Membrane potential and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying cell properties (cell pH, cell water, and cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red cell shape. Red cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal cell pH and cell water. By using assays that were rapid and accurate, cell pH, cell water, cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red cell shape and does not mediate the membrane curvature changes known to occur in red cells equilibrated at altered pH.     

Role of membrane sialic acid content in the adhesiveness of aged erythrocytes to human cultured endothelial cells

Following our previous observation that the oldest normal red blood cells were the most adherent to human cultured endothelial cells, we attempted to simulate this age-related adherence. Among all the membrane modifications experienced by erythrocytes during their life-span, loss of sialic acids has attracted considerable attention. Using two different preparations of neuraminidase, we performed a sialic acid depletion on the youngest erythrocytes to reach a sialic acid content similar to that observed in physiologically aged erythrocytes. These pretreated youngest cells displayed limited increase in the adhesiveness to endothelial cells, lower than that found with intact oldest cells. To obtain an adhesiveness of pretreated cells similar to that of naturally aged cells, it was necessary to exceed 80% of sialic acid depletion. At this extent of desialation, modifications of the electrophoretic pattern of glycophorins were observed as well as the appearance of peanut agglutinin reactivity which were never found in physiologically aged erythrocytes. Therefore, the sialic acid loss cannot be considered as being a single determinant factor of the naturally aged red cell adhesiveness.     

Human erythroid cells are affected by aluminium. Alteration of membrane band 3 protein.

There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10(6) cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12300/11200-20700 CFU-E/10(6) cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.     

Resolution of the paradox of red cell shape changes in low and high pH

The molecular basis of cell shape regulation in acidic pH was investigated in human erythrocytes. Intact erythrocytes maintain normal shape in the cell pH range 6.3-7.9, but invaginate at lower pH values. However, consistent with predicted pH-dependent changes in the erythrocyte membrane skeleton, isolated erythrocyte membranes evaginate in acidic pH. Moreover, intact cells evaginate at pH greater than 7.9, but isolated membranes invaginate in this condition. Labeling with the hydrophobic, photoactivatable probe 5-[125I]iodonaphthyl-1-azide demonstrated pH-dependent hydrophobic insertion of an amphitropic protein into membranes of intact cells but not into isolated membranes. Based on molecular weight and on reconstitution experiments using stripped inside-out vesicles, the most likely candidate for the variably labeled protein is glyceraldehyde-3-phosphate dehydrogenase. Resealing of isolated membranes reconstituted both the shape changes and the hydrophobic labeling profile seen in intact cells. This observation appears to resolve the paradox of the contradictory pH dependence of shape changes of intact cells and isolated membranes. In intact erythrocytes, the demonstrated protein-membrane interaction would oppose pH-dependent shape effects of the spectrin membrane skeleton, stabilizing cell shape in moderately abnormal pH. Stabilization of erythrocyte shape in moderately acidic pH may prevent inappropriate red cell destruction in the spleen.     

Membrane proteins in senescent erythrocytes.

The examination of erythrocyte senescence has been facilitated by recent advances in techniques for the isolation of aged red cells. One of these methods, which uses biotinylated rabbit erythrocytes, has been used to examine the state of membrane proteins in effete cells. These aged red cells were found to have normal ratios of alpha-spectrin and beta-spectrin as well as normal levels of ankyrin. The observation concerning ankyrin is particularly important due to the sensitivity of this protein to proteolysis and the postulated action of proteinases in the aging process. The senescent erythrocytes were also found to have an altered ratio of bands 4.1a and 4.1b without any apparent change in the total level of 4.1. In addition, the analysis of the aged cell membranes did not show any large-molecular-mass aggregated protein at the origin of the SDS/polyacrylamide gels, indicating a lack of transglutaminase activity in the senescence process for rabbit erythrocytes. These results indicate that aging of the rabbit erythrocyte is not accompanied by gross proteolytic degradation or transglutaminase-catalysed cross-linking of membrane components.     

Membrane skeletal protein structure and interactions in human erythrocytes after their treatment with diamide and calcium.

To analyse the role of native structures of membrane proteins in their structural modifications induced by the elevated intracellular free Ca2+ levels, we have studied the Ca(2+)-mediated effects on membrane skeletal proteins in human erythrocytes that were loaded with Ca2+ using the ionophore A23187 after their pretreatment with the sulphydryl oxidizing agent, diamide. The diamide treatment not only induced polymerization of the major membrane skeletal protein, spectrin, in the erythrocytes, but it also promoted intersubunit crosslinking within the tetramers and dimers of this protein. Loading of these diamide-treated cells with Ca2+ failed to induce significant structural modifications of spectrin as well as polypeptide 4.1, another major membrane skeletal protein, as compared to the erythrocytes that were loaded with Ca2+ without the diamide pretreatment. These results have been interpreted to suggest that the Ca(2+)-induced membrane skeletal protein changes in erythrocytes depend on both the shape and relative orientation of these proteins within the membrane skeleton.     

Alkaline hemolysis fragility is dependent on cell shape: results from a morphology tracker.

BACKGROUND: The morphometric analysis of red blood cells (RBCs) is an important area of study and has been performed previously for fixed samples. We present a novel method for the analysis of morphologic changes of live erythrocytes as a function of time. We use this method to extract information on alkaline hemolysis fragility. Many other toxins lyse cells by membrane poration, which has been studied by averaging over cell populations. However, no quantitative data are available for changes in the morphology of individual cells during membrane poration-driven hemolysis or for the relation between cell shape and fragility. METHODS: Hydroxide, a porating agent, was generated in a microfluidic enclosure containing RBCs in suspension. Automatic cell recognition, tracking, and morphometric measurements were done by using a custom image analysis program. Cell area and circular shape factor (CSF) were measured over time for individual cells. Implementations were developed in MATLAB and on Kestrel, a parallel computer that affords higher speed that approaches real-time processing. RESULTS: The average CSF went through a first period of fast increase, corresponding to the conversion of discocytes to spherocytes under internal osmotic pressure, followed by another period of slow increase until the fast lysis event. For individual cells, the initial CSF was shown to be inversely correlated to cell lifetime (linear regression factor R=0.44), with discocytes surviving longer than spherocytes. The inflated cell surface area to volume ratio was also inversely correlated to lifetime (R=0.43) but not correlated to the CSF. Lifetime correlated best to the ratio of cell inflation volume (Vfinal-Vinitial) to surface area (R=0.65). CONCLUSIONS: RBCs inflate at a rate proportional to their surface area, in agreement with a constant flux model, and lyse after attaining a spherical morphology. Spherical RBCs display increased alkaline hemolysis fragility (shorter lifetimes), providing an explanation for the increased osmotic fragility of RBCs from patients who have spherocytosis.     

The stress-free shape of the red blood cell membrane.

The two main proposals found in the literature for the stress-free shape of the red cell membrane are (a) the bioconcave shape and (b) the sphere of the same surface area. These possibilities are evaluated in this paper using theoretical modeling of equilibrium membrane shapes according to Zarda et al. (1977. J. Biomech. 10:211-221) and by comparison to experiments on red cells whose membrane shear modulus has been increased by treatment with diamide. Neither proposal is found to be compatible with all the experimental behaviour of native red cells. Neither proposal is found to be compatible with all the experimental behaviour of native red cells. To account for this discrepancy we propose that either the shear modulus of the native membrane is dependent on the membrane strain or that the bending stiffness is higher than estimated by Evans (1980. Biophys. J. 30:265-286). These studies suggest that the bioconcave disk is the more likely possibility for the stress-free shape.     

Quantitation of erythrocyte photohemolysis by light microscopy.

In a solution of erythrocytes and a photo-active compound, light activation of the compound produces hemolysis of the cells. This study describes a new assay in which focused light through a microscope is used to induce a circumscribed hemolysis of erythrocytes that have been mixed with fluorescein isothiocyanate-dextran 150,000 (FITC-DEX) and placed in a hemacytometer. The hemolytic response was monitored by detecting transmitted light intensity in the area (1.8 x 10(-4) cm2) of activation. Only cells within the specific microscopic field of activation hemolyze. This allows for multiple sites of activation within one sample. The hemolytic response was dependent on the concentration of FITC-DEX (0.5-4 mg/ml) and on light intensity (53-210 J/cm2) but not on small changes in hematocrit (3%-5%) or on the presence of platelets and leukocytes. Rabbit erythrocytes, however, were almost twice as sensitive as those from guinea pigs. Since the photohemolytic response will depend on the composition and strength of the erythrocyte membrane and presence of oxidant defense mechanisms, we suggest that this assay could be used to detect drug- or disease-induced changes in the red blood cell membrane.     

Human erythroid cells are affected by aluminium. Alteration of membrane band 3 protein

There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10⁶ cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12?300/11?200-20?700 CFU-E/10⁶ cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.     

Membrane raft actin deficiency and altered Ca-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to < 10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca²⁺-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.     

Aging of erythrocytes results in altered red cell surface properties in the rat, but not in the human. Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have not found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, countercurrent distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter. These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Aging of erythrocytes results in altered red cell surface properties in the rat,but not in the human Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have now found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, counter-current distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter.These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Structural effects in vitro of the anti-inflammatory drug diclofenac on human erythrocytes and molecular models of cell membranes

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its effects on cell membranes and particularly on those of human erythrocytes. In the present work, the structural effects on the human erythrocyte membrane and molecular models have been investigated and reported. This report presents the following evidence that diclofenac interacts with red cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that diclofenac interacted with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes it was observed that the drug induced changes different from the normal biconcave morphology of most red blood cells. This is the first time in which structural effects of diclofenac on the human erythrocyte membrane have been described.     

NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell

During the maturation process reticulocytes lose their intracellular organelles and undergo changes in membrane lipid composition and ion transport properties. While several reports indicate differences in the levels of magnesium, sodium and calcium in reticulocytes and erythrocytes, controversy remains concerning the actual magnitude and direction of ionic alterations during reticulocyte maturation. One problem with all of these studies is that the techniques used are invasive and are limited to measuring only the total cell ion content. We have used 31P, 23Na and 19F nuclear magnetic resonance (NMR) spectroscopy to compare the intracellular free ion and phosphometabolite levels in guinea pig reticulocytes and mature red blood cells. In contrast to a sharply decreased concentration of ATP in erythrocytes in comparison to reticulocytes, the intracellular free magnesium, measured using 31P-NMR, was increased by about 65% upon maturation (150 mumol/l cell water in reticulocytes in comparison to 250 mumol/l cell water in erythrocytes). Sizeable but opposite changes in intracellular sodium (5.5 mumol/ml cells in reticulocytes vs. 8.5 mumol/ml cells in erythrocytes) and intracellular free calcium (99 nM vs. 31 nM in reticulocytes and mature red cells, respectively) were also observed, suggesting that alterations in the kinetics of membrane ion transport systems, accompanying changes in phospholipid and cholesterol content, occur during the process of red cell maturation. However, in contrast to dog red blood cells, there was no evidence for the presence of a Na+/Ca2+ exchanger in guinea pig reticulocytes or erythrocytes.     

Erythrocyte morphological states, phases, transitions and trajectories

Morphological response (MR) of red blood cells represents a triphasic sequence of spontaneously occurring shape transformation between different shape states upon transfer the cells into isotonic sucrose solution in the order: S₀ (initial discoid shape in physiological saline) → S₁ (echinocytic shape at the beginning of MR, phase 1) → S₂ (intermediate discoid shape, phase 2) → S₃ (final stomatocytic shape, phase 3). In this paper, the dynamics of cell shape changes was investigated by non-invasive light fluctuation method and optical microscopy. Among 12 possible transitions between four main shape states, we experimentally demonstrate here an existence of nine transitions between neighbour or remote states in this sequence. Based on these findings and data from the literature, we may conclude that red blood cells are able to change their shape through direct transitions between four main states except transition S₁ → S₀, which has not been identified yet. Some shape transitions and their temporal sequence are in accord with predictions of bilayer couple concept, whereas others for example transitions between remote states S₃ → S₁, S₁ → S₃ and S₃ → S₀ are difficult to explain based solely on the difference in relative surface areas of both leaflets of membrane suggesting more complex mechanisms involved. Our data show that MR could represents a phenomenon in which the major role can play pH and chloride-sensitive sensor and switching mechanisms coupled with transmembrane signaling thus involving both cytoskeleton and membrane in coordinated shape response on changes in cell ionic environment.     

Cytoplasmic pH and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The mechanism of this transformation is unknown. The preceding companion study (Gedde and Huestis) demonstrated that these shape changes are not mediated by changes in membrane potential, as has been reported. The aim of this study was to identify the physiological properties that mediate this shape change. Red cells were placed in a wide range of physiological states by manipulation of buffer pH, chloride concentration, and osmolality. Morphology and four potential predictor properties (cell pH, membrane potential, cell water, and cell chloride concentration) were assayed. Analysis of the data set by stratification and nonlinear multivariate modeling showed that change in neither cell water nor cell chloride altered the morphology of normal pH cells. In contrast, change in cell pH caused shape change in normal-range membrane potential and cell water cells. The results show that change in cytoplasmic pH is both necessary and sufficient for the shape changes of human erythrocytes equilibrated in altered pH environments.     

Comparison of the effect of phenoxyherbicides on human erythrocyte membrane (in vitro)

The molecular mechanisms of phenoxyherbicides action in animals have been insufficiently studied. Now, we have investigated the interaction of sodium salts of phenoxyherbicides, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D-Na), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T-Na) and 4-chloro-2-methylphenoxyacetic acid (MCPA-Na) with human erythrocytes. In this study, we evaluated the effect of these compounds on erythrocyte membrane fluidity as well as changes in membrane proteins content. It was observed that all of the compounds studied altered membrane fluidity, changed the size and shape of the erythrocytes and provoked echinocytes formation. It was also revealed that 2,4-D-Na and 2,4,5-T-Na changed the content of erythrocyte membrane proteins mainly by a decrease in the level of spectrin and low molecular weight proteins. The comparison of the action of phenoxyherbicides examined showed that 2,4,5-T-Na caused the greatest changes in the erythrocytes membrane, whereas MCPA-Na induced the lowest alterations in the incubated cells. It must be noted that changes of the investigated parameters were observed only at presence of significant concentrations of these compounds that may penetrate human organism only as a result of acute poisoning.     

Effect of propionyl-L-carnitine treatment on membrane phospholipid fatty acid turnover in diabetic rat erythrocytes

In this work we have examined the effect of the oral administration of propionyl-L-carnitine (PLC) on the membrane phospholipid fatty acid turnover of erythrocytes from streptozotocin-induced diabetic rats. A statistically significant reduction in radioactive palmitate, oleate, and linoleate, but not arachidonate, incorporation into membrane phosphatidylcholine (PC) of diabetic rat erythrocytes with respect to control animals was found. Changes in radioactive fatty acid incorporation were also found in diabetic red cell phosphatidylethanolamine (PE), though they were not statistically significant. Oral propionyl-L-carnitine (PLC) treatment of diabetic rats partially restored the ability of intact red cells to reacylate membrane PC with palmitate and oleate, and reacylation with linoleate was fully restored. The analysis of the membrane phospholipid fatty acid composition revealed a consistent increase of linoleate levels in diabetic rat red cells, and a modest decrease of palmitate, oleate and arachidonate. The phospholipid fatty acid composition of diabetic red blood cells was not affected by the PLC treatment. Lysophosphatidylcholine acyl-CoA transferase (LAT) specific activity measured with either palmitoyl-CoA or oleyl-CoA was significantly reduced in diabetic erythrocyte membranes in comparison to controls. In addition LAT kinetic parameters of diabetic erythrocytes were altered. The reduced LAT activity could be partially corrected by PLC treatment of diabetic rats. Our data suggest that the impaired erythrocyte membrane physiological expression induced by the diabetic disease may be attenuated by the beneficial activity of PLC on the red cell membrane phospholipid fatty acid turnover.Abbreviations LAT lysophosphatidylcholine acyl-CoA transferase - PC phosphatidylcholine - PE phosphatidylethanolamine - PLC propionyl-L-carnitine - STZ streptozotocin