Refractoriness of cation transport in turkey erythrocytes to stimulation by cyclic adenosine 3':5'-monophosphate.

In turkey erythrocytes bidirectional fluxes of sodium and potassium develop a time-dependent refractoriness to stimulation by endogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP). The refractoriness of potassium influx and potassium outflux (both of which require extracellular sodium and potassium for stimulation by cyclic AMP) depends on the extracellular concentrations of sodium and potassium. In contrast, the refractoriness developed by sodium outflux (which does not require extracellular sodium or potassium for stimulation by cyclic AMP) does not depend on the extracellular concentrations of sodium or potassium. The refractoriness of these fluxes to cellular cyclic AMP reflects a decrease in the amount by which they can be maximally stimulated and appears to be proportional to the extent to which the transport system is utilized during the course of the incubation. Ouabain significantly reduces the rate at which cation transport in turkey erythrocytes becomes refractory to endogenous cyclic AMP. This effect of the glycoside is independent of the extracellular concentrations of sodium or potassium and does not correlate with how it alters the initial response of the transport systems to cyclic AMP.     

The effect of glycophorin A on oxidation of globoside by galactose oxidase

The interaction of galactose oxidase with native and desialylated glycophorin A was studies by oxidizing human erythrocytes and globoside/phospholipid vesicles with the enzyme. Oxidation of the glycolipid was improved in the presence of vesicle-incorporationted glycophorin A. Although galactose oxidase is a very basic protein, it was not adsorbed on native human erythrocytes. Instead, neuraminidase-treated cells bound a substantial amount of galactose oxidase, but the enzyme seemed to be released into the buffer when desialylated glycoproteins had been oxidized.Abbreviation PBS 0.01 M sodium phosphate-0.15 M NaCl, pH 7.4     

Reduced glutathione accelerates the oxidative damage produced by sodium n-propylthiosulfate, one of the causative agents of onion-induced hemolytic anemia in dogs

The oxidative effects of sodium n-propylthiosulfate, one of the causative agents of onion-induced hemolytic anemia in dogs, were investigated in vitro using three types of canine erythrocytes, which are differentiated by the concentration of reduced glutathione and the composition of intracellular cations. After incubation with sodium n-propylthiosulfate, the methemoglobin concentration and Heinz body count in all three types of erythrocytes increased and a decrease in the erythrocyte reduced glutathione concentration was then observed. The erythrocytes containing high concentrations of potassium and reduced glutathione (approximately five times the normal values) were more susceptible to oxidative damage by sodium n-propylthiosulfate than were the normal canine erythrocytes. The susceptibility of the erythrocytes containing high potassium and normal reduced glutathione concentrations was intermediate between those of erythrocytes containing high concentrations of potassium and reduced glutathione and normal canine erythrocytes. In addition, the depletion of erythrocyte reduced glutathione by 1-chloro-2, 4-dinitrobenzene resulted in a marked decrease in the oxidative injury induced by sodium n-propylthiosulfate in erythrocytes containing high concentrations of potassium and reduced glutathione. The generation of superoxide in erythrocytes containing high concentrations of potassium and reduced glutathione was 4.1 times higher than that in normal canine erythrocytes when the cells were incubated with sodium n-propylthiosulfate. These observations indicate that erythrocyte reduced glutathione, which is known as an antioxidant, accelerates the oxidative damage produced by sodium n-propylthiosulfate.     

Carbamylation of proteins leads to alterations in the membrane structure of erythrocytes

The effect of the sodium cyanate-induced carbamylation (carbamoylation) of proteins in erythrocytes was studied using spin labelling and spectrophotometric methods. The experiments were conducted in whole blood and in erythrocytes in phosphate buffer using 25 mmol/L of sodium cyanate. Lipid membrane fluidity was determined using three spin-labelled fatty acids: 5-, 12- and 16-doxylstearic acids (5-DS, 12-DS, 16-DS). Internal viscosity was measured with Tempamine, using also EPR spectroscopy. Osmotic fragility was determined spectrophotometrically. Incubation of whole blood with sodium cyanate led to an increase in lipid membrane fluidity in the deeper region of the lipid layer, indicated by 12- and 16-doxylstearic acid, and a decrease near the surface (5-DS). Statistically significant results were obtained for the internal viscosity and osmotic fragility of erythrocytes. An increase in internal viscosity and increase in osmotic fragility were found in erythrocytes after incubation of whole blood, as well as in erythrocytes incubated with sodium cyanate in buffer. Alterations in internal viscosity were stronger in erythrocytes incubated with sodium cyanate in blood than in erythrocytes in the buffer. On the other hand, higher osmotic fragility was observed for erythrocytes in the buffer.     

Effect of lysophosphatidylcholine on salt permeability through the erythrocyte membrane under haemolytic conditions

Human erythrocytes were incubated in haemolytic salt or sucrose media and the amount of potassium and haemoglobin released were monitored. In hypotonic NaCl and KCl solutions potassium release and haemolysis increased with time showing that the cell membrane had been injured and became permeable to intra- and extracellular cations which, due to intracellular haemoglobin, causes water influx and continuous haemolysis. Both potassium release and haemolysis remained, however, at their 2-minute level in the presence of LPC. Thus, LPC could reseal the membrane and prevent continuous salt fluxes. It protected erythrocytes from hypotonic haemolysis and the protection was more efficient in NaCl than in sucrose media. This suggests that the increase in the critical volume of erythrocytes caused by LPC occurs both in electrolyte and sucrose media, and the additional protection observed in electrolyte media is due to the resealing of the injured cell membrane by LPC. The repairing mechanism was mediated via the membrane lipids or integral proteins, since the time-course of haemolysis of erythrocytes swollen in NaCl media at the spectrin-denaturing temperature of 49.5 degrees C was similar to that at room temperature with and without LPC. LPC did not protect erythrocytes from colloid osmotic haemolysis caused by ammonia influx in an isotonic NH4Cl medium, but protected the cells from colloid osmotic haemolysis caused by sodium influx through nystatin-channels in NaCl media without any area or volume increase. Hence, LPC could not prevent ammonia influx through the lipid bilayer, but suppressed sodium influx through nystatin-channels presumably via LPC interference with cholesterol.     

Gel electrophoretic analysis of histones in lens epithelium, lens fiber, liver, brain, and erythrocytes of late chick embryos.

Histones from 19-day-old chick embryo lens epithelium, lens fibers, liver, brain, and erythrocytes were electrophoresed in polyacrylamide gels using buffers containing sodium dodecylsulfate, acetic acid urea, or mixtures of Triton X-100 acetic acid urea. In the last two buffer systems, histone bands were characterized by their apparent molecular weights determined by electrophoresis in the second dimension in sodium dodecylsulfate containing polyacrylamide gels. From the densitograms of the stained gels, the relative proportion of protein in different histone bands was estimated. With the exception of the erythrocyte-specific histone H5, all histones from different tissues examined at any of the gel systems migrated with the same mobilities. In lens epithelium and lens fibers, all histones were present in identical proportions. As compared to liver and brain, the total amount of histone Hl was significantly lower in lens cells and erythrocytes, possibly reflecting differences between the differentiated states. However, no tissue-specific differences were found in the relative distribution of histone Hl I and Hl II among lens epithelium, lens fiber, liver and, brain, but a threefold higher Hl I : Hl II ratio (0.5--0.7) was found in erythrocytes.     

Effects and mechanisms of action of polyene macrolide antibiotic nystatin on Babesia gibsoni in vitro

Nystatin is a membrane-active polyene macrolide antibiotic and a channel-forming ionophore. Nystatin exhibits in vitro activity against Babesia gibsoni infecting normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes. The calculated IC(50) value of nystatin against B. gibsoni infecting LK erythrocytes was 31.96 μg/ml. The anti-babesial activity of nystatin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations (HK). Moreover, when the parasites were harbored in canine HK erythrocytes, which contained high potassium and low sodium concentrations as a result of high Na-K-ATPase activity, the in vitro anti-babesial activities of nystatin also disappeared, apparently due to protection by HK erythrocytes. This suggested that nystatin could show in vitro anti-babesial activity against B. gibsoni by its ionophorous activity, the same as other ionophores such as valinomycin. Subsequently, the effects of nystatin on the host cells were observed. Nystatin could not modify the intracellular concentrations of potassium, sodium, adenosine triphosphate, or glucose in either LK or HK erythrocytes, although it caused weak hemolysis in HK erythrocytes. In addition, nystatin did not affect the survival of canine peripheral polymorphonuclear leukocytes. In conclusion, nystatin destroyed B. gibsoni by ionophorous activity but did not affect either canine erythrocytes or leukocytes in vitro.     

Gel Electrophoretic Analysis of Histones in Lens Epithelium, Lens Fiber, Liver, Brain, and Erythrocytes of Late Chick Embryos

Histones from 19-day-old chick embryo lens epithelium, lens fibers, liver, brain, and erythrocytes were electrophoresed in polyacrylamide gels using buffers containing sodium dodecylsulfate, acetic acid urea, or mixtures of Triton X-100 acetic acid urea. In the last two buffer systems, histone bands were characterized by their apparent molecular weights determined by electrophoresis in the second dimension in sodium dodecylsulfate containing polyacrylamide gels. From the densitograms of the stained gels, the relative proportion of protein in different histone bands was estimated. With the exception of the erythrocyte-specific histone H5, all histones from different tissues examined in any of the gel systems migrated with the same mobilities. In lens epithelium and lens fibers, all histones were present in identical proportions. As compared to liver and brain, the total amount of histone H1 was significantly lower in lens cells and erythrocytes, possibly reflecting differences between the differentiated states. However, no tissue-specific differences were found in the relative distribution of histone H1 I and H1 II among lens epithelium, lens fiber, liver and, brain, but a threefold higher H1 I: H1 II ratio (0.5–0.7) was found in erythrocytes.     

Nuclear Magnetic Resonance Studies on Intracellular Sodium in Human Erythrocytes and Frog Muscle

The nuclear magnetic resonance (NMR) spectrum of sodium was determined in muscle and erythrocytes using conventional continuous wave techniques. NMR spectra of fresh intact muscle revealed a single line with a width of about 38 Hz equivalent in intensity to about 53% of the total muscle sodium, in general agreement with previous work. Prolonged washing with sodium-free solutions led to a marked loss of both total and NMR-detectable sodium. The NMR-visible sodium remaining in the muscle was somewhat larger than the fraction calculated to remain extracellular and, presumably, was intracellular. The original sodium signal is thus interpreted as arising from both extracellular sodium and the narrow line portion of the signal from intracellular sodium. NMR spectra of sodium were also obtained for human erythrocytes under conditions preserving the sodium transport system. The intensity of the sodium signal in fresh cells was 98% of that present in the same samples after complete hemolysis of the cells. The NMR sodium present in intact cells was 92% of the sodium recovered by flame photometric determination of sodium from ashed samples. It is concluded that no NMR-“invisible” sodium occurs in human erythrocytes and that the presence of such sodium is not necessary for the normal functioning of the sodium transport system in erythrocytes.     

Action of Cortisol on Sodium Transport in Canine Erythrocytes

Incubation of blood from deoxycorticosterone-treated, adrenalectomized dogs with glucose, ²²NaCl, and cortisol, added in vitro, revealed log dose-related acceleration of sodium influx, of glucose utilization, and of lactate formation by cortisol in concentrations between 150 and 1000 µg/liter. Addition of 2-deoxyglucose, or preincubation of the blood until blood glucose concentration had fallen below 2.0 mg per 100 ml, reduced or abolished the acceleratory action of added cortisol on sodium influx but had no effect on sodium influx in the absence of added cortisol. Cortisol did not change the ATP or ATPase content of erythrocytes, or the metabolism of glucose via the pentose phosphate pathway, or the rate of efflux of ²²Na from the erythrocytes. The acceleratory actions of cortisol on sodium, influx, glucose utilization, and lactate formation were significantly correlated. Cortisol (1000 µg/liter) enhanced sodium influx by approximately 8.7 mmole per liter erythrocytes per hour for each 1 mmole cortisol-induced increment in ATP production. It is concluded that sodium influx in canine erythrocytes comprises a passive component, unchanged by cellular metabolism, and a second component which is accelerated and inhibited in proportion to prevailing plasma concentrations of cortisol and aldosterone, and which (for cortisol) depends upon accelerated ATP production via glycolysis. These steroid actions probably result from effects on enzyme activity rather than on new enzyme induction.     

Characteristics of sodium transport in pig (Sus scrofa) erythrocytes

Sodium content, sodium transport (ouabain-sensitive efflux rate of sodium, oMosNa; and ouabain-sensitive efflux rate constant of sodium, oKosNa), [3H]ouabain binding capacity, and Na+, K+-ATPase activity were measured in erythrocytes from young pigs (Sus scrofa). The sodium content, sodium transport, and the number of sodium pumps (assessed by ouabain binding capacity) were lower in the pig compared to the human erythrocytes. The efflux rate constant of sodium, oKosNa was 36% and the ouabain binding capacity was 60% of those in human, suggesting the degree of activation of sodium pump units is much lower.     

Measurement of a wide range of intracellular sodium concentrations in erythrocytes by 23Na nuclear magnetic resonance.

The accuracy of the 23Na nuclear magnetic resonance (NMR) method for measuring the sodium concentration in erythrocytes was tested by comparing the NMR results to those obtained by emission-flame photometry. Comparisons were made on aqueous solutions, hemolysates, gels, ghosts, and intact erythrocytes. The intra- and extracellular 23Na NMR signals were distinguished by addition of the dysprosium tripolyphosphate [Dy(PPP)7-2] shift reagent to the extracellular fluid. The intra- and extracellular volumes of ghosts and cells were determined by the isotope dilution method. Our results indicate that greater than 20% of the intracellular signal remains undetected by NMR in ghosts and cells. When the cells are hemolyzed, the amount of NMR-detectable sodium varies depending on the importance of gel formation. In hemolysates prepared by water addition, the NMR and flame photometry results are identical. The loss of signal in ghosts, cells, and undiluted hemolysates is attributed to partial binding of the Na+ ion to intracellular components, this binding being operative only when these components exist in a gel state. In a second part, 31P NMR was used to monitor the penetration of the shift reagent into the cells during incubation. Our data demonstrate that free Dy3+ can slowly accumulate inside the red cell.     

Characterization of a soluble form of the C3b/C4b receptor (CR1) in human plasma

A radioimmunoassay with the use of soluble 125I-Fab monoclonal anti-CR1 and rabbit IgG anti-CR1 bound to Staphylococcus aureus particles was employed to detect and quantitate CR1 antigen in human plasma. Among 16 normal individuals the concentration of soluble CR1 in plasma ranged from 13 to 81 ng/ml, and a similar range of concentration was found in plasma from 15 patients having systemic lupus erythematosus (SLE). The amount of plasma CR1 in normal donors, but not in SLE patients, significantly correlated with the number of CR1 sites on erythrocytes (r = 0.90, p less than 0.001), and was 7.1% of the amount of receptor that was present on erythrocytes in blood. The concentration of soluble CR1 was not diminished by ultracentrifugation or ultrafiltration of plasma, was not affected by various modes of anti-coagulation or even by clotting of blood, and did not change during incubation of blood at 4 degrees C for up to 4 hr. On sucrose density gradient ultracentrifugation of plasma the CR1 was distributed as a broad peak that overlapped the plasma protein profile. The Mr of plasma CR1 was identical to that of erythrocyte CR1 when assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and immunoblotting. In addition, the plasma form of CR1 exhibited the same structural phenotype as did receptor from erythrocytes of the same individual. CR1 antigen purified from plasma was as active as CR1 from erythrocytes in promoting the cleavage by factor I of C3b to iC3b, C3c, and C3dg. Therefore, a functionally and structurally intact form of soluble CR1 resides in plasma.     

Effects and mechanisms of action of ionophorous antibiotics valinomycin and salinomycin-Na on Babesia gibsoni in vitro

Valinomycin and salinomycin-Na, 2 ionophorous antibiotics, exhibited in vitro antibabesial activities against Babesia gibsoni that infected normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes, which completely lack Na,K-ATPase activity. The level of parasitemia of B. gibsoni was significantly decreased when the parasites were incubated in culture medium containing either 10(-1) ng/ml valinomycin or 10(2) ng/ml salinomycin-Na for 24 hr. Four-hour incubation in the culture medium containing 5 μg/ml salinomycin-Na led to the destruction of most parasites. In contrast, when the parasites infected canine erythrocytes containing high potassium (HK) and low sodium concentrations, i.e., HK erythrocytes, the in vitro antibabesial activities of both ionophorous antibiotics seemed to be weakened, apparently due to the protection by the host cells. Therefore, differential influences of ionophorous antibiotics on LK and HK erythrocytes were observed. In LK erythrocytes, the intracellular concentrations of potassium, sodium, and adenosine triphosphate (ATP) were not modified, and hemolysis was not observed after incubation in the medium containing each ionophorous antibiotic. These results suggested that these ionophorous antibiotics did not affect cells without Na,K-ATPase, and directly affected B. gibsoni. In HK erythrocytes, the ionophorous antibiotics increased the intracellular sodium concentration, and decreased the intracellular potassium and ATP concentrations, causing obvious hemolysis. Additionally, the decrease of the intracellular ATP concentration and the hemolysis in HK erythrocytes caused by valinomycin disappeared when the activity of Na,K-ATPase was inhibited by ouabain. These results indicate that modification of the intracellular cation concentrations by the ionophorous antibiotics led to the activation of Na,K-ATPase and increased consumption of intracellular ATP, and that the depletion of intracellular ATP resulted in hemolysis in HK erythrocytes. Moreover, the antibabesial activity of valinomycin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations. This showed that the intracellular cation concentration in the parasites was not modified in those media and would remain the same.     

The effect of nitroxyl radicals and sodium nitrite on the methemoglobin content of the blood and on the enzymatic activity of erythrocyte antioxidant protection]

Preliminary introduction of nitroxyl radicals to mice decreases methaemoglobin-forming effect of sodium nitrite and diminishes the content of total SH-groups and restored glutathione as well as the activity of glutathione reductase and total activity of dehydrogenases of the pentosophosphate path of erythrocytes. High level of lipids peroxidation in case of sodium nitrite intoxication remains unchanged under preliminary administration of nitroxyl radicals as well. Activity of the key enzymes of antioxidant protection of erythrocytes, superoxide dismutase and catalase, is not recovered with sodium nitrite intoxication in presence of nitroxyl radicals.     

Activation of sodium transport in human erythrocytes by beta-adrenoceptor stimulation in vivo

Beta-adrenoceptor stimulation in vivo shifts potassium into the cells. To examine whether human erythrocytes participate in this process, we measured, along with serum or plasma potassium, the concentrations of potassium and sodium in erythrocytes. Beta-adrenoceptor stimulation was obtained by infusion of either fenoterol or hexoprenaline into 6 volunteers at rest or by endogenous amines provoked in 14 volunteers during ergometric exercise. Metabolic effects were followed at rest on serum insulin, C-peptide, and growth hormone levels, and during exercise on pH on lactate concentration in blood. The potassium concentration (mean +/- S.E.M.) dropped (p less than 0.01) in serum from 4.64 +/- 0.37 to 3.19 +/- 0.43 mmol x l-1 in the first hour at rest and in plasma from 5.70 +/- 0.93 to 4.63 +/- 0.45 in 90 sec directly after exercise. The concentration of erythrocyte sodium dropped (p less than 0.001) from 9.68 +/- 0.73 to 8.81 +/- 0.62 mmol x l-1 in cells and from 9.62 +/- 1.16 to 8.55 +/- 1.24 during exercise for 90 s, respectively. Changes in the concentration ratio of cellular sodium to potassium confirmed this sodium shift. An increased sodium transport in erythrocytes due to beta-adrenoceptor stimulation in vivo appears to complement a shift of serum potassium into the cells and may be mediated by the membrane-bound sodium, potassium ATPase.     

A simple purification procedure for monomeric nucleosomes.

The soluble chromatin fragments from nuclei of avian erythrocytes digested with micrococcal nuclease were fractionated by the addition of sodium phosphate to 0.1 m. The supernatant consisted predominantly of monomeric nucleosomes, while most dimeric and larger nucleosomes precipitated. A variable percentage of monomers also precipitated, the exact amount depending upon the extent of digestion. The solubility properties can be used for the simple preparation of fractions that are highly enriched for monomers either containing, or deplete in, lysine-rich histone.     

大鼠红细胞作为SOD新型载体的细胞水平上的研究

用低渗透析 -等渗重封的方法制备了包埋超氧化物歧化酶 ( SOD)的大鼠载体红细胞 ,并从细胞水平上研究了透析条件对大鼠红细胞包埋 SOD的影响与载体红细胞的部分性质 .流式细胞计( FCM)研究表明 ,随透析时间延长和透析液渗透压降低 ,包埋 SOD的载体红细胞百分率升高 ,但载体细胞平均包埋 SOD的量无明显变化 ;SOD浓度对载体细胞百分率无明显影响 ,但与载体细胞平均包埋 SOD的量成线性关系 ;载体红细胞前向角散射 ( FLS)明显下降 ,但显微镜下观察到的载体细胞的大小无明显变化 ,当载体细胞反注射到大鼠体内后 FLS能迅速恢复 ;载体红细胞密度下降 ,其原因是低渗透析时红细胞膨胀未能完全恢复 ;载体红细胞未暴露与自身 Ig G结合的抗原位点 .激光扫描共聚焦显微镜 ( LSCM)分析表明 ,SOD在细胞内呈从细胞中心到细胞膜浓度逐步下降的辐射分布特征 .     

Role of erythrocyte membrane lipids in the antagonism between vitamin A and D3

The "in vitro" lytic effect of retinol (35 microM) on various animal species erythrocytes is partially inhibited by cholecalcipherol. The inhibition observed is not related with reduction of retinol bound by erythrocytes membranes while it can be related with the amount of glycolipids not gangliosides of these cells. Instead, we have observed that the amount of retinol molecules bound to erythrocytes membranes and their haemolysis can be related with the gangliosides contents of red cells membranes.     

Intracellular location of polyamines associated to red blood cells

The polyamines associated to human erythrocytes from healthy donors are mainly localized intracellularly. In fact chromatography of the erythrocytes on a resin which has a high affinity and capacity for polyamines does not affect the amount of polyamines associated to the erythrocytes. The low ability of spermine to adsorb to the external surface of erythrocytes at physiological ionic strength is suggested also by studies conducted with sealed ghosts. Also erythrocytes from patients with hematological and dermatological diseases which contain increased levels of polyamines show an intracellular location of these amines.