A spectrin-dependent ATPase of the human erythrocyte membrane

Removal of spectrin from erythrocyte membranes results in the simultaneous loss of a calcium-stimulated, magnesium-dependent ATPase with an apparent KD for Ca2+ of 1 microM. This ATPase activity with high Ca2+ affinity is specifically reconstituted by addition of purified spectrin to spectrin-depleted membranes, and the reconstituted activity is directly proportional to the amount of spectrin that is reassociated with the membranes. Spectrin binding and activation of the high Ca2+ affinity Mg2+-ATPase are proportionally inhibited by thermal denaturation, trypsin digestion, or treatment of the membranes with thiol-reactive reagents. Binding of calmodulin to the Ca2+ pump ATPase requires that calmodulin contains bound ca2+. By contrast, spectrin binding to the erythrocyte membrane is Ca2+-independent. Direct assay of calmodulin is purified spectrin and absence of chlorpromazine inhibition of reconstitution demonstrate that activation of the high Ca2+ affinity ATPase resulting from spectrin binding is not a result of contamination of spectrin by calmodulin. Additional evidence that the spectrin-activated ATPase is an entity separate and distinct from the Ca2+ pump is provided by other characteristics of the activation phenomenon. It is suggested that spectrin constitutes part of an ATPase which may function as a component of the "cytoskeleton" controlling erythrocyte shape and membrane flexibility.     

Variations in the relative activities of erythrocyte membrane ATPase with changes in severity of sickle cell anemia

Red blood cells from 31 patients with sickle cell anemia whose hemoglobins were ascertained as SS were assayed for Mg-, Ca-, Na-, and total ATPase activities. The ATPase activities were correlated with the various stages of severity in each patient as determined by clinical parameters. The results demonstrate that increases in ATPase activities were associated with increases in the percentage severity of sickle cell anemia. Severity correlated inversely with fetal hemoglobin levels in the sickle cell patients. ATPase activities were generally higher in SS genotypes than in AS and AA normal individuals.     

Relationship between the shape and the membrane potential of human red blood cells

Summary Microscopic observations of isotonic suspensions of human red blood cells demonstrate that cell shape is unaltered when the transmembrane electrical potential, orE _m , is set in the range –85 to +10 mV with valinomycin at varied external K⁺, or K _o .E _m was measured with the fluorescent potentiometric indicator, diS-C₃(5), as calibrated by a pH method. Repeating Glaser's experiments in which echinocytosis was attributed to hyperpolarization, we found that at low ionic strength the pH-dependent effects of amphotericin B appear to be unrelated toE _m . The effects of increased intracellular Ca²⁺, or Ca _o , on echinocytosis and onE _m are separable. With Ca ionophore A23187 half-maximal echinocytosis occurs at greater Ca _o than that which induces the half-maximal hyperpolarization associated with Ca-induced K⁺ conductance (Gardos effect). Thus, cells hyperpolarized by increased Ca _o remain discoidal when Ca is below the threshold for echinocytosis. With A23187 and higher Ca _o , extensive echinocytosis occurs in cells which are either hyperpolarized or at their resting potential. The Ca-activation curve for echinocytosis is left-shifted by low K _o , a new observation consistent with increased DIDS-sensitive uptake of⁴⁵Ca by hyperpolarized cells. These results support the following conclusions: (1) the shape and membrane potential of human red blood cells are independent under the conditions studied; (2) in cells treated with A23187, the Gardos effect facilitates echinocytosis by increasing Ca.     

Platelet and erythrocyte membrane changes in Alzheimer's disease

Previous reports have suggested that the physical properties of cell membranes and calcium homeostasis in both the central and peripheral nervous system are changed in Alzheimer's disease (AD). This study has examined the biophysical properties of erythrocyte and platelet membranes by measuring the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and possible related changes in lipid peroxidation. In addition, we have studied calcium homeostasis by measuring thrombin-stimulated changes in intraplatelet free calcium and Ca2(+)-ATPase activity in AD and healthy age and sex-matched controls. Our results show that there was no significant difference in the fluorescence anisotropy of DPH in erythrocyte membranes isolated from the three groups. There was also no significant difference in lipid peroxidation levels in erythrocytes and plasma of AD patients compared to controls. However, there was a significant reduction in the fluorescence anisotropy of DPH in platelet membranes from AD patients, compared with healthy controls. Recent evident suggests that the increase in platelet membrane fluidity results from alterations in internal membranes. We measured the specific activities of enzyme markers associated with intracellular and plasma membranes in platelets from AD patients and healthy controls. There was a significant reduction in the specific activity of antimycin A-insensitive NADH-cytochrome-c reductase (a specific marker for smooth endoplasmic reticulum (SER)), in AD patients compared to controls, but no change in the specific activity of bis(p-nitrophenyl)phosphate phosphodiesterase (a specific marker for plasma membrane). We have also shown that SER mediated [Ca2+] homeostasis is possibly impaired in AD platelets, i.e., the percentage of thrombin-stimulated increase in intraplatelet [Ca2+] above basal levels was significantly higher in AD compared to matched controls and there were significant reductions in the specific activities of Ca2+/Mg2(+)-ATPase and Ca2(+)-ATPase (but not Mg2(+)-ATPase) in AD platelets. Finally electron microscopic analysis of platelets showed that there was a significant increase in the incidence of abnormal membranes in AD patients compared to controls. The ultrastructural abnormalities seem to consist of proliferation of a system of trabeculated cisternae bounded by SER. These results suggest that both SER structure and function might be defected in AD platelets, which could explain the fluidity changes observed here.     

Role of the bilayer in the shape of the isolated erythrocyte membrane

Summary The determinants of cell shape were explored in a study of the crenation (spiculation) of the isolated erythrocyte membrane. Standard ghosts prepared in 5mm NaP_i (pH 8) were plump, dimpled disks even when prepared from echinocytic (spiculated) red cells. These ghosts became crenated in the presence of isotonic saline, millimolar levels of divalent cations, 1mm 2,4-dinitrophenol or 0.1mm lysolecithin. Crenation was suppressed in ghosts generated under conditions of minimal osmotic stress, in ghosts from red cells partially depleted of cholesterol, and, paradoxically, in ghosts from red cells crenated by lysolecithin. The susceptibility of ghosts to crenation was lost with time; this process was potentiated by elevated temperature, low ionic strength, and traces of detergents or chlorpromazine.In that ghost shape was influenced by a variety of amphipaths, our results favor the premise that the bilayer and not the subjacent protein reticulum drives ghost crenation. The data also suggest that vigorous osmotic hemolysis induces a redistribution of lipids between the two leaflets of the bilayer which affects membrane contour through a bilayer couple mechanism. Subsequent relaxation of that metastable distribution could account for the observed loss of crenatability.     

Radiation-induced changes in the structure of erythrocyte membrane proteins

Structural changes in proteins of erythrocyte membranes induced by gamma-radiation at doses of 10-10(3) Gy were studied using the method of tryptophan fluorescence quenching by acrylamide. It was found that the exposure to ionizing radiation leads to a decrease in intramolecular dynamics of membrane proteins.     

Further evidence for a membrane potential-dependent shape transformation of the human erythrocyte membrane

The influence of various factors (pH, temperature, sodium gluconate) on the ionic strength-dependent stomatocyte-discocyte-echinocyte transformation of the human erythrocyte membrane was investigated. The results give further evidence for a correlation between shape of erythrocyte membrane and the transmembrane potential of the cells.     

Influence of chlorpromazine on the transverse mobility of phospholipids in the human erythrocyte membrane: relation to shape changes

The influence of chlorpromazine (CPZ) on the transverse mobility of spin-labeled phospholipids incorporated into human erythrocytes was investigated by electron spin resonance. The very slow transverse diffusion of phosphatidylcholine, as well as the absence of transverse mobility of sphingomyelin were not modified even by sublytic concentrations (approximately equal to 1 mM) of CPZ. On the other hand, the rapid outside-inside translocation of the aminophospholipids (Seigneuret and Devaux (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755), was slightly hindered in CPZ containing membranes. If the spin-labeled aminolipids were incorporated in erythrocytes and allowed to flip to the inner monolayer before CPZ addition, a fraction of the spin labels (10-15%) flipped back instantaneously from the inner to the outer leaflet, upon incubation with CPZ. Similar experiments carried out with spin-labeled phosphatidylcholine and spin-labeled sphingomyelin showed that a fraction of the spin-labeled choline derivatives flip instantaneously to the inner leaflet if CPZ was added after the spin labels. Addition of lysophosphatidylcholine had no effect on the spin-labeled phospholipid redistribution nor on their transmembrane mobility. We interpret the immediate effect of CPZ addition as being due to a reorganization of the bilayer accompanying the rapid CPZ membrane penetration, phenomenon which is independent of the CPZ effect on the steady-state activity of the 'aminophospholipid translocase', the latter effect being probably a direct CPZ-protein interaction. By comparison of the time course of phosphatidylserine transverse diffusion in control discocyte cells and in CPZ-induced stomatocytes, we infer that the difference in cell shape is not a major factor in the regulation of the active inward transport of aminophospholipids in human erythrocytes.     

Relationship between erythrocyte membrane phase properties and susceptibility to secretory phospholipase A2

Normally, cell membranes resist hydrolysis by secretory phospholipase A(2). However, upon elevation of intracellular calcium, the cells become susceptible. Previous investigations demonstrated a possible relationship between changes in lipid order caused by increased calcium and susceptibility to phospholipase A(2). To further explore this relationship, we used temperature as an experimental means of manipulating membrane physical properties. We then compared the response of human erythrocytes to calcium ionophore at various temperatures in the range of 20-50 degrees C using fluorescence spectroscopy and two-photon fluorescence microscopy. The steady state fluorescence emission of the environment-sensitive probe, laurdan, revealed that erythrocyte membrane order decreases systematically with temperature throughout this range, especially between 28 and 45 degrees C. Furthermore, the ability of calcium ionophore to induce increased membrane order and susceptibility to phospholipase A(2) depended similarly on temperature. Both responses to calcium influx were enhanced as membrane fluidity increased. Analysis of the spatial distribution of laurdan fluorescence at several temperatures indicated that the ordering effect of intracellular calcium on fluid membranes generates an increase in the number of fluid-solid boundaries. Hydrolysis of the membrane appeared to progress outward from these boundaries. We conclude that phospholipase A(2) prefers to hydrolyze lipids in fluid regions of human erythrocyte membranes, but primarily when those regions coexist with domains of ordered lipids.     

Interaction between plant polyphenols and the erythrocyte membrane

The purpose of these studies was to determine the effect of polyphenols contained in extracts from apple, strawberry and blackcurrant on the properties of the erythrocyte membrane, treated as a model of the biological membrane. To this end, the effect of the substances used on hemolysis, osmotic resistance and shape of erythrocytes, and on packing order in the hydrophilic region of the erythrocyte membrane was studied. The investigation was performed with spectrophotometric and fluorimetric methods, and using the optical microscope. The hemolytic studies have shown that the extracts do not induce hemolysis at the concentrations used. The results obtained from the spectrophotometric measurements of osmotic resistance of erythrocytes showed that the polyphenols contained in the extracts cause an increase in the resistance, rendering them less prone to hemolysis in hypotonic solutions of sodium chloride. The fluorimetric studies indicate that the used substances cause a decrease of packing order in the hydrophilic area of membrane lipids. The observations of erythrocyte shapes in a biological optical microscope have shown that, as a result of the substances’ action, the erythrocytes become mostly echinocytes, which means that the polyphenols of the extracts localize in the outer lipid monolayer of the erythrocyte membrane. The results obtained indicate that, in the concentration range used, the plant extracts are incorporated into the hydrophilic area of the membrane, modifying its properties.     

Phenylhydrazine-induced changes in erythrocyte membrane surface lipid packing

Phenylhydrazine-induced oxidative damage in red cells results in increased binding of merocyanine 540, a fluorescence probe sensitive to changes in lipid packing. Fluorescence polarization studies with diphenylhexatriene did not reveal major changes in order parameters both in intact red cells and lysates treated with phenylhydrazine. These fluorescence studies indicate that major changes are observed in membrane lipids. Analytical studies of membrane phospholipids revealed a significant decrease in phosphatidylethanolamine. The results of the fluorescence and lipid studies, taken in association with our previously reported findings on spectrin and other cytoskeletal protein degradation in red cells exposed to phenylhydrazine, suggests that degradation of cytoskeleton membrane proteins is also responsible for changes in the lipid bilayer surface of the red cell membrane.     

Relationship between pH, sodium, and shape changes in chemotactic-factor-stimulated human neutrophils

The relationship between the chemotactic-factor-elicited changes in the intracellular pH and the shape of human neutrophils was investigated using simultaneous measurements of both parameters. The results demonstrate first that fMet-Leu-Phe and leukotriene B4 elicit qualitatively similar pH and shape change responses from the neutrophils. A relationship between the chemoattractant-elicited decrease in cytoplasmic pH and the shape changes is indicated by several findings including: 1) the similarities in the time courses of the two responses, 2) the ability of propionic acid to induce a transient and pertussis-toxin-sensitive shape change response, and 3) the ability of the calcium ionophore A23187 to similarly induce both responses under conditions when the degranulation is minimized. On the other hand, several other results indicate that the drop in pH is not a sufficient condition for the chemotactic-factor-stimulated shape changes. These include: 1) the ability of pertussis toxin to inhibit the shape changes induced by propionic acid and by A23187 without affecting the drop in pH, and 2) the observation that the drop in pH induced by propionic acid persists significantly longer than the shape change. Increasing the cytoplasmic pH by adding ammonium chloride was also found to cause shape changes in the neutrophils. The response to the base differs in two important aspects from that caused by propionic acid: it is pertussis-toxin-insensitive, and it is long-lived. Chemotactic factors have been found to induce a shape change under conditions when the internal pH was artificially increased or decreased, indicating that it is not the absolute cytoplasmic pH that represents the internal signalling parameter. The results are discussed in terms of the activation of the cytoskeletal network of the neutrophils by chemotactic factors.     

Bending undulations and elasticity of the erythrocyte membrane: effects of cell shape and membrane organization

The undulatory excitations (flickering) of human and camel erythrocytes were evaluated by employing the previously used flicker spectroscopy and by local measurements of the autocorrelation function K (t) of the cell thickness fluctuations using a dynamic image processing technique. By fitting theoretical and experimental flicker spectra relative values of the bending elastic modulus K _c of the membrane and of the cytoplasmic viscosity were obtained. The effects of shape changes were monitored by simultaneous measurement of the average light intensity I ₀ passing the cells and by phase contrast microscopic observation of the cells. Evaluation of the cellular excitations in terms of the quasi-spherical model yielded values of K _c /R _inf0 ^sup3 and · R ₀ (R ₀=equivalent sphere radius) and allowed us to account (1) for volume changes, (2) for effects of surface tension and spontaneous curvature and (3) for the non-exponential decay of K (t). From the long time decay of K (t) we obtained an upper limit of the bending elastic modulus of normal cells of K _c = 2–3 · 10^–19 Nm which is an order of magnitude larger than the value found by reflection interference contrast microscopy (RICT, K _c , = 3.4 · 10^–20 Nm, Zilker et al. 1987) but considerably lower than expected for a bilayer containing 50% cholesterol (K _c = 5 · 10^–19 Nm, Duwe et al. 1989). The major part of the paper deals with long time measurements (order of hours) of variations of the apparent K _c and values of single cells (and their reversibility) caused (1) by osmotic volume changes, (2) by discocytestomatocyte transitions induced by albumin and triflouperazine, (3) by discocyte-echinocyte transitions induced by expansion of the lipid/protein bilayer (by incubation with lipid vesicles) and by ATP-depletion in physiological NaCI solution, (4), by coupling or decoupling of bilayer and cytoskeleton using wheat germ agglutinin or erythrocytes with elliptocytosis and (5) by cross-linking the cytoskeleton using diamide. These experiments showed: (1) K _c and are minimal at physiological osmolarity and temperature and well controlled over a large range of these parameters. (2) Echinocyte formation does not markedly alter the apparent membrane bending stiffness. (3) During swelling the cell may undergo a transient discocyte-stomatocyte transition. (4) Strong increases of the apparent K _c and after cup-formation or strong swelling and deflation are due to the effect of shear elasticity and surface tension. Our major conclusions are: (1) The erythrocyte membrane exhibits a shear free deformation regime which requires ATP for its maintenance. (2) Shape transitions may be caused by relative area changes either of the two monolayers of the lipid/protein bilayer (corresponding to the bilayer coupling hypothesis) or of the bilayer and the cytoskeleton where the latter mechanism appears to be more frequent. (3) The low bending stiffness and the shear free deformation regime are explained in terms of a slight excess area of the lipid bilayer leading to a pre-undulated surface profile. Freeze fracture electron microscopy studies provide direct evidence for a pre-undulated bilayer with an undulation wavelength of approximately 100 nm. Offprint requests to: E. Sackmann     

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Ca-2+-stimulated membrane phosphorylation and ATPase activity of the human erythrocyte.

1. Human erythrocyte membranes were preincubated with ethyleneglycolbis-(beta-aminoethyl)-N,N' tetraacetate (EGTA) and subsequently labelled for short periods with micromolar concentrations of [8-3-H, gamma-32-P]ATP. Under these conditions, and at temperatures smaller than or equal to 22 degrees C, both ATP hydrolysis and membrane phosphorylation were stimulated by Ca-2+. 2. The properties of the Ca-2+-stimulated ATP hydrolysis and associated phosphorylation of a 150 000 molecular weight protein component, previously described (Knauf, P. A., Proverbio, F. and Hoffman, J. F. (1974) J. Gen. Physiol. 63, 324-336), have been studied. The behavior of the phosphorylated component, ECaP, has properties consistent with its role as a phosphorylated intermediate of Ca-2+-ATPase activity, including: (1) similar dependence of the steady-state level of ECaP and Ca-2+-ATPase on ATP concentration; (2) rapid turnover apparent upon the addition of excess non-radioactive ATP; and (3) good correlation between the steady-state levels of Ca-2+-dependent phosphorylation and Ca-2+-ATPase activity in separate preparations possessing variable specific activity. Addition of excess EGTA to ECaP caused only partial dephosphorylation. Sensitivity of Ca-2+-stimulated ATP hydrolysis and associated phosphorylation to micromolar concentrations of Ca-2+ implicates this activity in the "high-affinity" Ca-2+-pump system of the human erythrocyte (Schatzmann, H. J. (1973) J. Physiol. London 235, 551-569).     

A photometric method to analyze induced erythrocyte shape changes

In this paper, a photometric method was introduced to quantify biochemically-induced red blood cell (RBC) shape changes when no shear force was acting on the cells. To obtain the photometric RBC shape parameter (RF1), a monolayer of point-attached RBCs was prepared on the floor of a flat flow chamber and the transmission of light perpendicular to the monolayer plane was measured: 1) in phosphate buffered saline with 0.1% bovine serum albumin (PBS₊) and 2) in PBS₊, containing a shape changing compound (in both, the RBCs were not deformed due to shear flow). To normalize the data, a third transmission value at a shear stress of 3 Pa was measured in PBS₊ from the same RBC monolayer. To validate the photometric data, RF1 of RBCs exposed to shape changing agents was correlated by linear regression analysis with 1) data obtained with the tangent-counting technique (TC) and 2) the morphological index (MI). The coefficient of correlation was calculated at 0.95 for the TC data and 0.94 for the MI data, respectively. The sensitivity of the photometric method was tested with stomatocytogenic chlorpromazine (CP) and echinocytogenic sodium salicylate (SA). CP (2.5 μM) induced a significant decrease of RF1 to −0.045 (N = 6 donors, p < 0.01), whereas SA (2.5 mM) increased RF1 to +0.027 significantly (N = 6, donors, p < 0.01). Both the CP-induced and the SA-induced shape changes appeared less than 2 min after application of the shape changing agents, and changed gradually within another 30 min when the agent was present in PBS₊, partly disappearing within about 2 min after reincubation of the shape transformed RBCs in PBS₊ not containing the agent.