Erythrocyte membrane in Duchenne muscular dystrophy. II. Fluidity of lipids in dystrophic patients and their families

Electron spin resonance techniques was used to study the fluidity of intact and hemoglobin free erythrocyte membranes from patients with Duchenne muscular distrophy and from members of their family. A greater mobility of spin label motion was observed only at the surface of hemoglobin free membranes in the patients. These studies suggest alterations in lipid-protein interaction, indicating in Duchenne muscular distrophy a generalized membrane abnormality.     

The influence of metmyoglobin and ferrylmyoglobin on the human erythrocyte membrane

Preliminary experiments revealed that ferrylmyoglobin decayed more slowly in the absence than in the presence of intact erythrocytes and erythrocyte membranes. This suggested the existence of interactions between FerrylMb and the erythrocyte membrane. Subsequent studies examined the influence of FerrylMb on the membrane of intact erythrocytes and on isolated erythrocyte membranes. The incubation of intact erythrocytes with FerrylMb did not influence their osmotic fragility or the fluidity of their membranes; the level of peroxidation of the membrane lipids increased only slightly (there was only a slight increase in the level of membrane lipid peroxidation). The activity of acetylcholinesterase significantly increased after 15 minutes of incubation, whereas longer incubation did not lead to any changes in the activity of this enzyme. The incubation of isolated erythrocyte membranes with FerrylMb resulted in an increase in their fluidity and a significant rise in the level of lipid peroxidation.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

Effects of intramembrane particle aggregation on erythrocyte membrane fluidity: an electron spin resonance study in normal and in dystrophic subjects

Mobilization and aggregation of intramembrane particles (IMPs) are physiological events observed in various cells. In erythrocyte membranes, aggregation of IMPs can be induced by the exposure of partially desprectrinized erythrocyte membranes to acidic pH. We investigated the association between IMPs aggregation, protein mobility, and membrane fluidity in erythrocyte membranes of healthy controls and Duchenne muscular dystrophy (DMD) patients by using electron spin resonance and specific spin labels for membrane proteins and lipids. In erythrocyte membranes of control subjects, the partial spectrin removal induced a decreased segmental motion of protein spin label indicating an increase of protein-protein interactions. Stearic acid spin labels 5- and 16-(N-oxyl-4,4'-dimethyloxazolidine) showed that the treatment induces an increase of membrane fluidity. In DMD patients, both treated and untreated erythrocyte membranes showed changes of membrane fluidity when compared to those of the controls. Our results suggest that defects in the interactions between skeletal proteins and/or between membrane and skeleton components may contribute to the alterations of erythrocyte membranes in DMD.     

Spectroscopic characterization of vesicle formation on heated human erythrocytes and the influence of the antiviral agent amantadine

EPR investigations on the vesiculation process of heated human erythrocytes were performed, using different fatty acid spin labels. Spectrin denaturation and vesiculation do not influence the fluidity of the lipid phase of the remaining membrane of human erythrocytes: Vesicles released differ in chemical composition as well as in the lipid fluidity of their membrane from the intact human erythrocyte membrane. A reduced cholesterol-to-phospholipid ratio and a depletion of spectrin was found. By changing the ionic concentration of the suspension medium an effect on membrane spectra and on vesicle release was established. The adamantane derivative amantadine causes fluidization of the human erythrocyte membrane and inhibits vesicle release. Based on these results, a model for the mechanism by which adamantane-like molecules could interact with membranes is proposed.     

Leptin improves membrane fluidity of erythrocytes in humans via a nitric oxide-dependent mechanism--an electron paramagnetic resonance investigation

Abnormalities in physical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases. Recently, there has been an indication that leptin, the product of the human obesity gene, actively participates not only in the metabolic regulations but also in the control of cardiovascular functions. In the present study, to assess the role of leptin in the regulation of membrane properties, the effects of leptin on membrane fluidity of erythrocytes in humans are examined. The membrane fluidity of erythrocytes in healthy volunteers by means of an electron paramagnetic resonance (EPR) and spin-labeling method is determined. In an in vitro study, leptin decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (ho/h-1) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner in healthy volunteers. The finding indicated that leptin increased the membrane fluidity and improved the microviscosity of erythrocytes. The effect of leptin on the membrane fluidity was significantly potentiated by the nitric oxide (NO) donors, L-arginine and S-nitroso-N-acetylpenicillamine (SNAP), and a cyclic guanosine monophosphate (cGMP) analog, 8-bromo-cGMP. In contrast, the change evoked by leptin was significantly attenuated in the presence of the NO synthase inhibitors, N(G)-nitro-L-arginine-methyl-ester (L-NAME) and asymmetric dimethyl-L-arginine (ADMA). The results of the present study showed that leptin increased the membrane fluidity and improved the rigidity of cell membranes to some extent via an NO- and cGMP-dependent mechanism. Furthermore, the data also suggest that leptin might have a crucial role in the regulation of rheological behavior of erythrocytes and microcirculation in humans.     

Nitric oxide improves membrane fluidity of erythrocytes in essential hypertension: An electron paramagnetic resonance investigation

It has been shown that rheological abnormality might be an etiological factor in hypertension. Recent studies have revealed that human erythrocytes possess a nitric oxide (NO) synthase and that this activation might be involved in the regulation of rheological properties of erythrocytes. The present study was undertaken to investigate the role of NO in the regulation of membrane functions of erythrocytes in patients with essential hypertension by means of an electron paramagnetic resonance (EPR) and spin-labeling method. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (h(0)/h(-1)) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner. The finding indicated that the NO donor increased the membrane fluidity of erythrocytes. In addition, the effect of SNAP was significantly potentiated by 8-bromo-cyclic guanosine monophosphate. By contrast, the change of the fluidity induced by SNAP was reversed in the presence of L-N(G)-nitroarginine methyl ester and asymmetric dimethyl L-arginine. In patients with essential hypertension, the membrane fluidity of erythrocytes was significantly lower than in the normotensive subjects. The effect of SNAP was more pronounced in essential hypertension than in normotensive subjects. These results showed that NO increased the membrane fluidity and decreased the rigidity of cell membranes. Furthermore, the greater effect of NO on the fluidity in essential hypertension suggests that NO might actively participate in the regulation of rheological behavior of erythrocytes and have a crucial role in the improvement of microcirculation in hypertension.     

Fluidity of intact erythrocyte membranes. Correction for fluorescence energy transfer from diphenylhexatriene to hemoglobin

Membranes of intact erythrocytes were labeled by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) using an improved labeling procedure described previously (Plásek, J. and Jarolím, P. (1987) Gen. Physiol. Biophys. 6, 425-437). The relationship between the steady-state DPH fluorescence anisotropy r and the mean corpuscular hemoglobin concentration (MCHC) was studied. Fluorescence anisotropy increased with increasing MCHC. A linear dependence of r = 0.0026 (MCHC) + 0.113 was obtained which enabled us to measure the fluidity of intact red cell membranes. Without this correction for fluorescence quenching by hemoglobin, incorrect conclusions about membrane fluidity could be made. This fact is demonstrated in a group of pyruvate kinase deficient patients compared with a group of healthy blood donors.     

Changes in fluidity of erythrocyte membranes after storage of erythrocytes and regeneration of cellular ATP level

The membrane fluidity of freshly collected human erythrocytes, of erythrocytes stored for 3–4 weeks and of stored erythrocytes rejuvenated with glucose and inosine was investigated by measuring polarization of fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene and $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$. The fluidity of membranes prepared from stored erythrocytes was higher than that of fresh erythrocytes. After rejuvenation of erythrocytes with glucose and with or without inosine the membrane fluidity decreased. These changes were probably due to variations of ATP levels in the erythrocytes.     

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.     

Determination of Fluorescence Polarization of Membrane Probes in Intact Erythrocytes: Possible Scattering Artifacts

The anisotropy of the fluorescence of diphenylhexatriene has been reported to be less in the membranes of intact erythrocytes than in erythrocyte ghost membranes or in membranes prepared from erythrocyte lipids. Evidence is presented that this may be an artifact due to the intense light scattering by the intact erythrocytes.     

Fluidity properties and liquid composition of erythrocyte membranes in Chediak-Higashi syndrome

We have earlier shown through electron spin resonance (ESR) studies of leukocytes that membranes of cells from both Chediak-Higashi syndrome (CHS) mice and humans have abnormally high fluidity. We have extended our studied to erythrocytes. Erythrocytes were labeled with the nitroxide-substituted analogue of stearic acid, 2-(3-carboxypropyl)-4,4- dimethyl-2-tridecyl-3-oxazolidinyloxyl, and ESR spectra were obtained. Order parameter, S, at 23 degrees C, was 0.661 and 0.653 for erythrocytes of normal and CHS mice (P less than 0.001). S was 0.684 for normal human erythrocytes and 0.675 (P less than 0.001) for CHS erythrocytes at 25 degrees C. Because S varies inversely to fluidity, these results indicate that CHS erythrocytes tend to have higher fluidity than normal. In vitro treatment of both mice and human CHS erythrocytes with 10 mM ascorbate returned their membrane fluidity to normal. We prepared erythrocyte ghosts and extracted them with CHCl3:CH3OH (2:1). Gas-liquid chromatography analysis showed a greater number of unsaturated fatty acids for CHS. The average number of double bonds detected in fatty acids for mice on a standard diet was 1.77 for normal and 2.02 for CHS (P less than 0.04); comparison of human erythrocytes from one normal control and one CHS patient showed a similar trend. Our results suggest that an increased proportion of unsaturated fatty acids may contribute to increased fluidity of CHS erythrocytes. Our observation that both leukocytes and erythrocytes of CHS have abnormal fluidity indicates that CHS pathophysiology may relate to a general membrane disorder.     

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

Changes in membrane properties of erythrocytes and polymorphonuclear cells in psoriasis

Using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and its cationic derivative, 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene, we evaluated membrane fluidity in living polymorphonuclear leukocytes and in erythrocytes of psoriatic patients. Our results have shown that erythrocyte membranes of psoriatic patients exhibit a decrease of fluidity. These changes were not associated with any relevant modifications of the cholesterol to phospholipid molar ratio. Moreover, we observed a decrease in polymorphonuclear leukocytes membrane fluidity associated with changes in chemotactic migration. Our results indicate changes of membrane fluidity involving membranes different from the epidermal cells and suggest the hypothesis of a defective membrane-cytoskeleton interaction in psoriasis.     

Electron paramagnetic resonance studies of membrane fluidity in ozone-treated erythrocytes and liposomes

Doxyl stearate spin probes which differed in the attachment of the nitroxide free radical to the fatty acid have been used to study membrane fluidity in ozone-treated bovine erythrocytes and liposomes. Analysis of EPR spectra of spin labels incorporated into lipid bilayer of the erythrocyte membranes indicates an increase in the mobility and decrease in the order of membrane lipids. In isolated erythrocyte membranes (ghosts) the most significant changes were observed for 16-doxylstearic acid. In intact erythrocytes statistically significant were differences for 5-doxylstearic acid. The effect of ozone on liposomes prepared from a lipid extract of erythrocyte lipids was marked in the membrane microenvironment sampled by all spin probes. Ozone apparently leads to alterations of membrane dynamics and structure but does not cause increased rigidity of the membrane.     

Ionic strength-dependent alterations of membrane structure of red blood cells

Electron paramagnetic resonance (EPR) measurements using various fatty acid spin labels were performed on membranes of intact human erythrocytes at physiological, and at low ionic strength. In the case of spin probes bearing the nitroxide near the polar head group, a less restricted motion at low ionic strength was seen than with those labels with a nitroxide deeper within the hydrophobic tail of the membrane. Although these data clearly show an influence of ionic strength on membrane structure, and possibly a modified protein-lipid interaction, they cannot be simply discussed in terms of an altered membrane fluidity.     

Dielectric breakdown measurements of human and bovine erythrocyte membranes using benzyl alcohol as a probe molecule

Dielectric breakdown of intact erythrocytes and subsequent haemolysis in the presence of increasing concentrations of benzyl alcohol were investigated by means of an electrolytical discharge chamber and a hydrodynamic focusing Coulter Counter.Low concentrations of the drug stabilized human and bovine erythrocytes against haemolysis induced by dielectric breakdown of the cell membrane in isotonic solutions, while high concentrations caused lysis similar to hypotonic and mechanical haemolysis. The stabilizing effect of the drug on electrically induced haemolysis depends on the pulse length of the applied electric field. The critical dielectric breakdown voltage of the membranes of intact cells decreases progressively with increasing benzyl alcohol concentrations, at which the membrane is also more stabilized against electrical and osmotic haemolysis. Occasionally, an increase in the dielectric breakdown voltage is observed at drug concentrations at which lysis occurs. A similar dependence of the breakdown voltage on drug concentration was found for human erythrocyte ghost cells prepared by dielectric breakdown.The results are consistent with the electromechanical model suggested for the dielectric breakdown mechanism and with the assumption of Metcalfe, using NMR and ESR techniques, that the fluidity of the membrane increases with increasing benzyl alcohol concentration.     

Enhanced sensitivity to calcium in Duchenne muscular dystrophy.

The ionophore A23187 causes an increase in the Ca content of human erythrocytes and a Ca-dependent increase in K efflux (Gardos effect). These changes are associated with a reduction in osmotic fragility and cell size. Treatment of erythrocytes from patients with Duchenne muscular dystrophy with A23187 results in ⁴⁵Ca uptake comparable to that of erythrocytes from control subjects. However, the reduction in osmotic fragility and K content observed in dystrophic erythrocytes is twofold greater than in control erythrocytes. These results indicate that an alteration in the regulation of erythrocyte membrane function by Ca occurs in Duchenne muscular dystrophy. This alteration may be responsible for other changes in erythrocyte membrane properties observed in Duchenne muscular dystrophy.     

Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study.

Membrane fluidity of erythrocytes obtained from 15 children with trisomy 21 and 20 healthy controls were studied by measuring steady-state fluorescence anisotropy and fluorescence lifetime of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in hemoglobin-free erythrocyte membranes. Our results demonstrate a significant decrease in DPH fluorescence anisotropy and a significant increase in TMA-DPH fluorescence anistropy in erythrocytes from subjects with trisomy 21. No significant differences between the two groups were observed in the fluorescence lifetime of DPH and TMA-DPH. These data suggest an increase in membrane fluidity in the interior part of the membrane and a decrease in fluidity at the lipid-water interface region. This could be in part attributed to an increased oxidative damage in trisomy 21.     

Adiponectin and membrane fluidity of erythrocytes in normotensive and hypertensive men

Objective: Abnormalities in physicochemical properties of the cell membranes may underlie the defects that are strongly linked to hypertension. Recent evidence indicates that adiponectin may have protective effects against cardiovascular diseases. The purpose of the present study was to assess the possible link between plasma adiponectin and membrane fluidity in normotensive (NT) and hypertensive (HT) men. Research Methods and Procedures: We measured the membrane fluidity (a reciprocal value of membrane microviscosity) of erythrocytes in NT and HT men by using an electron paramagnetic resonance and spin‐labeling method. Results: The order parameter (S) for the spin label agent (5‐nitroxide stearate) and the peak height ratio (h₀/h_?1) for 16‐nitroxide stearate in the electron paramagnetic resonance spectra of erythrocytes were significantly higher in HT men than in NT men, indicating that membrane fluidity of erythrocytes was decreased in HT men compared with NT men. Both of plasma adiponectin and nitric oxide (NO) metabolite levels were significantly lower in HT men than in NT men. The plasma adiponectin levels were correlated with plasma NO metabolites. The S and the h₀/h_?1 of erythrocytes were inversely correlated with the plasma adiponectin and NO metabolite levels, indicating that the decreased membrane fluidity of erythrocytes was associated with hypoadiponectinemia and reduced plasma NO metabolites. Discussion: The results of the present study demonstrated that plasma adiponectin levels were lower in HT men than in NT men and that hypoadiponectinemia was associated with decreased membrane fluidity of erythrocytes. The finding suggests that adiponectin may be linked to the rheologic behavior of the erythrocytes and the microcirculation in men, at least in part, by the NO‐dependent mechanism.