Erythrocyte Membrane Function Research Articles

Plasma lipids and physicochemical properties of the erythrocyte plasma membrane throughout pregnancy.

The physiopathological relevance of plasma lipid concentrations is supported by the observation that they might affect the physicochemical properties of the plasma membrane of circulating cells and might be crucial in the pathological conditions complicating pregnancy. Plasma and erythrocyte membrane lipid composition, membrane fluidity and function [membrane-bound enzyme sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) activity] were studied in 24 healthy women in the nonpregnant condition and at 12, 24 and 38 weeks' gestation. The plasma showed an increase in total and high density lipoprotein (HDL) cholesterol, triglyceride (TG) and phospholipid (PL) levels. In the erythrocyte membrane we found the cholesterol/phospholipid (C/PL) ratio increased and the saturated to unsaturated fatty acid (FA) ratio (Sat/Unsat) significantly reduced; fluorescence polarization showed an initial increase during pregnancy and a progressive decrease afterwards; Na+/K+-ATPase activity was progressively reduced. A generalized alteration in the composition and function of the maternal erythrocyte membrane is present during a physiological pregnancy, and these modifications may involve a more complex physiopathogenetic mechanism.

Plasma lipids and physicochemical properties of the erythrocyte plasma membrane throughout pregnancy

Background. The physiopathological relevance of plasma lipid concentrations is supported by the observation that they might affect the physicochemical properties of the plasma membrane of circulating cells and might be crucial in the pathological conditions complicating pregnancy. Methods. Plasma and erythrocyte membrane lipid composition, membrane fluidity and function [membrane-bound enzyme sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) activity] were studied in 24 healthy women in the nonpregnant condition and at 12, 24 and 38 weeks' gestation. Results. The plasma showed an increase in total and high density lipoprotein (HDL) cholesterol, triglyceride (TG) and phospholipid (PL) levels. In the erythrocyte membrane we found the cholesterol/phospholipid (C/PL) ratio increased and the saturated to unsaturated fatty acid (FA) ratio (Sat/Unsat) significantly reduced; fluorescence polarization showed an initial increase during pregnancy and a progressive decrease afterwards; Na+/K+-ATPase activity was progressively reduced. Conclusions. A generalized alteration in the composition and function of the maternal erythrocyte membrane is present during a physiological pregnancy, and these modifications may involve a more complex physiopathogenetic mechanism.

Effect of L-Carnitine and Palmitoyl-L-Carnitine on Erythroid Colony Formation in Fetal Mouse Liver Cell Culture

The administration of L-carnitine to patients undergoing hemodialysis increases hematocrit and improves the response to recombinant human erythropoietin (rhEPO). This suggests a contribution by carnitine to erythrocyte membrane function or erythropoiesis. We investigated the effect of L-carnitine and palmitoyl-L-carnitine on erythropoiesis by assessing erythroid colony formation in in vitro fetal mouse liver cell cultures. L-Carnitine or palmitoyl-L-carnitine was added with rhEPO to fetal mouse liver cell cultures. Doses of L-carnitine of up to 200 µmol/l to the culture had no effect on colony formation. In contrast, the addition of above 12.5 µmol/l palmitoyl-L-carnitine into the culture increased colony formation significantly. These results suggest that long-chain acyl carnitine may have an effect on erythropoiesis.

Effects of La3+ on lipid fluidity and structural transitions in human erythrocyte membranes.

The effects of La3+ on the structure and function of human erythrocyte membranes were investigated by fluorescence polarization, spin-labeled electron spin resonance (ESR) and differential scanning calorimetry (DSC). The results showed that increasing concentrations of La3+ inhibited (Na(+) + K+)-ATPase and Mg(2+)-ATPase activities. La3+ lowered the lipid fluidity of erythrocyte membranes and induced structural transitions in erythrocyte membranes.

Abnormalities in Adenosine Triphosphatase of the Erythrocyte Membrane in Iron Deficiency Anaemia

During iron deficiency rat and human erythrocyte membrane enzyme activities (Total ATPase and Ouabainsensitive Na+.K+ ATPase) showed significant (P < 0.001) decrease. The influence of iron deficiency on erythrocyte Na+ and K+ was also studied in rats and humans. The former parameter showed a significant (P < 0.01) increase while the latter showed a downward trend. Plasma Fe and Total Iron Binding capacity (TIBC) in iron deficiency varied significantly (P < 0.05) from normal values. These results suggest a defect in erythrocyte membrane function and a possible potentiating effect of intracellular Na+, plasma Fe and TIBC on ATPase activity in iron deficiency. Values obtained for rats and humans showed differences in the activities of membrane ATPase in iron deficiency anaemia.

Thiol group control of sodium-lithium countertransport kinetics in uraemia: evidence of a membrane abnormality affected by haemodialysis.

Uraemia affects erythrocyte metabolism and membrane function but no consistent effect on Na/Li countertransport (CT) has been reported. We report only small differences in Na/Li CT at 150 mmol/l Na over haemodialysis, but major differences in other properties of Na/Li CT. The Km for external sodium and Vmax both increased during haemodialysis but the Vmax/Km ratio, which was greater than normal, was not affected. The thiol reagent, N-ethylmaleimide (NEM), which causes a decrease in Km and Vmax in normal subjects, had no effect on Km in the predialysis erythrocytes. After haemodialysis, the sensitivity of Na/Li CT to NEM was improved. The changes in Na/Li CT kinetics were not related to changes in membrane lipid fluidity or plasma lipids. These observations suggest that uraemia affects a thiol group that controls Na/Li CT kinetics and that haemodialysis temporarily improves this aspect of membrane function.

Modulation of Erythrocyte Membrane Mechanical Function by β-Spectrin Phosphorylation and Dephosphorylation

The mechanical properties of human erythrocyte membrane are largely regulated by submembranous protein skeleton whose principal components are alpha- and beta-spectrin, actin, protein 4.1, adducin, and dematin. All of these proteins, except for actin, are phosphorylated by various kinases present in the erythrocyte. In vitro studies with purified skeletal proteins and various kinases has shown that while phosphorylation of these proteins can modify some of the binary and ternary protein interactions, it has no effect on certain other interactions between these proteins. Most importantly, at present there is no direct evidence that phosphorylation of skeletal protein(s) alters the function of the intact membrane. To explore this critical issue, we have developed experimental strategies to determine the functional consequences of phosphorylation of beta-spectrin on mechanical properties of intact erythrocyte membrane. We have been able to document that membrane mechanical stability is exquisitely regulated by phosphorylation of beta-spectrin by membrane-bound casein kinase I. Increased phosphorylation of beta-spectrin decreases membrane mechanical stability while decreased phosphorylation increases membrane mechanical stability. Our data for the first time demonstrate that phosphorylation of a skeletal protein in situ can modulate physiological function of native erythrocyte membrane.

Effect of alpha-interferon on erythrocyte and hepatocyte plasma membranes derived from cirrhotic rats.

Interferons are recognized to inhibit collagen production. Since fibrosis has been associated with liver dysfunction, we investigated the effects of alpha-interferon on the function and lipid composition of hepatocyte and erythrocyte plasma membranes derived from CCl4-cirrhotic male Wistar rats. In both cell types, CCl4 decreased Na+/K+ and Ca(2+)-ATPase activity and increased the cholesterol to phospholipids (CH/PL) ratio (p < 0.05). Administration of interferon (80,000 IU/kg s.c. for 8 weeks) increased survival from 40 to 90%, and preserved normal ATPase activity and CH/PL ratio. Our results show that administration of alpha-interferon to CCl4-cirrhotic rats improves survival, and liver and erythrocyte membrane function and composition, probably as a result of its antifibrogenic effect.

Erythrocyte sodium lithium countertransport in normal and hypertensive pregnancy: relation to haemodynamic changes.

To establish the changes in erythrocyte sodium lithium countertransport (SLC) with advancing normal pregnancy and to determine if these changes were different in pregnancy induced hypertension (PIH). The changes in both groups were assessed in relation to haemodynamic changes. SLC, mean arterial pressure (MAP), cardiac output (CO) and total peripheral vascular resistance (TPVR) were determined serially during normal pregnancy and cross-sectionally in PIH. Women were studied again 20 weeks after delivery where possible. Routine antenatal clinic and antenatal ward of a regional reference centre. Fifty-one normal primigravid women were studied serially and 41 primigravid women with PIH were studied at time of diagnosis. During normal pregnancy SLC (mmol Li/h/l cells) increased from a nonpregnant value of 0.24 +/- 0.02 (mean +/- SEM) to 0.32 +/- 0.02 at 14 weeks, and 0.37 +/- 0.02 at 20 weeks gestation. This was maintained until 38 weeks (0.40 +/- 0.02). The increase until 20 weeks occurred at the time of greatest change in CO (5.10 +/- 0.18 to 6.79 +/- 0.20 l/min) and TPVR (1327 +/- 58 to 969 +/- 33 dyn/s/cm-5). The decrease in TPVR with a rise in SLC is opposite to the relation reported in essential hypertension so that a functional relation is unlikely. However, the changes within pregnancy were positively correlated (r = 0.43, P < 0.01). In hypertensive pregnancies TPVR was elevated compared with normotensive pregnancies (1543 +/- 100 vs 1090 +/- 37) but the SLC was not different from that found in normotensive pregnancies (0.43 +/- 0.02 vs 0.40 +/- 0.02). The changes in SLC activity suggest dynamic effects on erythrocyte membrane function during pregnancy. However, no differences could be found between normal and hypertensive pregnancy and SLC is unlikely to be of value as a marker of hypertensive risk during pregnancy.

Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. I. Impairment of resealing and formation of aqueous pores in the ghost membrane after modification of SH groups

Resealed human erythrocyte ghosts prepared by a two-step procedure were shown to have small residual barrier defects with the properties of aqueous pores, such as size discrimination of hydrophilic nonelectrolytes (erythritol to sucrose), indicative of an apparent pore radius of about 0.7 nm, and a low activation energy (about 12–20 kJ/mol (mannito, sucrose)) of the leak fluxes. As in other cases (Deuticke et al. (1991) Biochim. Biophys. Acta 1067, 111–122) these leak fluxes can be inhibited by phloretin. Treatment of such resealed ghosts with the mild SH oxidizing agent, diamide, induces additional membrane leaks to the same extent and with the same properties as in native erythrocytes (Deuticke et al. (1983) Biochim. Biophys. Acta 731, 196–210), including reversibility of the leak by SH reducing agents, inhibition by phloretin and stimulation by alkanols. In contrast, resealed ghosts prepared either from diamide-treated erythrocytes or by adding diamide to the ‘open’ membranes prior to reconstitution of high ionic strenght and raising the temperature, exhibit a state of greater leakiness. This leakiness is somewhat different in its origin from the former class of leaks, since it can also be produced by N-ethylmaleimide, which is essentially ineffective when added to the membrane in its ‘tight’ state. The leaks induced in the ‘open’ state of the membrane, which can be regarded as a consequence of an impaired resealing, are nevertheless reversible by reducing agents added after resealing and are comparable in many, but not all their characteristics to leaks induced in the ‘tight’ state of the membrane. Resealing in the presence of the isothiocyanostilbenes DIDS or SITS mimicks the leak forming effect of diamide by modifying a small population of SH groups, while amino groups seem not to be involved. The findings indicate and substantiate an important role of the redox state of membrane skeletal protein sulfhydryls in the maintenance and the re-establishment of the barrier function of the erythrocyte membrane.

Human erythrocyte protein 4.2, a high copy number membrane protein, is N-myristylated.

Band 4.2 is a major protein of the erythrocyte membrane which has been immunologically detected in a variety of cell types and is apparently essential for normal erythrocyte membrane function. Since band 4.2 has unusual solubility and membrane binding properties and has an N-terminal glycine following the initiating methionine, we explored the possibility that band 4.2 is myristylated. When Sf9 cells infected with a recombinant band 4.2 Baculovirus were incubated with [3H]myristic acid, label became incorporated into recombinant band 4.2 protein and resisted extraction with hydroxylamine. Consistent with N-terminal myristylation, the incorporation of label was dependent upon protein synthesis. The fatty acid covalently bound to recombinant band 4.2 was definitively identified as myristic acid by recovering the fatty acid after hydrolysis of band 4.2 and examining its migration relative to standards in thin layer chromatography. It was determined that native erythrocyte band 4.2 is an N-myristylated protein by reverse phase high performance liquid chromatography detection of an azlactone derivative of N-myristylglycine after mild acid hydrolysis and azlactone derivatization of the purified protein. Study of myristylation of band 4.2, an abundant normal cellular protein, and its role in membrane binding may produce insights relevant to other myristylated cellular proteins.

Calcium-stressed erythrocyte membrane structure and function for assessing glipizide effects on transglutaminase activation.

A proposed mechanism of action of hypoglycemic sulfonylureas is the prevention of transglutaminase-mediated endocytosis of insulin receptors. When activated by high levels of intracellular calcium, transglutaminase (TG) catalyzes the cross-linking of intracellular proteins to membrane proteins and modifies membrane structure and function. This study examined the effects of the sulfonylurea glipizide on TG activity in an erythrocyte model by assessing various membrane ATPase activities and high molecular weight protein polymer formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To activate TG, red blood cells were exposed to 1 mM intracellular Ca2+ using 10(-5) M Ca2(+)-ionophore A23187. In Ca2(+)-stressed cells, calmodulin stimulation (0.1 micrograms/ml) of (Ca2+ + Mg2+)-ATPase was decreased to 21.2% of control activity. Increasing concentrations of calmodulin (0.1-3.0 micrograms/ml) could not overcome the inhibitory effects of TG on the (Ca2+ + Mg2+)-ATPase in Ca2(+)-stressed cells with or without glipizide. An increased Ca2+ sensitivity of calmodulin-independent (Ca2+ + Mg2+)-ATPase due to Ca2+ stress was seen in all Ca2(+)-stressed cells even in the presence of 1 mM glipizide. Structural changes were observed in the form of high molecular weight polymer formation. Cells exposed to high Ca2+ and glipizide (3 x 10(-5)-10(-3) M) showed no improvement in ATPase activity or protection from protein cross-linking compared with cells without the drug. We conclude that in this model glipizide fails to inhibit TG induced protein cross-linking and does not prevent the decrease in (Ca2+ + Mg2+)-ATPase activation in Ca2(+)-stressed red blood cells. This finding considerably weakens the proposal that sulfonylureas act by inhibiting TG activity.

Riboflavin Deficiency and the Function and Fluidity of Rat Erythrocyte Membranes

The effect of riboflavin deficiency on the fluidity and function of the red blood cell (RBC) membrane and on the activity of some enzymes involved in antioxidant defense mechanisms was studied. Growing male rats were fed an experimental (riboflavin-deficient) or a control (riboflavin-supplemented) diet. Following 7 wk of feeding, RBC from riboflavin-deficient rats contained higher levels of peroxidation products, most likely due to decreased glutathione reductase activity. An elevation in glutathione peroxidase activity was also observed whereas the activity of catalase and superoxide dismutase was not affected. Membrane fluidity was studied by fluorescence polarization, using 1,6-diphenyl-1,3,5 hexatriene (DPH) as a probe. The fluidity of RBC membranes isolated from riboflavin-deficient rats was significantly lower than that of the controls. This decreased fluidity was accompanied by an increase in the activity of the membrane-bound enzyme acetylcholinesterase. This study demonstrated that a decrease in cells' ability to cope with peroxidative damage as a result of riboflavin deficiency may lead to changes in the fluidity and function of membranes.

Excess of red cell membrane proteins in hereditary high‐phosphatidylcholine hemolytic anemia

Previous descriptions of hereditary high-phosphatidylcholine hemolytic anemia (HPCHA) have highlighted the association of increased erythrocyte membrane phosphatidylcholine with abnormal membrane cation permeability. We studied the function and composition of erythrocyte membranes from three individuals with HPCHA to characterize further the membrane abnormalities in this disorder. Despite significant macrocytosis, HPCHA red cells were dehydrated and showed an increased surface area to volume ratio compared to normal red cells. The passive efflux of K+ from HPCHA erythrocytes was increased fourfold at 37 degrees C. Total membrane phospholipid was increased 7-42%, largely due to excess phosphatidylcholine, which made up 35.8-37.2% of total phospholipid. Membrane cholesterol:phospholipid ratios were in the normal range. It appears that the excess phosphatidylcholine was not acquired during circulation, since plasma lipids were normal and all subpopulations of density-separated HPCHA erythrocytes were similarly abnormal. The ratio of total protein to phospholipid in white ghosts was increased, indicating that membrane protein was increased to an even greater extent than membrane lipids. No abnormal membrane proteins were identified by Coomassie or periodic acid Schiff (PAS) staining. Quantitation of the major membrane proteins indicated that the total protein excess in HPCHA membranes was due to a proportional increase in all major proteins. We conclude that HPCHA erythrocytes have excess membrane proteins and hypothesize that the changes in lipid composition and cation permeability are secondary to underlying protein abnormalities, which remain to be defined.

Effects of gossypol on the activity of kidney ( Na+ + K+)-ATPase and the functions of erythrocyte membrane

Gossypol extracted from cottonseed oil, reputed to exert contraceptive action in males, may induce a side effect of hypokalemia. The cellular and molecular mechanisms by which gossypol produces hypokalemia are not quite understood. We have examined the inhibitory effect of gossypol on the activity of ( Na + + K +)-ATPase purified from the outer medulla of rabbit kidney, the half maximal inhibitory concentration (IC 50) is 6.5 μm. The kinetic studies using this enzyme preparation show that gossypol is noncompetitive with ATP, Mg 2+, Na + and K + with apparent Ki of 15.0, 13.0, 14.0 and 4.4 μm, respectively. On the other hand, in order to estimate the effects of gossypol on membrane transport of intact cells, we have investigated the effects of gossypol on the activity of ( Na + + K +)-ATPase, membrane integrity and permeability of human erythrocytes. It shows that gossypol inhibited the enzyme activity (<5 μm) and expressed the hemolysis (<50 μm) in vitro in a concentration-dependent manner, and increased the K +-efflux of the cells (10–40 μm). The above effects are antagonized by 1–2% bovine serum albumin. The data demonstrate that gossypol is a specific and potent membrane active agent. These results may be relevant to the in vivo actions of gossypol.

Non-Insulin Dependent Diabetes Mellitus (NIDD) Has Different Effects on Erythrocyte (RBC) Membrane Function in Hypertensives (HT) Compared with Normotensives (NT)

Non-Insulin Dependent Diabetes Mellitus (NIDD) Has Different Effects on Erythrocyte (RBC) Membrane Function in Hypertensives (HT) Compared with Normotensives (NT)

Erythrocyte membrane: structure, function, and pathophysiology.

Erythrocytes are unique among mammalian cells. They contain no nucleus or subcellular metabolic structures, yet they survive for 3 to 7 rn~nths .~ ' During their lifetime, erythrocytes travel thousands of miles through tubes of various sizes while delivering oxygen to the tissues.2o Survival of practically all other cells in the body depends upon the proper functioning of erythrocytes. They travel as individual cells, containing a concentrated solution of hemoglobin surrounded by a membrane. In spite of their apparent simplicity, they have limited (but necessary) metabolic capability. They are exposed to a high oxygen content and come in contact with a variety of endogenous and exogenous chemicals. Their ability to survive depends on the ability of the membrane to remain intact and flexible.20 This paper focuses on the erythrocyte membrane structure and function and the pathophysiological changes that can occur when the membrane is exposed to an unfavorable environment.

Effect of sodium fluoride — On erythrocyte membrane function — With reference to metal ion transport in rabbits

Effect of NaF on membrane function was examined with reference to metal ion transport in rabbit's erythrocytes. Activities of membranal enzymes [Na + + K + − ATPase & Mg ++ - ATPase], plasma enzymes [Acid and Alkaline Phosphatases] and concentrations of Ca, Mg, Na and K were estimated in erythrocyte membrane, hemolysate and plasma. A 17% decrease in Na + + K + − ATPase and 37% increase in Mg ++ - ATPase have been observed after fluoride ingestion. Plasma, Acid and Alkaline Phosphatases are reduced by 27% and 34% respectively. Fluoride in circulation and in erythrocyte membrane do not effect the concentration of Na and K, however, Ca and Mg are significantly altered in erythrocyte membrane, hemolysate and plasma. It is concluded that fluoride is affecting the normal erythrocyte membrane function.

Investigation of membrane proteins in rat erythrocytes in spontaneous hypertension by means of spin-label technique.

It has been suggested that alterations in cell membrane proteins may play a role in changes of erythrocyte membrane structure and function in hypertension. In order to characterize the structure of membrane proteins of erythrocytes from spontaneously hypertensive rats (SHR) the spin-label technique with a maleimide spin-label was used. A significant difference was observed in the characteristic electron-spin resonance (e.s.r.) spectrum of the label between samples from normotensive rats and SHR. The difference was eliminated and the spectrum significantly changed after treatment of the labelled membrane with EDTA followed by washing out the EDTA extracts, whereas the same treatment with EDTA without the following washing had no effect on the e.s.r. spectrum. It is concluded that the EDTA extracts different substances in the different rat groups. The spin-label technique is a useful method for distinguishing cell membrane properties in SHR and normotensive rats.

Protein kinase C in the human erythrocyte. Translocation to the plasma membrane and phosphorylation of bands 4.1 and 4.9 and other membrane proteins.

Protein kinase C (PK-C) was demonstrated in human erythrocytes using the exogenous substrate histone H1. The enzyme was dependent on the simultaneous presence of micromolar Ca2+ and phosphatidylserine. The Ca2+ requirement was reduced in the presence of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Activity was normally recovered in cytoplasmic extracts, but treatment of intact cells with TPA (EC50 = 40 nM) prior to lysis caused a rapid translocation of activity from the cytoplasm to the membrane. Following translocation, PK-C activity in the isolated membrane was independent of Ca2+ and phosphatidylserine and could not be removed by manipulating ionic strength. Ghosts from TPA-treated cells showed a marked increase in the phosphorylation of five proteins (termed PK-C-1-5) of Mr 120,000, 110,000, 80,000, 78,000, and 49,000. Addition of purified bovine brain PK-C to ghosts from untreated cells resulted in the phosphorylation of the same five proteins. PK-C-3/4 corresponded to Band 4.1 and PK-C-5 to Band 4.9. Both proteins were isolated from ghosts and shown to be substrates for PK-C in vitro. PK-C-1 and -2 appear to be minor peripheral membrane proteins as both were released from the membrane by alkaline solutions. Incubation of 32P-prelabeled erythrocytes with TPA (EC50 = 40 nM) also resulted in a dose-dependent phosphorylation of PK-C-1-5. These results suggest that PK-C may play an important role in erythrocyte membrane function.