Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.     

Rheological Properties of Blood in Athletes

Rheological properties of blood were studied in subjects with high physical activity (athletes). It was found that the whole blood viscosity decreased under conditions of relative rest. The use of the concept of hemorheological profiles made it possible to reveal different profiles in subjects exposed to different courses of training exercise. The relationship between the rheological properties of blood and the total work capacity in humans was established. The typical parameter of profiles in subjects with high physical activity was high erythrocyte deformability associated mainly with erythrocyte form and viscoelastic properties of its membrane.     

杜仲绿原酸对高脂小鼠血液流变学作用研究

旨以研究杜仲绿原酸对高脂高胆固醇诱导的高血脂模型小鼠血液流变学的影响,以昆明小鼠为实验动物,随机分成5组:阴性对照组,模型对照组和低剂量(25 mg/kg BW)、中剂量(50 mg/kg BW)、高剂量(100 mg/kg BW)杜仲绿原酸组,每组10只.后4组饲以高脂饲粮,同时小鼠灌胃杜仲绿原酸4周,实验结束,分别测定各组小鼠血液流变学参数、血清和肝脏的抗氧化酶活性和脂质过氧化产物MDA含量及其总抗氧化能力和羟自由基清除率.高脂血症小鼠的全血粘度、血浆粘度、红细胞压积、血沉、纤维蛋白原、红细胞刚性指数和聚集指数显著降低(P＜0.05),红细胞变形指数显著提高(P＜0.05),小鼠血清和肝脏SOD、GSH-Px水平、总抗氧化能力和羟自由基清除能力均显著升高(P＜0.05),MDA水平显著降低(P＜0.05).在高脂膳食条件下,杜仲绿原酸能有效提高血液的抗氧化防御功能(包括抗氧化力、抗氧化酶活性)、改变血液流变学参数等,降低血液粘度、红细胞刚性和聚集,增强变形能力,使细胞膜的流动性增高,其中以中剂量效果相对较好.     

The Rho kinase inhibitor Y-27632 increases erythrocyte deformability and low oxygen tension-induced ATP release

Low oxygen (O(2)) tension and mechanical deformation are stimuli for ATP release from erythrocytes. It has been shown previously that rabbit erythrocytes made less deformable with diamide, a thiol cross-linking agent, release less ATP in response to low O(2) tension, suggesting a link between these two stimuli. In nonerythroid cells, activation of the Rho/Rho kinase signaling pathway has been reported to decrease cell deformability by altering Rho kinase-dependent cytoskeleton-protein interactions. We investigated the hypothesis that the Rho kinase inhibitor Y-27632 would increase erythrocyte deformability and thereby increase low O(2) tension-induced ATP release from erythrocytes. Here we show that Y-27632 (1 μM) increases erythrocyte deformability (5%) and increases low O(2) tension-induced ATP release (203%) from healthy human erythrocytes. In addition, we found that, when erythrocytes were made less deformable by incubation with diamide (100 μM), Y-27632 restored both deformability and low O(2) tension-induced ATP release to levels similar to those measured in the absence of diamide. These findings suggest that the Rho kinase inhibitor Y-27632 is able to reverse the diamide-induced decrease in erythrocyte deformability and rescue low O(2) tension-induced ATP release. These results further support a link between erythrocyte deformability and ATP release in response to low O(2) tension.     

Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX

Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, which may result from treatment with the Ca2+ ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.     

Spin label study of erythrocyte deformability. Ca2+-induced loss of deformability and the effects of stomatocytogenic reagents on the deformability loss in human erythrocytes in shear flow

The Ca2+-induced loss of deformability in human erythrocytes and the recovery of the lost deformability by stomatocytogenic reagents were investigated by means of a new flow electron paramagnetic resonance (EPR) spin label method, which provides information on deformation and orientation characteristics of spin labeled erythrocytes in shear flow. The Ca2+-induced loss of deformability is attributed mainly to the increase in intracellular viscosity resulting from efflux of intracellular potassium ions and water (Gardos effect). Partial recovery of the lost deformability is demonstrated in the presence of stomatocytogenic reagents, such as chlorpromazine, trifluoperazine, W-7, and calmidazolium (R24571). The recovery can not be explained solely by suppression of the Gardos effect due to the reagents. Incorporation of an optimal amount of the reagents into the membrane appears to compensate for the membrane modification due to Ca2+ ions to restore a part of the lost deformability.     

Effects of Interval and Continuous Exercise Training on CD4 Lymphocyte Apoptotic and Autophagic Responses to Hypoxic Stress in Sedentary Men

Exercise is linked with the type/intensity-dependent adaptive immune responses, whereas hypoxic stress facilitates the programmed death of CD4 lymphocytes. This study investigated how high intensity-interval (HIT) and moderate intensity-continuous (MCT) exercise training influence hypoxia-induced apoptosis and autophagy of CD4 lymphocytes in sedentary men. Thirty healthy sedentary males were randomized to engage either HIT (3-minute intervals at 40% and 80%VO_2max, n=10) or MCT (sustained 60%VO_2max, n=10) for 30 minutes/day, 5 days/week for 5 weeks, or to a control group that did not received exercise intervention (CTL, n=10). CD4 lymphocyte apoptotic and autophagic responses to hypoxic exercise (HE, 100W under 12%O₂ for 30 minutes) were determined before and after various regimens. The results demonstrated that HIT exhibited higher enhancements of pulmonary ventilation, cardiac output, and VO₂ at ventilatory threshold and peak performance than MCT did. Before the intervention, HE significantly down-regulated autophagy by decreased beclin-1, Atg-1, LC3-II, Atg-12, and LAMP-2 expressions and acridine orange staining, and simultaneously enhanced apoptosis by increased phospho-Bcl-2 and active caspase-9/-3 levels and phosphotidylserine exposure in CD4 lymphocytes. However, five weeks of HIT and MCT, but not CTL, reduced the extents of declined autophagy and potentiated apoptosis in CD4 lymphocytes caused by HE. Furthermore, both HIT and MCT regimens manifestly lowered plasma myeloperoxidase and interleukin-4 levels and elevated the ratio of interleukin-4 to interferon-γ at rest and following HE. Therefore, we conclude that HIT is superior to MCT for enhancing aerobic fitness. Moreover, either HIT or MCT effectively depresses apoptosis and promotes autophagy in CD4 lymphocytes and is accompanied by increased interleukin-4/interferon-γ ratio and decreased peroxide production during HE.     

Effect of penicillin G-induced epileptic seizures on hemorheological parameters in rats

Normally, cerebral blood flow (CBF) is quantitatively coupled to cerebral metabolic rate like other tissues and maintained basically by altering vascular geometry and appropriate perfusion pressure. However, the rheological properties of the blood are important factors for effective tissue perfusion. Although a lot of studies have reported that hemorheological parameters are affected by a wide range of pathophysiological conditions, to our knowledge no research related to the effects of epileptic seizures on hemorheological parameters has been carried out. Thus, the aim of this study was to explore possible changes in rheological parameters including red blood cell (RBC) deformability, rigidity and aggregation, whole blood and plasma viscosity during epileptic seizures induced by penicillin G in rats. Eighteen female albino rats were divided into three groups that included sham operated controls (Group S), epileptic group (Group E), intraperitoneal penicillin group (Group IPP). Epilepsy was induced by intracortical injections of penicillin G. Hemorheological studies had been carried out 3 h after the induction of epilepsy. Among the studied hemorheological parameters, only RBC deformability was found to be different in the E group compared to S group. Epileptic seizures led to an increase in RBC deformability in the E group. In conclusion, these results suggest that in addition to an increase in CBF, RBC deformability may also improve to better match brain metabolic demands during seizures.     

Changes of Rheological and Hematological Parameters in Rats of Wistar and SHR Lines at Single and Multiple Tredmill Running

In experiments on rats of the Wistar line and on spontaneously hypertensive animals of the SHR line, a complex study was performed of rheological (deformability and aggregation properties) and hematological parameters of erythrocytes at the single (40 min) and multiple (20–30 min for 7 and 14 days) exercise load (a forced tredmill running at a rate of 14 m/min). It has been shown that rats of both lines respond to the single, sufficiently light exercise load by a manifestation of stress-reaction. After the repeated exercise load, all stress-induced changes were leveled and replaced by adaptation to this action in Wistar rats, but not in the SHR line rats. The hypertensive rats were characterized by a greater fatigue and refusal of the tredmill running; therefore, this load to them had to be shortened to 20 min. For the 1st week of running, no statistically significant differences in the studied parameters were found between rats of the Wistar and SHR lines; however, by the 14th day of training, substantial differences between these lines were noticed: the Wistar rats were easily adapted to the tredmill running, whereas the SHR animals completely refused to run. At the single running, the stress-polycythemia, an increased hematocrit, a decreased hemoglobin concentration per erythrocyte, an increased plasma corticosterone level, and changes of blood rheological characteristics were shown, the erythrocyte deformability and the spontaneous aggregation rate under the stress conditions being in the direct positive correlational dependence that was absent in control. The Wistar rat training to the exercise load for 14 days led to normalization of the parameters, which indicates a favorable effect of trainings on the animal health state.__________Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 3, 2005, pp. 272–276.Original Russian Text Copyright © 2005 by Katukhin, Khama-Murad, Kislyakova, Kazennov, Maslova, Tavrovskaya.     

Changes of physiological and biochemical characteristics of rat erythrocytes after blood loss

In experiments of Wistar male rats, changes are studied of erythrocyte hematological, biochemical (activitities of transport ATPases), and rheological properties (capability for aggregation and deformability) 7 days after bloodletting of 12-15 % of the total blood mass. It has been shown that alongside with an elevation of erythrocyte volume and of the number of immature cells - reticulocytes, there was a statistically significant increase of Na,K-ATPase and Ca-ATPase activities in the whole erythrocytes and their membrane preparations - ghosts, the increment of activity in the case of Na,K-ATPase being essentially higher in the whole cells. This indicates the appearance of an enzyme activator inside the erythrocytes. There are also revealed a decrease of firmness of erythrocyte aggregates, a deceleration of spontaneous aggregation, and an increase of index of erythrocyte deformability. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are directed to optimization of blood circulation in large vessels and capillary network.     

Changes of physiological and biochemical characteristics of rat erythrocytes after blood loss

In experiments on Wistar male rats, changes are studied of erythrocyte hematological, biochemical (activities of transport ATPases), and rheological properties (capability for aggregation and deformability) 7 days after bloodletting of 12–15% of the total blood mass. It has been shown that alongside with an elevation of erythrocyte volume and of the number of immature cells—reticulocytes, there was a statistically significant increase of Na,K-ATPase and Ca-ATPase activities in the whole erythrocytes and in their membrane preparations—ghosts, the increment of activity in the case of Na,K-ATPase being essentially higher in the whole cells. This indicates the appearance of an enzyme activator inside the erythrocytes. There are also revealed a decrease of firmness of erythrocyte aggregates, a deceleration of spontaneous aggregation, and an increase of index of erythrocyte deformability. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are aimed at optimization of blood circulation in large vessels and capillary network.     

Effect of hypoxia on erythrocyte deformability in different species

The contribution of erythrocyte deformability to the pulmonary vascular resistance during hypoxia in different animal species has not been examined. We hypothesized that the increase in pulmonary vascular resistance during hypoxia was partially due to erythrocytes (RBC's) becoming less deformable during hypoxia, and therefore their transit in the capillaries becomes restricted. To test this, we measured an index of deformability of RBC's from six animal species (dog, pig, cat, rabbit, hamster, rat) during normoxic and hypoxic condition, and compared the changes in deformability with the pulmonary hypoxic pressor response (HPR) which has been reported in the same species. Deformability was indexed as the resistance that a Hemafil polycarbonate membrane (Nucleopore filter, 4.7 micron pores) offers to a 10% suspension of RBC's. The RBC suspension was either normoxic (PO2 = 150 torr) or hypoxic (PO2 = 50 torr). We found that hypoxia decreased RBC deformability; the largest decrease occurred in rat RBC's, a small but significant decrease was observed in the RBC's of cats, rabbits and hamsters, but no change was detected in RBC's of dogs or pigs. In general, such changes in deformability do not correlate well with the HPR in intact or in isolated lungs, for example the pig, had the largest HPR but the smallest change in RBC deformability. In some species, however, there was a positive correlation between RBC deformability and HPR, for example rats, rabbits and cats are usually better responders than dogs and hamsters, similarly the deformability of RBC's in rats, rabbits and cats were also more influenced by hypoxia than RBC's from dogs. The limiting factors in this relationship are the artificial conditions which were used to measure deformability and HPR, both may be different than in the intact conditions. Nevertheless the present data show that erythrocytes of some species can become less flexible during hypoxia, and hence may impede the transit in the capillaries. Therefore we propose that the hypoxic pressor response in the pulmonary vasculature may be partly due to smooth muscle contraction (vasoconstriction) and partly due to a decrease in erythrocyte deformability (capillary obstruction). Both components are likely to be species dependent.     

Erythrocyte Membrane Phosphatidylserine Exposure in Obesity

It has been suggested that increased erythrocyte membrane phosphatidylserine (PS) exposure could contribute to hypercoagulability and hemorheological disturbances in obesity. The aim of our study was to evaluate PS exposure in obese patients and in a control group and to correlate this with hemorheological properties, i.e., erythrocyte aggregability (EA) and deformability, and to evaluate the effect of weight loss on these parameters. An anthropometric and analytical evaluation was performed at baseline and after 3 months on a diet (very low‐calorie diet for 4 weeks and low‐calorie diet for 2 months) on 49 severe or morbid obese patients (37 women, 12 men) and 55 healthy volunteers (39 women, 16 men). PS exposure on erythrocyte membrane was performed by flow cytometry. Erythrocyte aggregation was measured using the Myrenne MA₁ and the Sefam aggregometer. Erythrocyte deformability was determined in a stress diffractometer. Prothrombin fragment F1+2 (F1+2) was determined as a marker of the hypercoagulable state, and plasma malondialdehyde (MDA) as an indicator of oxidative stress. Obese patients had a higher EA index, higher PS exposure on erythrocyte membranes and higher levels of MDA and F1+2. The differences in erythrocyte aggregation and F1+2 between obese patients and the control group were maintained after adjusting for PS exposure. After 3 months of diet, a significant reduction in PS exposure on erythrocyte membrane was observed. Obese patients show increased PS exposure on erythrocyte membrane, with no effect on rheological properties. Increased PS exposure could contribute to hypercoagulability in these patients. Weight loss obtained with diet treatment reduces PS exposure on erythrocyte membrane.     

Red blood cell dysfunction in septic glucose-6-phosphate dehydrogenase-deficient mice

Glucose-6-phosphate dehydrogenase (G-6-PDH) deficiency is the most common known human genetic polymorphism. This study tested the hypothesis that G-6-PDH deficiency worsens sepsis-induced erythrocyte dysfunction. Sepsis (24 h) was induced by cecal ligation and puncture in wild-type (WT) and G-6-PDH-deficient (G-6-PDH activity 15% of WT) mice. Erythrocyte responses were tested in whole blood as well as in subpopulations of circulating erythrocytes. Whereas erythrocyte deformability was similar in unchallenged deficient and WT animals, sepsis decreased erythrocyte deformability that was more pronounced in deficient than WT animals. Sepsis also resulted in anemia and hemolysis in deficient compared with WT animals. Mean corpuscular hemoglobin content and erythrocyte deformability decreased in younger erythrocyte subpopulations from septic deficient compared with WT animals. Sepsis decreased the reduced-to-oxidized glutathione ratio in erythrocytes from both deficient and WT animals; however, plasma glutathione increased more in deficient than in WT animals. Erythrocyte content of band 3 associated with the cytoskeleton was elevated in deficient compared with WT erythrocytes. The antioxidant N-acetyl-l-cysteine in vivo alleviated the sepsis-induced decrease in erythrocyte deformability in deficient animals compared with sham-operated control animals. This study demonstrates that a mild degree of G-6-PDH deficiency (comparable to the human class III G-6-PDH deficiencies) worsens erythrocyte dysfunction during sepsis. Increased erythrocyte rigidity and tendency for hemolysis together with alterations in band 3-spectrin interactions may contribute to the immunomodulatory effects of G-6-PDH deficiency observed after major trauma and infections in humans.     

Stimulation of erythrocyte phosphatidylserine exposure by paclitaxel.

Side effects of cytostatic treatment include development of anemia resulting from either decreased generation or accelerated clearance of circulating erythrocytes. Recent experiments revealed a novel kind of stress-induced erythrocyte death, i.e. eryptosis, which is characterized by enhanced cytosolic Ca(2+) levels, increased ceramide formation and exposure of phosphatidylserine at the cell surface. The present study explored whether cytostatic treatment with paclitaxel (Taxol) triggers eryptosis. Blood was drawn from cancer patients before and after infusion of 175 mg/m2 Taxol. The treatment significantly decreased the hematocrit and significantly increased the percentage of annexin-V-binding erythrocytes in vivo (by 37%). In vitro incubation of human erythrocytes with 10 microM paclitaxel again significantly increased annexin-V-binding (by 129%) and augmented the increase of annexin-V-binding following cellular stress. The enhanced phosphatidylserine exposure was not dependent on caspase-activity but paralleled by erythrocyte shrinkage, increase of cytosolic Ca(2+) activity, ceramide formation and activation of calpain. Phosphatidylserine exposure was similarly induced by docetaxel but not by carboplatin or doxorubicin. Moreover, eryptosis was triggered by the Ca(2+) ionophore ionomycin (10 microM). In mice, ionomycin-treated eryptotic erythrocytes were rapidly cleared from circulating blood and sequestrated into the spleen. In conclusion, our data strongly suggest that paclitaxel-induced anemia is at least partially due to induction of eryptosis.     

慢性常压缺氧和缺氧伴CO_2潴留大鼠红细胞变形能力与能量代谢的研究

本工作研究了慢性常压缺氧和缺氧伴ＣＯ＿２潴留肺动脉高压大鼠红细胞变形能力和红细胞内ＡＴＰ含量的变化。结果表明,慢性常压缺氧和缺氧伴ＣＯ＿２潴留大鼠不同切应力下的红细胞变形指数和红细胞内ＡＴＰ含量均明显低于其对照组,且该两组红细胞内ＡＴＰ含量与不同切应力下的红细胞变形指数呈显著正相关。提示慢性缺氧和伴ＣＯ＿２潴留大鼠红细胞内ＡＴＰ含量降低可能是导致红细胞变形能力降低的诸因素之一,后者又可导致和加重肺动脉高压的形成。     

Effect of some nickel compounds on red blood cell characteristics

The effect of certain inorganic and coordinated nickel compounds on the resistance to different destructive substances, rheological properties, and functional activity of healthy human red blood cells (RBC), was investigated. It is shown that nickel compounds affect the erythrocyte membrane lipid bilayer, as well as membrane proteins to various extents, depending on the type of compounds used. In general, the acceleration of erythrocyte aging was observed to be more pronounced in young erythrocytes. The observed results suggest that nickel compounds decrease water permeability across erythrocyte membranes. Almost all the investigated nickel compounds decrease erythrocyte thermostability, deformability, and the rate of O₂ release by erythrocytes.     

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.     

Role of Ca2+-activated K+ channels in human erythrocyte apoptosis

Exposure of erythrocytes to the Ca²⁺ ionophore ionomycin has recently been shown to induce cell shrinkage, cell membrane blebbing, and breakdown of phosphatidylserine asymmetry, all features typical of apoptosis of nucleated cells. Although breakdown of phosphatidylserine asymmetry is thought to result from activation of a Ca²⁺-sensitive scramblase, the mechanism and role of cell shrinkage have not been explored. The present study was performed to test whether ionomycin-induced activation of Ca²⁺-sensitive Gardos K⁺ channels and subsequent cell shrinkage participate in ionomycin-induced breakdown of phosphatidylserine asymmetry of human erythrocytes. According to on-cell patch-clamp experiments, ionomycin (1 µM) induces activation of inwardly rectifying K⁺-selective channels in the erythrocyte membrane. Fluorescence-activated cell sorter analysis reveals that ionomycin leads to a significant decrease of forward scatter, reflecting cell volume, an effect blunted by an increase of extracellular K⁺ concentration to 25 mM and exposure to the Gardos K⁺ channel blockers charybdotoxin (230 nM) and clotrimazole (5 µM). As reflected by annexin binding, breakdown of phosphatidylserine asymmetry is triggered by ionomycin, an effect again blunted, but not abolished, by an increase of extracellular K⁺ concentration and exposure to charybdotoxin (230 nM) and clotrimazole (5 µM). Similar to ionomycin, glucose depletion leads (within 55 h) to annexin binding of erythrocytes, an effect again partially reversed by an increase of extracellular K⁺ concentration and exposure to charybdotoxin. K-562 human erythroleukemia cells similarly respond to ionomycin with cell shrinkage and annexin binding, effects blunted by antisense, but not sense, oligonucleotides against the small-conductance Ca²⁺-activated K⁺ channel isoform hSK4 (KCNN4). The experiments disclose a novel functional role of Ca²⁺-sensitive K⁺ channels in erythrocytes, i.e., their participation in regulation of erythrocyte apoptosis. cell volume; charybdotoxin; osmolarity; phosphatidylserine; annexin     

Reduced erythrocyte deformability in Krushinsky-Molodkina rats with acute hemorrhagic stroke

Acute hemorrhagic stroke in Krushinsky-Molodkina rats was used to assess the ability of erythrocytes to change their shape in a shear flow. Membrane rigidity and internal viscosity of erythrocytes were measured by laser diffractometry (i.e., obtaining a diffraction pattern from a thin layer of an erythrocyte suspension in a shear flow followed by computer processing of the image). The results testify to reduced deformability of erythrocytes under hemorrhagic stroke.