslinked complexes were subsequently identified by two-dimensional SDS PAGE. Catalytic oxidation of ghosts from both fresh and anaerobically ATP-depleted red cells produced crosslinked oligomers of spectrin (heterodimers, trimers, and tetramers), oligomers of band 3 (dimers, trimers, and tetramers), complexes of 2 + 4.9, and dimers of 4.1, 4.5, and 5 (actin). In addition, crosslinked ghosts from ATP-depleted cells, but not from fresh red cells, contained an additional large molecular weight (>106 daltons) complex that was enriched in spectrin, suggesting a rearrangement of spectrin in the membrane to closer contacts. Maintenance of intracellular ATP stores during anaerobic incubation prevented the formation of this complex after subsequent crosslinking. In contrast, the propensity of ghosts to form this complex after cross-linking was not reversed by restoration of ATP level or by EDTA extraction of membrane-bound calcium. In addition to reducible membrane protein complexes, introduction of calcium (>0.5 mM) into fresh red cells by ionophore A23187 or into their ghosts produced a nonreducible large molecular weight polymer, which was shown by others to result from membrane protein crosslinking catalized by Ca2+ -stimulated transglutaminase. This crosslinking is markedly decreased in ATP-depleted cells due to inactivation of the cytoplasmic transglutaminase during ATP depletion. Our data indicate that the organization of spectrin and other polypeptides in red cell membranes depends on intracellular ATP and calcium levels.

Metabolic dependence of protein arrangement in human erythrocyte membranes. II. Crosslinking of major proteins in ghosts from fresh and ATP-depleted red cells

Metabolic dependence of protein arrangement in human erythrocyte membranes. II. Crosslinking of major proteins in ghosts from fresh and ATP-depleted red cells

Polymerisation of red cell membrane protein contributes to spheroechinocyte shape irreversibility.

THE discocyte–spheroechinocyte transformation of red cells undergoing ATP depletion and calcium accumulation is reversible after restoration of normal ATP and calcium levels or exposure of cells to stomacytogenic agents such as cationic anaesthetics1–3. This transformation becomes irreversible after a prolonged incubation without glucose or an introduction of high calcium concentrations into the cells.4 In our studies of membrane protein composition of metabolically depleted red cells, we have noted that aerobically ATP-depleted erythrocytes contained a >1 × 106-dalton reducible membrane protein polymer which was selectively enriched in spectrin, the major protein at the cytosol membrane interface5. We suggested that the formation of this complex was due to changes in the assembly of spectrin in ATP-depleted red cell membranes to form closer contacts, allowing a spontaneous crosslinking of the nearest spectrin neighbours through disulphide couplings5. Another polymer differing from that in ATP-depleted red cells by an absence of cleavage with reducing agents was recently noted in fresh red cells, into which Ca2+ (>0.5 mM) was introduced by ionophore A23187 (ref. 6); it has been attributed to a crosslinking of γ-glutamyl e-lysine residues of spectrin and other membrane proteins, catalysed by a Ca2+-activated cytoplasmic transglutaminase6,7. As both the above membrane protein polymers were found in cells which transformed into spheroechinocytic shape, we have studied and report here results which show that such spontaneous membrane protein crosslinking contributes to a permanent fixing of the red cells to their abnormal shape.

Metabolic Dependence of Protein Arrangement in Human Erythrocyte Membranes. I. Analysis of Spectrin-rich Complexes in ATP-depleted Red Cells

Abstract The discocyte-echinocyte transformation and the decrease in deformability associated with red cell ATP depletion have been attributed to changes in the physical properties of spectrin and actin, membrane proteins located at the membrane-cytosol interface. We investigated the spontaneous formation of spectrin-rich complexes in human erythrocyte membranes, employing two-dimensional SDS- polyacrylamide gel electrophoresis. Membranes of red cells depleted in ATP under aerobic conditions exhibited (1) an increase in components 4.5 and 8 and globin subunits, (2) a spontaneous formation of heterodimers of spectrin 1 + 2 and spectrin 2 + component 4.9, and (3) a large molecular weight (greater than 10(6) daltons) protein complex with a high spectrin to band 3 ratio. These complexes were dissociated with dithiothreitol and were prevented by anaerobic incubation or the maintenance of red cell ATP and GSH levels with glucose, adenine, and inosine. The complexes 1 + 2 and 2 + 4.9 were also seen in acetylphenylhydrazine-treated, glucose-6-phosphate dehydrogenase- deficient fresh erythrocytes that showed marked GSH depletion but preserved greater than 70% of the original ATP level. However, membranes of these cells did not contain the greater 10(6) dalton aggregate with a high spectrin to band 3 ratio. We concluded that the formation of the latter complex results from rearrangement of spectrin and other polypeptides in membranes of ATP-depleted red cells. Under aerobic conditions, the rearranged proteins undergo spontaneous intermolecular crosslinkings through disulfide couplings.

Metabolic dependence of protein arrangement in human erythrocyte membranes. I. Analysis of spectrin-rich complexes in ATP-depleted red cells

The discocyte-echinocyte transformation and the decrease in deformability associated with red cell ATP depletion have been attributed to changes in the physical properties of spectrin and actin, membrane proteins located at the membrane-cytosol interface. We investigated the spontaneous formation of spectrin-rich complexes in human erythrocyte membranes, employing two-dimensional SDS-polyacrylamide gel electrophoresis. Membranes of red cells depleted in ATP under aerobic conditions exhibited (1) an increase in components 4.5 and 8 and globin subunits, (2) a spontaneous formation of heterodimers of spectrin 1 + 2 and spectrin 2 + component 4.9, and (3) a large molecular weight (greater than 10(6) daltons) protein complex with a high spectrin to band 3 ratio. These complexes were dissociated with dithiothreitol and were prevented by anaerobic incubation or the maintenance of red cell ATP and GSH levels with glucose, adenine, and inosine. The complexes 1 + 2 and 2 + 4.9 were also seen in acetylphenylhydrazine-treated, glucose-6-phosphate dehydrogenase-deficient fresh erythrocytes that showed marked GSH depletion but preserved greater than 70% of the original ATP level. However, membranes of these cells did not contain the greater 10(6) dalton aggregate with a high spectrin to band 3 ratio. We concluded that the formation of the latter complex results from rearrangement of spectrin and other polypeptides in membranes of ATP-depleted red cells. Under aerobic conditions, the rearranged proteins undergo spontaneous intermolecular crosslinkings through disulfide couplings.

An approach to hypothermic renal preservation

Abstract A selective review of the literature concerning hypothermic renal storage suggests that two major interrelated factors control the success of the various procedures that have been advocated; these are the preservation of an intact vascular endothelium and the maintenance of adequate adenine nucleotide reserves. The damaging effect of warm ischemia is ascribed primarily to microcirculatory occlusion by rigid, ATP-depleted red cells, and secondarily to other effects of adenine nucleotide deficiency. It is suggested that continuous perfusion is a valuable technique when there has been significant warm ischemia, because it provides an effective method of restoring a patent microcirculation and is also useful when prolonged storage is required, since adenine nucleotide levels are better maintained than by the available alternatives. However, careful selection of perfusates is necessary to avoid additional perfusion injury to the endothelium. The criteria for effective preservation without continuous perfusion appear to be: little if any warm ischemic injury, effective removal of blood prior to storage, and the inclusion in the washout fluid of relatively impermeant solutes that will prevent cell swelling (particularly endothelial swelling) during preservation. An integrated approach involving both “washout” and perfusion techniques is suggested.

Influence of Ca2+ on red cell deformability and adaptation to sphering agents

The following results indicate the close connection between Ca2+ and red cell deformability: ATP-depleted red cells have a lowered deformability, but chlorpromazine and papaverine return the deformability to normal despite unchanged low ATP-concentrations of the cells. Both drugs are known to displace membrane-bound calcium. Without significantly decreasing red cell ATP-content, the calcium-ionophore A23187 reduces the deformability in the presence of 10(-4) to 10(-3) mol external calcium. The ionophore is ineffective in reducing the red cell deformability when the cells are suspended in a calcium-free medium. The sphering agent lysolecithin is less effective in reducing red cell deformability, when the external calcium-concentration is kept low. Without lysolecithin external Ca2+ is ineffective in reducing red cell deformability for several h. These results have been interpreted as showing a calcium-induced reduced mobility of the protein moiety in the red cell membrane.

Effect of Procaine HCl on ATP: Calcium-dependent Alterations in Red Cell Shape and Deformability

Procaine hydrochloric acid, a cationic anesthetic, although unable to prevent the effect of calcium ionophore A23187 on erythrocytes, inhibited the discocyte--echinocyte transformation, increased viscosity, and decreased filterability of red cells undergoing ATP depletion. The effects were abolished by washing ATP-depleted, procaine HCl-treated red cells prior to these determinations. Procaine HCl had no effects on volume, incubated osmotic fragility, or monovalent cation composition of ATP-depleted red cells. The drug increased 45Ca uptake by ATP-depleted red cells but did not change the fraction of membrane-bound calcium. Sodium dodecyl sulfate acrylamide gel electrophoresis of membrane proteins from ATP-depleted red cells revealed formation of high molecular weight protein complexes, which were not formed when biconcave shape and ATP content were maintained by incubation with adenine (0.54 mM) and inosine (12.7 mM); Formation of these complexes was not prevented when the biconcave shape was maintained by procaine HCl. It was concluded that the maintenance of the biconcave shape and normal deformability during ATP depletion by procaine HCl was not related to a displacement of membrane-bound calcium and inhibition of ATP-dependent rearrangement of red cell membrane proteins.

Effect of procaine HCLl on ATP: calcium-dependent alterations in red cell shape and deformability

Procaine hydrochloric acid, a cationic anesthetic, although unable to prevent the effect of calcium ionophore A23187 on erythrocytes, inhibited the discocyte--echinocyte transformation, increased viscosity, and decreased filterability of red cells undergoing ATP depletion. The effects were abolished by washing ATP-depleted, procaine HCl-treated red cells prior to these determinations. Procaine HCl had no effects on volume, incubated osmotic fragility, or monovalent cation composition of ATP-depleted red cells. The drug increased 45Ca uptake by ATP-depleted red cells but did not change the fraction of membrane- bound calcium. Sodium dodecyl sulfate acrylamide gel electrophoresis of membrane proteins from ATP-depleted red cells revealed formation of high molecular weight protein complexes, which were not formed when biconcave shape and ATP content were maintained by incubation with adenine (0.54 mM) and inosine (12.7 mM); Formation of these complexes was not prevented when the biconcave shape was maintained by procaine HCl. It was concluded that the maintenance of the biconcave shape and normal deformability during ATP depletion by procaine HCl was not related to a displacement of membrane-bound calcium and inhibition of ATP-dependent rearrangement of red cell membrane proteins.