Formation Of Stomatocytes Research Articles

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 greater than tri-n-propyltin greater than tetra-n-butyltin greater than triphenyltin chloride greater than tri-n-ethyltin bromide greater than dibutyltin dichloride greater than stannous chloride greater than 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 greater than tri-n-propyltin greater than tetra-n-butyltin greater than 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.

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.

The effect of phorbol esters on human erythrocyte morphological discocyte-echinocyte transitions

12- O-Tetradecanoylphorbol-13-acetate (TPA) (100 nM) when incubated with human erythrocytes under conditions of ATP depletion, delayed the onset of the morphological transition from discocytes to echinocytes so that at 2 h, when control incubations were estimated to contain 65% echinocytes, those treated with TPA contained 23% echinocytes. TPA did not alter the subsequent rate of the transition which was complete by 3 h in control cells and 5 h in TPA-treated cells. Addition of 100 nM TPA to ATP-depleted erythrocytes at 2.5 h (>80% echinocytes) for 0.5 h at 37 °C resulted in 17% reversal to a discocyte morphology, but as the time of incubation under conditions of ATP depletion was extended, the level of the reversal fell. TPA had no significant effect on the fall in ATP concentrations over the time course of the experiments (5 h). Preincubation of discocytes with TPA for 10 min also prevented, by approx. 50%, the echinocytosis induced by the calcium (0.2 mM) loading of discocytes using 5 μM A23187. TPA was unable to reverse the echinocyte morphology of calcium-loaded cells back to discocytes. The less potent tumour promotor 4-phorbol-12,13-didecanoate had no effect on this discocyte-echinocyte transition. Incubation of discocytes with the diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) (1–10 μM) had complex effects on morphology, and the ATP-induced morphological transition, ranging from stomatocyte formation to echinocyte formation, depending upon the concentration of the agent and the time of incubation.

A spin-label study of the correlation between stomatocyte formation and membrane fluidization of erythrocytes

Change in the membrane fluidity of human erythrocytes on transformation to stomatocytes was observed by ESR spectroscopy using 12-doxyl stearic acid or its methyl ester as a probe. When the transformation to stomatocytes was induced by four qualitatively different methods, i.e. (a) addition of cationic amphiphilic agents such as chloropromazine, tetracaine, chloroquine or primaquine, (b) addition of Triton X-100, a non-ionic detergent, (c) lowering the pH, and (d) depleting membrane cholesterol, membrane fluidization was always observed. This indicates the existence of a close correlation between stomatocyte formation and increase in the membrane fluidity. Furthermore, since the stomatocytes fixed by diamide treatment exhibited membrane fluidization only in the presence of the reagent, the enhanced membrane fluidity was a direct consequence of the reagent interacting with, and changing the state of, the lipid bilayer itself, and not through the influence of some structural alteration of spectrin. These results provide experimental support for the theoretical prediction made by Brailsford et al. [ J. theoret. Biol. 86, 531 (1980)]. Plausible mechanisms for the discocyte-stomatocyte transformation are discussed.

Selective removal of lipids from the outer membrane layer of human erythrocytes without hemolysis. Consequences for bilayer stability and cell shape

(1) Treatment of erythrocytes with phospholipase A 2 from bee venom cleaves about 55% of the phosphatidylcholine in the outer membrane lipid layer without changing the discoid shape of the cells. All of the fatty acids and 80% of the lysophosphatidylcholine produced under this conditions can be sequentially extracted by bovine serum albumin without hemolysis of the cells. (2) The cells remain discoid up to extraction of all of the fatty acids and 15% of the lysophosphatidylcholine. Removal of a higher fraction of lysophosphatidylcholine induces formation of stomatocytes and sphero-stomatocytes, probably going along with an internalization of membrane vesicles. Stomatocytosis can be explained on the basis of the ‘bilayer couple hypothesis’ (Sheetz, M.P. and Singer, S.J. (1974) Proc. Natl. Acad. Sci. 71, 4457–4461). The shape change will compensate for the differences in surface pressure between the two leaflets induced by selective removal of material from the outer leaf of the bilayer. (3) Increasing the shear modulus of the membrane by diamide prevents this compensatory shape change even after extraction of up to 80% of the lysophosphatidylcholine, which amounts to a loss of 34% of the phospholipids of the outer membrane layer or 22% of its area. This leads to the interesting situation of a membrane possibly having a strikingly diminished ratio of the numbers of phospholipid molecules in the outer to that in the inner lipid layer. A marked difference in surface pressures should arise in this situation, unless other compensatory mechanisms become operative. Evidence for a compensation for outer lipid loss by a constriction of the inner layer has been obtained. A compensation by transbilayer reorientation of phospholipids could not be demonstrated. This latter observation supports the concept of a stabilisation of the asymmetric phospholipid arrangement by proteins such as spectrin.

Rapid transbilayer diffusion of 1,2-diacylglycerol and its relevance to control of membrane curvature.

SHEETZ AND SINGER have proposed that the actions of certain amphipathic drugs in producing curvature of the membrane of human red blood cells (RBCs) could be explained by assuming that these drugs selectively entered one of the leaflets of the lipid bilayer, thus causing lateral expansion of that leaflet relative to the other1. The membrane was therefore forced to curve in order to accommodate this extra material and the direction of this curvature was predictable, leading to production of either stomatocytes or echinocytes. Membrane curvature, leading to the formation of stomatocytes or echinocytes, can also be produced by subjecting human RBCs to phospholipase C attack2–5. In this report we attempt to explain these phospholipase C-induced changes in terms of Sheetz and Singer's model, taking into account the observation that 1,2-diacylglycerol (the product of phospholipase C action), unlike phospholipids, can migrate rapidly across the membrane bilayer. Rapid transbilayer migration of 1, 2-diacylglycerol has previously been inferred from the observed increase in the rate of synthesis of phosphatidate in intact RBCs that have been exposed to phospholipase C: this phosphatidate is formed from diacylglycerol and cytosolic ATP by diacylglycerol kinase, presumably at the inner surface of the membrane2. We have now used this procedure, in which the endogenous diacylglycerol kinase of the erythrocyte is used to trap 1, 2-diacylglycerol appearing on the inner surface of the red cell membrane, to compare the time courses of phospholipase C-catalysed production of diacylglycerol and of the appearance of phosphatidate, and thus obtain an estimate of the rate of transbilayer migration of 1, 2-diacylglycerol.

Mediterranean stomatocytosis: A possible red cell membrane defect

Stomatocytes are red cells in which the usual central pale area becomes elongated, so that the cell has the appearance of a pair of lips forming a mouth. In cases referred to as showing 'stomatocytosis' more than 10% of the red cells are stomatocytes. The authors searched for such cases over 18 mth., after one of them had gained the impression that stomatocytosis was frequently seen in Mediterranean migrants to Australia. The haematological investigation of 50 such cases was described and included family studies which suggested its inheritance as an autosomal dominant. Thirty-five had haemoglobin concentrations within the normal range, the remainder having mild to moderate anaemia, the lowest haemoglobin being 8g./100 ml. The anaemia appeared to be haemolytic in type. Thrombocytopenia was observed in 20 of 30 cases where platelet counts were performed, varying from 38- to 130,000mm.³. Reticulocytes varied from 2.2 to 7.4% in 19 of 26 cases, and slight splenomegaly in 10 out of 13 cases examined. Splenectomy was undertaken in one anaemia case with good result. Most patients presented to their doctors with a variety of complaints, none of which were clearly haematological. Acyltransferase activity was measured in 3 cases and significant reduction observed. It is possible that stomatocytosis may be caused by an abnormal red cell lipid partem resulting at least in part from a lowered level of acyltransferase. However, incubation of normal compatible red cells for 24hr. with plasma from patients with stomatocytosis did not induce stomatocyte formation and conversely stomatocytosis was not reversed when incubated for 24hr. at 37° in normal plasma. The plasma and red cells of 6 cases were analysed for sodium and potassium concentration and no evidence was found of the increased potassium/sodium flux across the red cell membrane such as has been described in two previously published studies of stomatocytosis.