Shape Of Human Erythrocytes Research Articles

Factors of the shape change of human erythrocytes induced with lidocaine.

We studied the molecular mechanism of the shape change of erythrocytes with a local anesthetic, lidocaine. The shape of human erythrocytes changed from discocytes to stomatocytes in the presence of lidocaine when ATP was present. But, the shape of resealed cells which were prepared with 10 mM Tris-HCl buffer (pH 7.4) containing 2 mM ATP-MgCl2 and various substances was not changed from discocytes to stomatocytes with lidocaine. When intact cells and resealed cells which were prepared with various concentrations of Tris-HCl buffer (pH 7.4) were incubated with various concentrations of lidocaine and their membrane proteins were analyzed by SDS-PAGE, the densities of bands 62K, 28K and 22K depended on lidocaine concentration: in particular, that of band 28K changed remarkably. These membranous 62K-, 28K- and 22K-proteins agreed with cytoplasmic 62K-, 28K- and 22K-proteins in molecular weight. We propose that not only ATP but also the 62K-, 28K- and 22K-proteins in the cytoplasm are concerned with the shape change of human erythrocytes induced with lidocaine.

Structure-activity relationships of 4′-O-substituted 1-benzylisoquinolines with respect to their actions on the cell membrane of blood platelets and erythrocytes

4′-O-Substituted 1-benzyl-2-methyl-6, 7-dimethoxy-1,2,3,4-tetrahydroisoquinolines inhibited the collagen-induced activation (aggregation and ATP secretion) of rabbit platelets and transformed the shape of human erythrocytes in a dose-dependent manner. Both effects increased with increasing number of carbon atoms of the 4′-substituted hydrocarbon moiety. The incorporation of these compounds into the erythrocyte membrane was also dependent on the number of carbon atoms of the 4′-substituted radical. The most potent phenoxy derivative suppressed arachidonic acid release from membrane phospholipids but had no effect on arachidonic acid metabolism in platelets. This indicated that the effects are comparable to those of a natural bisbenzylisoqguinoline, cepharanthine. These effects appear to be due to a perturbing action on the membrane lipid bilayer.

Inhibition of calmodulin stimulation of phosphodiesterase and Ca 2+Mg 2+-ATPase activities and shape change of erythrocyte ghosts by chloroquine

The effects of chloroquine on calmodulin (CaM)-related enzyme activities and the shape of human erythrocytes have been studied. It was found that the CaM activation of rat brain phosphodiesterase was abolished by the addition of chloroquine. CaM was included in the assay of phosphodiesterase activity at the concentration that gave half-maximal activation. The concentration of chloroquine that caused 50% inhibition of CaM stimulation of phosphodiesterase was 7 × 10 −5 M. The type of inhibition was competitive with respect to CaM. The CaM-stimulated Ca 2+, Mg 2+-ATPase in erythrocyte membrane was also inhibited by chloroquine, the 50% inhibitory concentration of which was about 2 × 10 −4 M. Its mode of action was also competitive with respect to CaM. The shapes of erythrocyte ghosts prepared by hypotonic hemolysis were examined in a solution consisting of 2 mM MgCl 2, 154 mM NaCI and 10 mM Tris-HCl (pH7.4); they were discocytic in the presence of 2mM ATP and in its absence. They were converted to the invaginated form by the addition of chloroquine in the concentration range of 1 x 10 −4–5 × 10 −4 M. This concentration is similar to that which caused the inhibition of CaM activation of Ca 2+,Mg 2+-ATPase.

Ethanol-induced alterations in human erythrocyte shape and surface properties: modulatory role of prostaglandin E1.

Exposure of human erythrocytes to ethanol (1 to 20% by vol) in Ca2+ and Mg2+-free phosphate-buffered saline, pH 7.4, transformed biconcave discs into spiculated echinocytes within 3 min at 25 degrees C. The effects of ethanol were concentration-and time-dependent, but reversible by washing in the incubation buffer system within 60 min of initial exposure to ethanol. After prolonged ethanol exposure (180 min), washing of cells resulted in the formation of stomatocytes (cup-forms). Ethanol-induced echinocytosis was also accompanied by a 30% enhancement in the agglutinability of erythrocytes by ligands with high affinity for negative surface charge (poly-L-lysine and wheat germ agglutinin, 20 microliters/ml) without any alterations in surface charge topography. Concomitant exposure of erythrocytes to prostaglandin E1 (100 nM) selectively prevented the enhancement of ligand-mediated agglutinability, but did not modify cell shape. These data indicate that certain erythrocyte surface properties may not be directly influenced by cell shape and suggest a unique modulatory action of prostaglandin E1 on shape-transformed cells.

Modulation of human erythrocyte shape and fatty acids by diet

A semi-synthetic diet (Vivonex) was administered via nasogastric tube to three cystic fibrosis patients with pancreatic exocrine deficiency for 14 days to gain weight. Dietary essential fatty acids were provided as safflower oil, which constituted 1.3% of total calories. Plasma and red blood cells were analyzed for the content and composition of lipids at the start of the diet and at days 7 and 14 of the dietary period, and the results were correlated with the morphology of the cells. Feeding Vivonex to the patients led to an essential fatty acid deficiency, which was manifested in a 50% decrease in the linoleic acid content of the phosphatidylcholine of plasma and red blood cells at days 7 and 14 and in a 20% decrease in the linoleic acid content of red cell phosphatidylethanolamine at day 14. There was no significant alteration in the levels or composition of the other phospholipid classes and in the free cholesterol/phospholipid ratio. The decrease in the linoleic acid content of the erythrocytes was accompanied by a dramatic increase in the proportion of cells as echinocytes. We conclude that restricted linoleic acid availability in cystic fibrosis patients causes a change in red blood cell shape either directly by decreasing the linoleoylphosphatidylcholine content of the membrane or indirectly by affecting enzyme activity.

Charge-independent effects of drugs on erythrocyte morphology

The effects of chlorpromazine, tetracaine, indomethacin, barbitone and benzyl alcohol on human erythrocyte shape have been examined. Cationic and anionic drugs produced stomatocytes and echinocytes respectively as expected for cells in isotonic saline. Particular attention has been directed here to some features of drug induced morphology change which are independent of the charge of the drug. It was found that (i) the direction (increase or decrease) of the extent of morphological change as temperature was increased from 20 to 37°, (ii) the exposure time for maximum shape change (0–2 min), and (iii) the time course of cell morphology (0–30 min) were different for similarly charged drugs. The influence of low concentrations of the drugs on the thermal fragmentation patterns of the cells has been determined. A single index has been derived which allows comparison of the morphological effects of cationic and anionic drugs. It was concluded that, while the type (stomatocyte or echinocyte) of shape change observed was dependent on the charge of the drug, cell morphology at drug concentrations high enough to produce marked shape change at 37° was strongly influenced by charge independent drug-specific effects.

Transformation of human erythrocyte shape by endotoxic lipopolysaccharide.

Human erythrocytes were observed to undergo a discocyte to echinocyte to spheroechinocyte shape transformation during brief incubation with endotoxic lipopolysaccharide. It was concluded that lipopolysaccharide-membrane interactions alter the curvature of erythrocyte membranes.

Interaction of cholesterol and lysophosphatidylcholine in determining red cell shape

The effect of lysolecithin on the shape of human erythrocytes of varied cholesterol content was examined by scanning electron microscopy. Under the conditions of these experiments, all of the [14C]lysolecithin incubated with cells was shown to be located in the external membrane leaflet. The membrane lysolecithin required to induce echinocytosis (spiculation) in normal cells (0.8 mol cholesterol/mol phospholipid) was approximately 0.08-0.10 mumol/10(10) cells, which contributed 1.6-2.0 micrometer 2 or 1% of the cell surface area. This value is consistent with the premise that echinocytosis was caused by a slight differential expansion of the outer surface of the bilayer. The lysolecithin required for echinocytosis decreased as the membrane cholesterol content increased; from 0.14 mumol/10(10) cells at 0.5 mol cholesterol/mol phospholipid to 0.03 mumol/10(10) cells at 1.4 mol cholesterol/mol phospholipid. These data were interpreted in terms of a bilayer couple mechanism. Assuming that the two amphipaths acted additively, the amount of lysolecithin required to induce echinocytosis was used to estimate the partition of cholesterol between the two leaflets of the red cell membrane. A value of about 51:49% in favor of the outer leaflet was found at all cholesterol levels.

Alterations in human erythrocyte shape and the state of spectrin and phospholipid phosphorylation induced by cholesterol depletion

Cholesterol depletion of erythrocytes, obtained after incubation with phosphatidylcholine vesicles, induces in most of the experiments: (1) a discocytestomatocyte transformation as observed by scanning electron microscopy; (2) a specific decrease in spectrin phosphorylation of intact erythrocytes; (3) an increase in lipid phosphorylation. It is concluded that the effect of cholesterol on erythrocyte shape is probably mediated through its action on the activity o of membrane-bound enzymes, proteases or kinases.

Effects of Tetrachlorobiphenyls on the Osmotic Fragility and Shape of Human Erythrocytes

The effects of tetrachlorobiphenyl (TCB) isomers on the osmotic fragility and shape of human erythrocytes were examined. Low concentrations of 2, 3, 2', 3'-, 2, 4, 2', 4'-and 2, 5, 2', 5'-TCB protected erythrocytes from hypotonic hemolysis, but at high concentrations, these compounds greatly promoted hypotonic hemolysis. Along with the anti-hemolytic effect, the compounds caused alterations in cell shape that ranged from normal discocytes to cup-formed cells and to spherocytes. Thus, these compounds can be classified as cup-formers. The alterations in shape were brought about by conditions in which the membranes were protected from hypotonic hemolysis. The break point in the Arrhenius plot for hypotonic hemolysis was decreased by about 3°C when 2, 3, 2', 3'-TCB was added. Therefore, these compounds increase fluidity of the bilayer, which is clearly demonstrated by the ability of 2, 3, 2', 3'-TCB to lower the break point temperature, which enables it to protect erythrocytes from osmotic swelling. By contrast, 2, 6, 2', 6'-and 3, 4, 3', 4'-TCB neither protected erythrocytes from hypotonic hemolysis nor altered cell shape. The relation between these phenomena and chemical structure is discussed.

381 - The erythrocyte membrane electric field and its functional role

Results are demonstrated and discussed stressing that the electric field of the cell membrane is an important regulatory parameter of various functions. The electrostatics of the membrane is due both to transmembrane potential and to surface potentials on both sides of the membrane. The transmembrane potential of human erythrocytes is determined by the ionic conditions of the outside solution as well as by the cell state. The outside surface potential depends on the spatial distribution of fixed charges in the glycocalyx and its dynamics. To some extent, both parameters are calculable and measurable. It is demonstrated here that the shape of human erythrocytes depends on the sign of transmembrane potential. The ouabain-sensitive sodium efflux indicates a minimum under conditions were transmembrane potential is near zero. Rubidium efflux, on the other hand, is determined by the properties of surface charges.

The erythrocyte membrane electric field and its functional role

Summary Results are demonstrated and discussed stressing that the electric field of the cell membrane is an important regulatory parameter of various functions. The electrostatics of the membrane is due both to transmembrane potential and to surface potentials on both sides of the membrane. The transmembrane potential of human erythrocytes is determined by the ionic conditions of the outside solution as well as by the cell state. The outside surface potential depends on the spatial distribution of fixed charges in the glycocalyx and its dynamics. To some extent, both parameters are calculable and measurable. It is demonstrated here that the shape of human erythrocytes depends on the sign of transmembrane potential. The outbain-sensitive sodium efflux indicates, a minimum under conditions where transmembrane potential is near zero. Rubidium effux, on the other hand, is determined by the properties of surface charges.

Equilibrium and kinetic effects of drugs on the shapes of human erythrocytes.

We have previously proposed that if the two half-layers of a membrane are different in their protein and lipid compositions, they may respond differently to some membrane perturbation (the bilayer couple hypothesis). This hypothesis has been applied to explain the changes in shape of human erythrocytes that are produced by a variety of amphipathic compounds. These compounds are presumed to intercalate by their hydrophobic ends into the lipid portions of the membrane; if the compounds are anions, the binding is preferentially to the outer half of the bilayer, if cations, to the inner half. It is proposed that such preferential binding causes an expansion of one half-layer relative to the other, with a corresponding change in cell shape. The predicted sidedness of these shape changes is now demonstrated in experiments with methochlorpromazine and 2,4,6-trinitrophenol. Under appropriate nonequilibrium or equilibrium or equilibrium conditions, both of these compounds are shown to be either crenators or cup-formers of the intact erythrocyte, depending upon which side of the membrane they are concentrated in. These results therefore strongly support the bilayer couple hypothesis.

The Dependence of Shape of Human Erythrocyte Ghosts on Calcium, Magnesium, and Adenosine Triphosphate

Abstract ATP depletion of erythrocytes is associated with discocyte-echinocyte transformation and accumulation of calcium. This study was undertaken to evaluate whether this shape transformation results from interaction of Ca2+, Mg2+, and ATP with structural proteins of the membrane. ATP, Mg2+, or Ca2+ were incorporated into ghosts during hypotonic hemolysis followed by restoration of isotonicity with NaCl and incubation. Reconstituted ghosts were examined by electron microscope after osmium tetroxide fixation followed by platinum and carbon shadowing and in-phase contrast microscope. Four ghost configurations were observed: (1) flat, discoidal electron-penetrable ghosts were produced when ghosts were prepared without any additives, or with ATP alone, Mg2+ alone, or EDTA; (2) biconcave and cup-shaped less electron-penetrable ghosts predominated when equimolar concentrations of Mg2+ and ATP (0.5-2 mM) were introduced into ghosts during hemolysis; (3) spherical, spiculated, or markedly deformed electron-dense ghosts of smaller volume were the prevalent shape forms when Ca2+ alone was introduced during hemolysis; (4) small, smooth, electron-dense spheroids were present when both Ca2+ and Mg ATP (2 mM) were added during hemolysis. The above shapes were independent of ouabain, monovalent cations (NaCl, KCl, or choline chloride), transmembrane osmotic gradient, and ghost volume. The shape effects of Ca2+ and Mg ATP were prevented by preincubation of red cells with SH-blocking agent N-ethylmaleimide (4 mM) and by repeated ghost washing, which was associated with removal of water-soluble fibrous proteins from membranes. It is concluded that the interaction of Ca2+, Mg2+, and ATP with membrane structural proteins exerts a significant role in the control of shape of human erythrocytes.

Structure and function of erythrocytes. I. Relation between the energy metabolism and the maintenance of biconcave shape of human erythrocytes: Gardos, G., I. Szasz, and I. Arky. Acta Biochim. Biophys. Acad. Sci. Hung., 1: 253–266, 1966. (36 ref.)

Structure and function of erythrocytes. I. Relation between the energy metabolism and the maintenance of biconcave shape of human erythrocytes: Gardos, G., I. Szasz, and I. Arky. Acta Biochim. Biophys. Acad. Sci. Hung., 1: 253–266, 1966. (36 ref.)

Transformation and restoration of biconcave shape of human erythrocytes induced by amphiphilic agents and changes of ionic environment

1. 1. The biconcave discoid shape of normal human erythrocytes can be altered reversibly by a great number of amphiphilic agents. Crenated cells are induced, as a rule, by anionic or non-ionized compounds; cup-like cells by cationic substances. Formation of crenated and cup cells probably is induced by interaction of the transforming agents with sites charged oppositely within the membrane. 2. 2. Compounds inducing crenation or cup formation can be regarded as true antagonists, since either group of compounds regularily brings about complete reversal of shape transformatins induced by the other one. 3. 3. Suspending erythrocytes in isotonic solutions of non-penetrating anions leads to the occurrence of cup cells, the extent of transformation becoming more pronounced with lowering of extracellular pH. Restoration of normal shape under these conditions is accomplished by addition of penetrating anions. The concentrations of these anions necessary to produce 90% normalisation increase with fall of pH. This type of cup formation supposedly is induced by the increase above a critical value of the pH difference between erythrocyte membrane and adjacent media. 4. 4. The results point to a great lability of the interactions of structural elements of the erythrocyte membrane responsible for the maintenance of biconcave shape. They suggest this shape to result from an equilibrium between opposite forces directed towards formation of crenated or cup cells, respectively.