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.     

Role of membrane lipid distribution in chlorpromazine-induced shape change of human erythrocytes

This is a study of the morphology and transbilayer lipid distribution of human erythrocytes treated with chlorpromazine (CPZ) over extended time courses. At 0°C, treatment of dilauroylphosphatidyl[1-¹⁴C]choline-labeled erythrocytes with 120 μM CPZ produced an immediate stomatocytic transformation (t_1/2<5 min) with no concurrent change in transbilayer distribution of radiolabeled lipid, as determined by bovine serum albumin extractability. At 37°C, CPZ treatment of cells produced two sequential morphological effects: immediate stomatocytosis (t_1/2<1 min) with no concurrent change in radiolabel transbilayer distribution, followed by gradual increase in stomatocytic extent over several hours, with concurrent redistribution of radiolabeled lipid to the inner monolayer. Cells pretreated with vanadate at 37°C exhibited a triphasic morphological response: CPZ produced immediate stomatocytosis, followed by a transient reversion to echinocytes lasting about 2 h, before returning to stomatocytic morphologies over the next several hours. The echinocytic reversion was accompanied by exposure of phosphatidylserine on the cell surface, as indicated by increased activation of exogenous prothrombinase. These findings suggest that while CPZ induces transbilayer lipid redistribution over extended time periods (which may mediate the complex morphological transformations observed), the early stomatocytic response elicited by addition of CPZ is not due to lipid reorganization.     

Calcium does not mediate the shape change that follows ATP depletion in human erythrocytes

Crenation, the shape change that follows ATP depletion in human erythrocytes, also follows ionphore-mediated Ca2+-loading. Experiments designed to test whether Ca2+ mediates metabolic crenation showed that: (1) an influx of extracellular Ca2+ is not required for metabolic crenation; (2) metabolic crenation is accompanied by a 70% increase in 86Rb+ permeability, a change much smaller than the increase expected if crenating concentrations of Ca2+ were released from bound intracellular pools; (3) A23187 plus EGTA, a treatment that depletes intracellular Ca2+ and stops Ca2+ crenation, does not affect metabolic crenation; (4) calmodulin inhibitors do not slow metabolic crenation. We conclude that Ca2+ does not mediate metabolic crenation. Albumin washes reverse Ca2+ crenation and metabolic crenation involve the accumulation of some amphiphilic species (e.g., lysolipid or diacylglycerol) in the cell membrane outer monolayer, and that ATP depletion induces a second crenating process which might be a reorganization of the cytoskeleton.     

The relationship between the osmotic fragility of human erythrocytes and cell age

Erythrocytes in a normal blood sample are hemolyzed over a range of hypotonic salt concentrations. In order to investigate the relationship between the distribution of osmotic fragilities and the distribution of cellular ages, the osmotic fragility has been compared with three indices of cellular age. The activity of glutamic-oxalacetic transaminase (GOT) and the percentage hemoglobin A_1C were measured in samples hemolyzed in different hypotonic salt concentrations. The osmotic fragility curve was also obtained for cells of different density separated by centrifugation. These experiments indicate that the fragility distribution is not an accurate reflection of the distribution of cellular ages. The mean fragility for older cells is higher than that of younger cells. However, cellular aging does not produce a gradual increase in osmotic fragility. Instead, it seems to produce changes which can both increase and decrease the fragility, resulting in a broader distribution of fragilities with some of the older cells actually less fragile than the younger ones.     

Early stage shape change of human erythrocytes after application of electric field pulses.

Erythrocytes which receive electric field pulses are subject to poration, fusion and shape changes due to electrodynamic forces, aminophospholipid perturbation and influences on the normal flip-flop process. The shape change characteristics of cells suspended in different media were analysed after application of rectangular electric field pulses from t=11-44 micros and from E=4-8 kV/cm. Albumin is shown to decelerate the echinocyte shape change within the first few seconds after pulse application. The addition of fluoride and vanadate accelerates the shape change due to their inhibiting influence on the aminophospholipid translocase. For both the duration of the field pulse and its field strength, there exist lower threshold values under which no early stage shape change is observable. The activation energy calculated from the dissipative influence of the electric field alone is smaller than expected, indicating the electrodynamic influence on the flip-flop process. Cell shapes were additionally analysed by contour tracing to focus on the echinocyte spicule distribution after pulse application. This image analysis revealed that, with an increase of both pulse duration and field strength, the shape change velocity and the shape change intensity increase.     

Degranulation, membrane addition, and shape change during chemotactic factor-induced aggregation of human neutrophils

Neutrophils stimulated by the chemotactic factor formyl-methionyl- leucyl-phenyl-alanine (FMLP) undergo a transient change in surface properties that permits the cells to adhere more readily to surfaces and to each other. This transient change can be monitored by light scattering as stimulated neutrophils form aggregates while stirred in a platelet aggregometer. Maximum change in light scattering occurs within 1 min and correlates with an increase in the percentage of cells that are in aggregates of four or more cells and a decrease in the percentage of single cells. With time (3-5 min), small aggregates disappear and single cells reappear. The transient change in adhesiveness is accompanied by a persistent change in cell shape; the cells become polarized and protrude ruffles from one sector of the cell surface. During aggregation the cells adhere to one another with smooth sides together and ruffles pointed outward. During disaggregation the cells dissociate laterally with the simultaneous internalization of membrane in the region opposite the ruffles. Particle bound to the surface by charge (thorotrast, cationized ferritin) are concentrated and internalized in this region. The change in cell shape from round to ruffled occurs within seconds, suggesting that membrane is added to the cell surface from an intracellular store. We therefore quantified surface membrane by electron microscopy morphometry and measured a 25% increase within 10 s of adding FMLP. The source of new membrane appeared to be the specific granule membrane since the kinetics of granule discharge (between 30% and 50% of all release occurs in the first 10 s) correlate with the appearance of new membrane. Furthermore, the amount of membrane that appears at the cell surface at 10 s correlates with that lost from intracellular granules in that time. Chemotaxin-induced aggregation thus begins with granule discharge and membrane addition followed by protrusion of ruffles. Adherence is maximal at 60 s and the gradual loss of adhesiveness that follows is associated with uropod formation and enhanced endocytic activity.     

Interaction of added amphiphilic lipids with the membrane of intact human erythrocytes to induce change in the cell shape

Addition of an appropriate amount of amphiphilic lipid, such as fatty acid, lysophospholipid and medium-chain phospholipid, into a suspension of human erythrocytes (pH 7.4) at 37 degree C resulted in their incorporation into the membrane and induction of a cell shape change of crenation (echinocyte-spherocyte) type without causing hemolysis. The extent of the shape change was dependent on the amount of the lipid incorporated and the crenation disappeared on removing the incorporated molecules from the membrane. The crenation induced by acidic lipids was further altered drastically by resuspending the treated cells in media of pH 6, 7, and 8, whereas that induced by choline-phospholipid or -lysophospholipid was not so pH-dependent. Based on these results, the mechanism of this shape change is discussed.     

Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2-loaded human platelets

The adenylate cyclase stimulator forskolin was used to study the inhibitory effects of elevated cAMP on the activation of washed human platelets loaded with the fluorescent Ca2+ indicator quin2. In the presence of 10 microM isobutylmethylxanthine forskolin inhibited rises in [Ca2+]i evoked by thrombin and platelet-activating factor (PAF) due to both Ca2+ influx and release from internal stores with similar potency. Aggregation evoked by thrombin and PAF was suppressed whilst partial shape-change persisted, even in the absence of a measurable rise in [Ca2+]i. Forskolin did not affect the rise in [Ca2+]i evoked by Ca2+ ionophore; aggregation was suppressed but shape-change persisted.     

Studies on the relationship between lectins from Axinella polypoides agglutinating bacteria and human erythrocytes

A new lectin, called Axinella IV, with bacteria agglutinating activity was detected in the crude extract of the sponge Axinella polypoides. Immunofluorescence studies and immunoelectrophoresis revealed no cross-reactivities with the already known lectins Axinella I. II, and III. d-Galactose and oligosaccharides with d-Galactose in terminal nonreducing position are inactive in agglutination inhibition tests demonstrating also different specificity of Axinella IV lectin when compared with the three aforementioned lectins.     

Relationship between the transverse distribution of phospholipids in plasma membrane and shape change of human platelets

ESR spectroscopy was used to investigate the distribution of spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the presence of human platelets. Three rates were determined: hydrolysis of the ester bond at position 2, reduction of labels by cytoplasm, and internalization of labels situated in the outer leaflet of the plasma membrane. We found that the half-time for transverse diffusion of added phospholipids was shorter for aminophospholipids (40 min and less than 10 min for PE and PS, respectively) than for the choline derivatives (greater than 120 min for PC, not measurable for SM). Addition of any of the phospholipids led to a considerable change in the initial platelet shape (assessed by electron microscopy) from a discoid form to a smaller body with very long pseudopods. When aminophospholipids were used, the platelets quickly returned to the initial shape [half-time of 20 min and less than 5 min for (0.2)PE and (0.2)PS, respectively]. Conversely, there was no relaxation after (0.2)PC or (0.2)SM was added. We conclude that there is a relationship between the excess of phospholipids in the outer leaflet of the plasma membrane and cytoskeletal organization presumably via actin polymerization, which is responsible for platelet shape.     

Organotin-induced hemolysis,shape transformation and intramembranous aggregates in human erythrocytes

Organotin compounds examined in this study exhibited a relative order of potency for induction of in vitro hemolysis in human erythrocytes as follows: tri-n-butyltin > tri-n propyltin > tetra-n-butyltin > triphenyltin chloride > tri-n-ethyltin bromide > dibutyltin dichloride > stannous chloride > tri-n-methyltin chloride = butyltin chloride dihydroxide. All of the organotin compounds induced erythrocyte shape transformation from the normal discocyte to an echinocyte and, in addition, triphenyltin chloride, tetra-n-butyltin and tri-n-ethyltin bromide also elicited stomatocyte formation at higher concentrations. Select organotin compounds also formed tin-containing aggregates within the plasma membrane. The relative order of effectiveness for organotin induction of intramembranous aggregates was tri-n-butyltin > tri-npropyltin > tetra-n-butyltin > tri-n-ethyltin bromide, which was based upon the lowest concentration at which they were observed. These results support the previously suggested theory that organotins are membrane effectors because of their comparatively high hydrophobic, lipid partitioning properties. The relatively lipophilic compound, triphenyltin chloride, appeared to be anomalous because it did not readily promote hemolysis or induce the formation of intramembranous aggregates in human erythrocytes. A log-linear statistical model demonstrated an association of hemolysis with both tri-n-butyltin aggregate formation and shape transformation. Select organotin compounds should be useful probes in membrane studies because of their numerous effects.Abbreviations DBT dibutylin dichloride - MBT butyltinchloride dihydroxide - SnCl₂ stannous chloride - TBT tri-n-butyltin - TET tri-n-ethyltin bromide - TMT tri-n-methyltin chloride - TPhT triphenyltin chloride - TPT tri-n-propyltin - TTBT tetra-n-butyltin     

Effect of potassium leakage on reversible aggregation of human erythrocytes

Changes in the aggregation of human erythrocytes caused by polydextrane were studied under conditions influencing the rate of potassium leakage from cells to medium. It was shown that aggregation decreases as the leakage of potassium ions increases and is completely abolished at leakage rates higher than 2.5-3.0 mmol/l of erythrocytes per hour. The involvement of nonequilibrial electrokinetic phenomena in the inhibition of erythrocyte aggregation by ionic fluxes across erythrocyte surface is discussed. It is proposed that potassium leakage affects the erythrocyte sedimentation rate in clinical investigations.     

Glutathione reductase from human erythrocytes. Catalytic properties and aggregation.

The catalytic properties of glutathione reductase from human erythrocytes have been studied over a range of buffer conditions and substrate concentrations. This study provides optimal conditions for determining the basic kinetic parameters of the enzyme. The catalytic behaviour of glutathione reductase is consistent with spatially separated binding sites for its substrates. In certain assays anomalies were observed which are correlated with an inactivation of the enzyme by NADPH. Concurrent sedimentation experiments showed that NADPH promoted aggregation of the enzyme. Both inactivation and aggregation could be connected with oxidation of thiols at the active site. The relation of the properties of glutathione reductase to cellular conditions is discussed.     

Electric field pulses induce reversible shape transformation of human erythrocytes

Electric field pulses >2-3 kV cm¹ long known to induce membrane poration and fusion of erythrocytes as well as to enhance the transbilayer mobility of phospholipids and to perturb aminophospholipid asymmetry, are shown to induce, at 0 C., transformation of the discocytic cells into echinocytes and spheroechinocytes. The extent of transformation increases with strength, duration and number of pulses. Its time course is biphasic., a major rapid phase (t/2 ～ 5 s) being followed by a minor one, lasting for 2-3 h. Shape transformation goes along with the exofacial exposure of phosphatidylserine (PS), detected by FITC-annexin V binding and quantified by a calibration curve established via externally inserted dilauroylphosphatldylserine. Incubation of these echinocytes at 37 C leads to a rapid recovery of the discocytic shape followed by slower formation of stomatocytes. Shape recovery is temperature dependent (E_a ～100 kJ/mol), and can be impaired by depletion of ATP or Mg⁺⁺ and by addition of vanadate or fluoride. Shape recovery and stomatocyte formation go along with a rapid loss of annexin binding in about 45% of the cells while the rest maintains its binding capacity. In the presence of vanadate, annexin binding increases in all cells. The results are discussed in the light of the bilayer couple concept of erythrocyte shape and the enhanced transverse mobility of phospholipids. Echinocyte formation is most likely caused by the reorientation of endofacial aminopho-spholipids to the outer leaflet of the bilayer. Shape recovery and stomatocyte formation probably result from a continuous reinternalization of PS via the ATP dependent aminophospholipid translocase, but may also be supported by downhill movement of PC to the inner leaflet and by other yet unidentified processes.