Erythrocyte Shape Changes Research Articles

Shape changes of GSH-treated erythrocytes during complement-induced hemolysis.

Shape changes of GSH-treated human erythrocytes during the acidified serum test were examined by a differential interference microscope. A time-course study of the erythrocytes undergoing complement-mediated hemolysis revealed two kinds of hemolytic processes. One is rapid hemolysis, in which GSH-treated erythrocytes are transformed into spherical forms prior to hemolysis. The other process is gradual or slow hemolysis of a small proportion of GSH-treated erythrocytes. In this process, the erythrocytes are transformed into echinocytes and then into spheroechinocytes prior to hemolysis. However, echinocytic shape changes are not an absolute prerequisite to hemolysis, since echinocytes are not always transformed into a prelytic spherical form, but some of them are reversed to discocytes. Under the experimental conditions of this study, rapid hemolysis of spherical erythrocytes is the main mechanism of hemolysis.

Shape changes of human erythrocytes induced by various amphipathic drugs acting on the membrane of the intact cells

Most of the amphipathic drugs acting on nerves (general and local anesthetics, neuroleptics. antipyretics-analgesics, non-steroidal anti-inflammatory agents, etc.) and some other drugs (anti-histaminic agents, plant alkaloids, etc.) were found to induce instantly one of two typical types of shape changes of human erythrocytes when added into the cell suspension. It was confirmed that, generally, drugs with an anionic polar group induced membrane externalization (crenation), and those with a cationic group induced membrane internalization (invagination). The drugs in the former group are antagonistic against the shape-transforming effects of the drugs in the latter group, and vice versa. Drugs with a neutral polar group or with a quarternary ammonium group induced a complex type of shape change, suggesting the intramembraneous translocation of these drugs.

両親媒性薬物によるヒト赤血球の変形と浸透圧抵抗性変化との関連

Many of the amphipathic drugs tested, which induced shape changes in intact human erythrocytes, produced biphasic effects on hypotonic hemolysis of cells in a similar concentration range, protection at low concentrations and stimulation at higher concentrations. Both the exvaginators and invaginators, which induced shape changes of the membrane exvagination and invagination type, respectively, showed the same type of effect on osmotic resistance. When two drugs of either the exvaginator or invaginator type are present together in a hemolyzing medium, their effects on osmotic resistance appeared to be additive, as was the case with their effects on cell shape. In contrast, when an exvaginator and an invaginator are present together, their effects, both on osmotic resistance and cell shape, seemed to be antagonistic. The present results suggest that these drug effects may be caused, at least partly, by an asymmetric expansion of the membrane lipid bilayer due to asymmetric distribution of the drugs incorporated into the membrane.

Diminished spectrin extraction from ATP-depleted human erythrocytes. Evidence relating spectrin to changes in erythrocyte shape and deformability.

We measured spectrin "extractability" in erythrocytes which were metabolically depleted by incubation at 37 degrees C in plasma or glucose-free buffers. Membranes were extracted with 1 mM EDTA (pH 8, 40 h, 4 degrees C) and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This procedure solubilized 85--90% of the spectrin, actin, and residual hemoglobin from ghosts of fresh erythrocytes. In incubated erythrocytes, inextractable spectrin rapidly accumulated when ATP concentrations fell below 0--15% of normal. In severely depleted cells, 60--90% of the total ghost spectrin became inextractable. Inextractability was not abolished by physically disrupting the ghost before extraction, but was reversed when erythrocyte ATP was replenished with adenosine. The accumulation of inextractable spectrin correlated temporally with the increase in apparent membrane deformability and the increases in erythrocyte vicosity, calcium content, sodium gain, and potassium loss characteristic of ATP-depleted erythrocytes. No change in integral membrane protein topography (assessed by the distribution of intramembranous particles and concanavalin A surface-binding sites) was detected in depleted cells. Analogous changes were observed in erythrocytes exposed to extremes of pH and temperature. When the pH in the erythrocyte interior fell below 5.5, a pH where spectrin was aggregated and isoelectrically precipitated, erythrocyte and ghost viscosity increased coincident with a marked decrease in spectrin extractability. Similarly above 49 degrees C, a temperature where spectrin was denatured and precipitated, erythrocyte viscosity rose as inextractable spectrin accumulated. These observations provide direct evidence of a change in the physical state of spectrin associated with a change in erythrocyte shape and deformability. They support the concept that erythrocyte shape and deformability are largely determined by the shape and deformability of the spectrin-actin protein meshwork which laminates the inner membrane surface.

B-21. 高脂血症と赤血球変形

B-21. 高脂血症と赤血球変形

Chlorotetracycline induces calcium mediated shape changes in human erythrocytes. Is Ca asymmetrically distributed in the red cell membrane?

Calcium was localized in the red cell membrane by light microscopy using chlorotetracycline hydrochloride (CTC) as chelate probe. Treating human erythrocytes with CTC dissolved in saline free of divalent cations, leads to a 530 nm fluorescence emission in the cell border and to characteristic cell shape changes which were evaluated to assess intramembrane calcium distribution. CTC prevented and reverted erythrocyte crenation induced either by washing or superfusing the cells with saline. The ionophore A23187, EGTA and glucose depletion depressed the shape modifying effect of CTC. Thus, CTC appears to act on red cell shape by complex formation with membrane associated calcium. This is further confirmed by the failure of degraded CTC, devoid of metal binding capacity, to modify the crenated shape. The CTC effect can be reverted by superfusing the erythrocytes with CTC-free medium. Thus, calcium binds more tightly to the membrane than to CTC and is not displaced by the antibiotic. If the bilayer couple hypothesis [Sheetz, M.P., Singer, S.J., Proc. Natl. Acad. Sci. USA 71, 4457-4461 (1974)]applies, crenation is reverted by expansion of the inner membrane half relative to the outer membrane half. Expansion of the inner membrane half results from intercalation of CTC which binds to calcium. Thus, calcium in the red cell membrane preferentially occupies the inner leaflet of the bilayer.

Relationships of the spectrin complex of human erythrocyte membranes to the actomyosins of muscle cells.

Important similarities are reported between human smooth muscle actomyosin and the human erythrocyte spectrin complex, primarily components 1, 2, and 5 (Fairbanks G., Steck, T.L., and Wallach, D.F.H. (1971), Biochemistry 10, 2606). The actin-like protein, component 5, is identical with human uterine actin in its ability to form 50-70-A filaments to stimulate myosin ATPase activity, and to bind rabbit heavy meromyoson specit heavy meromyosin specifically. Antibodies to human smooth muscle myosin(uterine) were prepared which were monospecific. A weak but specific cross-reaction of these antisera with components 1 and/or 2 (spectrin) was characterized and at least 25% of the antimyosin antibodies showed a low affinity reaction iwth spectrin. Antibodies generated against a soluble complex of spectrin components 1 and 2 reacted only with component 1 and did not cross-react with myosin. In addition to these structural similarities between smooth muscle actomyosin and the spectrin complex, we have found that spectrin is involved in ATP-dependent erythrocyte shape changes (Sheetz, M.P., Painter, R.G., AND Singer, S.J. (1976B), Cold Spring Harbor Symp. Cell Motility (in press) and, therefore, the spectrin complex is also a mechanochemical protein system.

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.

Conductimetric investigation of erythrocyte behaviour during shear flow of concentrated suspensions through a large tube.

The behaviour of canine erythrocytes in isotonic saline suspensions undergoing steady unidirectional shear flow in the vertical direction, at cell volume concentrations (ρ) ranging between 0.017 and 0.840, is studied by measuring suspension resistivities in three mutually orthogonal directions. Results are compared with the predicted behaviour of rigid spheroids and liquid drops. Analyses of suspension resistivities show that there are significant changes in erythrocyte shape with increasing shear rate ( k ) at all k covered (2.6 x 10 – 10 3 s -1 ) and at all ρ, very large changes occurring at ρ > 0.65. However, the changes in the equivalent principal axis-ratios of erythrocytes with k deviate substantially from the flow behaviour expected of liquid drops, the average ‘shape factor’ for the cells decreasing rather than increasing with k . Neither the simple liquid drop nor the rigid spheroid model can adequately explain some aspects of the results obtained. Results show that cell concentration is a major determinant of erythrocyte behaviour and point to the existence of three concentration regimes in the range of flow rates investigated, with different mechanisms of particle orientation during shear flow predominating in each. At ρ < 0.08 no significant change in orientation distribution with k is observed; but results show that erythrocytes tend to assume the orientation corresponding to the maximum dissipation of energy in a shear field or corresponding to the maximum energy dissipation of settling disks (the effect increasing as ρ is reduced) indicating a predominance of inertial effects. Between ρ ≃ 0.08 and ρ ≃ 0.65 the orientation distribution changes towards alignment of erythrocytes parallel to the plane of flow, the degree of alignment increasing with k and with ρ. Here, particle-particle interactions facilitate the orientation of erythrocytes by shear stresses. Above ρ ≃ 0.65 the change in cell shape with k increases sharply, suggesting that erythrocyte deformation becomes the principal mechanism of suspension flow.

Accumulation of 1,2-diacylglycerol in the plasma membrane may lead to echinocyte transformation of erythrocytes.

THE change in shape of erythrocytes from the normal biconcave disk to a spiculed sphere (echinocyte1) can be caused by an increased intracellular concentration of Ca2+ (refs 2 to 5). We have found that an immediate biochemical effect of raising intracellular [Ca2+] is to increase the production of 1,2-diacylglycerol and cause its accumulation in the plasma membrane. This change in membrane composition may alter the structure of the membrane and thus cause the change in shape. Energy-depleted cells also show increased 1,2-diacylglycerol and similar but less extreme morphological changes, even in the absence of Ca2+.

Changes in shape and osmotic resistance of human erythrocytes resulted from changes in the lysolecithin content of the membranes.

Incubation of human erythrocytes in lysolecithin-containing medium, prepared by an addition of purified lysolecithin to blood plasma or to phosphate-buffered saline, induced the increases in lysolecithin content of the erythrocyte membranes as well as morphological changes of the cells from normal biconcave disc to crenated or spherical forms. The erythrocytes with slight excess of lysolecithin incorporated, which take shapes of crenated disc or crenated sphere, rather have increased osmotic resistance, whereas those with much more lysolecithin, taking almost spherical form, show reduced osmotic resistance. After washing these erythrocytes with heated normal plasma to remove excess lysolecithin from the membrane, the increased osmotic resistance observed in the former erythrocyte preparation was restored to the normal level, whereas the decreased resistance in the latter cell preparation was never reversed, in spite of the restoration to the normal morphological shape.

The effect of triton X-100 on the permeability of erythrocytes to non-electrolytes.

AbstractStudies of the effect of triton X‐100 on the fragility of rabbit erythrocytes and on shape and volume changes of rabbit and sheep erythrocytes suspended in an isotonic salt solution suggest that this subtance alters the erythrocytes of other species in a manner similar to that reported previously for human red cells. Using a densimeter technique it was found that the permeability of relatively slowly moving non‐electrolyte‐carrier‐complexes is decreased by triton X‐100 but more rapidly penetrating systems are not affected by this substance. The permeability of some molecules which cross the membrane by diffusion is increased by triton X‐100. It is suggested that triton X‐100 affects both the aqueous channels and the portion of the membrane across which carriers move. Its action is less than that of tannic acid or butanol.

Shape and volume changes in frog erythrocytes following ultraviolet radiation.

Frog erythrocytes in Ringer's solution were exposed to ultraviolet radiation and then followed in camera lucida drawings for changes in shape and dimension. Cell thickness was found to increase while cell width remained constant throughout the period prior to hemolysis. The cell shortened and bulged at the ends during the middle third of the prolytic period while a region around the cell center remained constricted. When this constricted region gave way, the cell became spherical and hemolyzed. Cell volume as calculated from the cell's dimensions increased linearly with time throughout the prolytic period to hemolysis then dropped rapidly to a constant value somewhat higher than the original cell volume. These changes in shape and volume are consistent with a colloid osmotic type of hemolysis but with other factors acting to limit the rate of swelling and the forms assumed during the swelling process. The relationship between the time of hemolysis and the cell surface area exposed to the ultraviolet is discussed as it applies to the site of ultraviolet damage.