Erythrocyte ATP Research Articles

The general concept of a patient-oriented approach to the use of drugs with cytoprotective activity in patients with coronary heart disease

The purpose of the study: to develop a general concept of a patient-oriented approach to the use of drugs with cytoprotective activity in patients with coronary heart disease (CHD). Materials and methods. Examination of 60 patients with CHD: stable angina pectoris of I-III functional classes was performed. According to the standard general clinical methods for verifying the diagnosis were used: ECG, Doppler echocardiography, coronary angiography, lipid profile, complete blood count, coagulogram, renal, hepatic complex. Also the condition of stress-realizing and stress-limiting systems of the functional system of adaptation was deeply examined in patients: determination of the level of personal anxiety by the method of questioning, determination of the concentration of cortisol, insulin, cAMP and cGMP in blood serum by the method of enzyme immunoassay; determination of the endothelial and inducible nitric oxide synthase levels in erythrocyte lysate by the method of enzyme immunoassay; the study of ATP and ADP concentrations in blood serum and erythrocytes, also the study of 2,3-DFG concentration in erythrocytes by spectrophotometric methods. The individual reactivity of blood leukocytes’ mitochondria of patients was estimated in vitro under the influence of metabolic drugs (trimetazidine, meldonium, cytoflavin) by confocal microscopy according to the technique developed and patented by us (mitochondria were stained with pyrene). The materials were processed statistically. Results. Two variants of the leukocyte mitochondrial response in patients with CHD to the introduction of metabolic correctors in vitro were found — in the form of their activation or inhibition, depending on numerous parameters of the initial state of patients, which served as the basis for the development of a general concept of a patient-oriented approach to the use of drugs with cytoprotective activity in patients with coronary heart disease. According to this concept, the individual reactivity of a patient with CHD to the administration of a metabolic drug depends on the initial state of the functional adaptation system, which can be determined by the activity of stress-realizing and stress-limiting systems (cortisol/insulin ratio, serum cAMP/cGMP, eNOS, iNOS of erythrocyte lysate) and by the degree of preservation of the structure and function of effector organs. It is appropriate to prescribe drugs that stimulate energy metabolism in cells only to patients with initial hypoergosis, reduced mitochondrial activity at the phases of activation or resistance of the general adaptation syndrome, while maintaining reserves for energy adaptation, by short courses. Conclusion. A general concept of a patient-oriented approach to the use of drugs with cytoprotective activity in patients with coronary artery disease has been developed, according to which metabolic correctors should be prescribed in short courses, provided that individual reserves for energy adaptation are preserved in patients with initial mitochondrial hypoergosis in the activation or resistance phase of the general adaptation syndrome.

Adaptive changes of cows blood under technological stress

The creation of livestock complexes with the introduction of new methods of organization and technologies has led to a wide manifestation of technological stresses in cattle. Stress reduces the natural resistance and the level of humoral immunity, as a result of which conditions are created for the activation of conditionally pathogenic microflora, which leads to digestive disorders, respiratory diseases, decreased productivity, increased morbidity and mortality of animals. Therefore, the analysis of adaptive reactions of blood in cows under technological stress will allow us to assess the state of the body and timely develop measures to prevent emerging disorders. The marker of the general adaptation process is the blood system. The study of the content of leukocytes, erythrocytes, ATP in erythrocytes, electrophoretic mobility of erythrocytes (EFPE) of cattle blood under technological stress.It was shown that in animals after technological stress there was a significant increase in the number of leukocytes on the 3rd- 14th day of the study compared with the control group of animals, the development of neutrophilosis, monocytosis and lymphopenia was noted. The number of red blood cells, hemoglobin concentration, EFPE and ATP content after technological stress were lower compared to the control group for 3 days. Thus, to correct technological stress in cows, means are needed that increase the overall adaptive capabilities of the animal organism.

RED BLOOD CELL DEFORMABILITY AND PHYSICAL ACTIVITY

Physical exertion and sport cause changes of different nature and degree (positive or negative, strong, weak, moderate) in blood and blood cells’ biomechanical and fluid properties. The aim of this minireview was to attempt to summarize the main red blood cell deformability alterations during and after physical activity classified as follows: erythrocyte geometric shape; erythrocyte membrane rheological properties; biochemical and structural properties of erythrocyte membrane lipids and proteins; hemoglobin concentration; calcium ion concentration; intracellular diffusion and osmosis; nitric oxide and erythrocyte nitric oxide synthase; erythrocyte ATP synthase; erythrocyte age and degree of erythrocyte cell maturation; body and erythrocyte temperature. It is essential to study and evaluate the individual hemorheological response profile of erythrocyte deformability during physical activity because it is strongly personal. This is one of the major erythrocyte properties determining oxygen supply to working muscles. The multiple hemorheological changes during physical activity and their interconnectedness and strong personality make their differentiation very difficult. It is reasonable to weigh their effects (with specific quantitative measurements) to simplify the analysis and increase the likelihood of obtaining valid conclusions. But there will always be the probabilistic nature of these conclusions, determined by the complex and strictly individual picture of the hemorheological effects of physical exercises and activities.

Dynamics of Metabolic and Oxidative Parameters of Erythrocytes during Treatment of Chronic Heart Failure with Molecular Hydrogen.

We studied the effect of molecular hydrogen (H2) on the content of 2,3-diphosphoglyceric acid (2,3-DPG), ATP, malondialdehyde, and catalase activity in erythrocytes in chronic heart failure. Inhalation of 2% molecular hydrogen H2 was carried out for 40 min repeatedly (5 days) or once. Inhalation of H2 caused an increase in ATP concentration in both research groups, but was more pronounced after repeated inhalation. The content of 2,3-DPG increased after repeated exposure to H2. The increase in metabolic activity under the effect of H2 was accompanied by a decrease in malondialdehyde concentration and an increase in catalase activity. Thus, the application of H2 in chronic heart failure reduced oxidative stress and improved metabolism of erythrocytes, which contributes to improvement of microcirculation. This allows us to recommend H2 for protection against ischemic and reperfusion damage to the myocardium.

Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (PKLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients.

Electrical Propagation of Vasodilatory Signals in Capillary Networks.

We have developed a computational model to study electrical propagation of vasodilatory signals and arteriolar regulation of blood flow depending on the oxygen tension and agonist distribution in the capillary network. The involving key parameters of endothelial cell-to-cell electrical conductivity and plasma membrane area per unit volume were calibrated with the experimental data on an isolated endothelial tube of mouse skeletal feeding arteries. We have estimated the oxygen saturation parameters in terms of erythrocyte ATP release from the data of a left anterior descending coronary blood perfusion of dog. Regarding the acetylcholine-induced upstream conduction, our model shows that spatially uniform superfusion of acetylcholine attenuates the electrical signal propagation, and blocking calcium-activated potassium channels suppresses that attenuation. On the other hand, a local infusion of acetylcholine induces enhanced electrical propagation that corresponds to physiological relevance. Integrating the electrophysiology of endothelial tube and the electrophysiology/mechanics of a lumped arteriole, we show mechanistically that endothelial purinergic oxygen sensing of ATP released from erythrocytes and local infusion of acetylcholine are individually effective to induce vasodilatory signals to regulate blood flow in arterioles. We have recapitulated the upstream vasomotion in arterioles from the elevated oxygen tension in the downstream capillary domain. This study is a foundation for characterizing effective pharmaceutical strategies for ascending vasodilation and oxygenation.

Old cells die hard - sympatholysis and the ageing erythrocyte.

Old cells die hard - sympatholysis and the ageing erythrocyte.

Pyruvate kinase deficiency: epidemiology, molecular analyses and modern diagnostic approaches (literature review)

Red cell pyruvate kinase deficiency is the most common glycolytic defect causing congenital nonspherocytic hemolytic anemia. Pyruvate kinase is the enzyme involved in the last step of glycolysis – the transfer of a phosphate group from phosphoenolpyruvate producing the enolate of pyruvate and ATP (50 % of total energy ATP of erythrocytes). ATP deficiency directly shortened red cell lifespan. Affected red blood cells are destroyed in the splenic capillaries, leading to the development of chronic hemolytic anemia. It is an autosomal recessive disease, caused by homozygous and compound heterozygous mutations in the PKLR gene. There are no exact data on the incidence of pyruvate kinase deficiency, but the estimated frequency varies from 3: 1,000,000 to 1:20,000. The clinical features of the disease and the severity are highly variable. Diagnosis of pyruvate kinase deficiency is based on the determination of pyruvate kinase activity and molecular genetic study of the PKLR gene. The variety of clinical manifestations, possible complications, as well as the inaccessibility of diagnostic methods complicate the diagnosis.

Pyruvate Kinase Deficiency Cases Mimicking Congenital Dyserythropoietic Anemia

Introduction Pyruvate kinase (PK) deficiency is the most common glycolytic pathway defect that leads to hereditary hemolytic anemia with autosomal recessive inheritance pattern, which may present with severe hemolysis in the neonatal period, frequent blood transfusion requirement during infancy or with milder course of anemia. The prevalence reported among the Caucasians is 51 per million. Herein we present two cases of PK deficiency who were clinically and morphologically mimicking congenital dyserythropoietic anemia (CDA) at presentation and were diagnosed molecularly. Case 1 A three-year-old male patient was admitted with jaundice. Personal history revealed that he was born prematurely from a non-consanguineous family, and he did not need phototherapy at neonatal period. By the third month, he received first erythrocyte transfusion; then he needed transfusions every month. Strikingly, transfusion requirements increased in febrile episodes. Physical examination at admission revealed jaundice and splenomegaly. Complete blood count showed hemoglobin (Hb) 7.2 g/dL, mean corpuscular volume (MCV) 86.1 fL, mean corpuscular hemoglobin (MCH) 27.3 pg, mean corpuscular hemoglobin concentration (MCHC) 31.7 pg, and reticulocytes 12.3%. A blood film examination showed macrocytosis, hypochromia, anisocytosis, and rare spherocytes. Hemoglobin electrophoresis was normal, and direct Coombs' test was negative. The PK enzyme level was ignored since the patient was regularly transfused. Bone marrow aspiration showed erythroid hyperactivity and severe megaloblastic changes with double and multi-nucleated erythroid precursors suggestive for CDA. However, no genetic mutation detected concerning CDA (C15orf41, F8, F9, KLF1, CDAN1, VWF, SEC23B, GATA1, G6IBA genes). After negative molecular testing for CDA, sequencing of the PK-LR gene revealed the presence of a homozygous c.1151C> T mutation. Case 2 A three-month-old male patient presented with pallor and jaundice. Personal history revealed that he was born at term from a non-consanguineous family, and he required phototherapy and exchange transfusion due to icterus and anemia at the neonatal period. He needed erythrocyte transfusions during follow-up. Hemogram revealed Hb 6.7 g/dL, MCV 81.2 fL, MCH 25.2 pg, MCHC 31.1 pg, and reticulocytes 2.6%. Blood film examination showed polychromasia, rare spherocytes, and acanthocytes. Direct Coombs' test and Parvovirus B19 PCR analysis were negative. Bone marrow aspiration showed erythroid hyperactivity along with double and multi-nucleated erythroid precursors, which suggested CDA. However, no genetic mutation detected concerning CDA (SEC23B, CDAN1). The PK enzyme level was normal, which was 2 ½ months after the last transfusion. Finally, the molecular analysis of the PK-LR gene revealed the presence of a novel homozygous c.880G>A mutation. The mutation predicted as possibly damaging by in Silico analysis. Discussion The diagnostic approach to transfusion-dependent hereditary hemolytic anemia could be challenging related to false normal erythrocyte enzyme studies and osmotic fragility tests in previously transfused patients. It was reported that PK deficiency cases with inappropriately low reticulocytes might mimic CDA, which was discerned in cases where NGS performed for CDA but revealed PK-LR mutation. PK deficiency may lead to ineffective erythropoiesis due to erythrocyte ATP depletion, as well as CDA-like morphological findings (Cases 1 and 2) and low reticulocyte counts (Case 2). We suggest that in cases where CDA is suspected, PK deficiency should be considered in the differential diagnosis. Furthermore, instead of the PK enzyme level in patients with regular transfusions, PK-LR genetic analysis would be appropriate. Disclosures No relevant conflicts of interest to declare.

Effect of compound electrolyte injection on phosphatidylserine exposure in erythrocytes after blood salvage-retransfusion in dogs

Objective To evaluate the effect of compound electrolyte injection on phosphatidylserine (PS) exposure in erythrocytes after blood salvage-retransfusion in dogs. Methods Twenty healthy mongrel dogs, weighing 10-15 kg, aged 3-5 weeks, were divided into 2 groups (n=10 each) using a random number table method: normal saline group (group NS) and compound electrolyte injection group (group MEI). The process of intraoperative blood salvage-retransfusion was simulated in both groups: femoral vein was cannulated for blood withdrawal until the volume of blood lost was 400 ml, and the shed blood was salvaged by a blood recovery machine.The washing solution was normal saline in group NS and compound electrolyte injection in group MEI.The erythrocytes were retransfused after being labeled with fluorescein isothiocyanate.Blood samples were obtained before and after blood salvage for determination of erythrocyte ATP content by enzyme-linked immunosorbent assay.Blood samples were obtained at 24, 48 and 72 h after blood retransfusion, and the PS exposure rate of the salvaged erythrocytes was determined by flow cytometry.The spleen was taken at 72 h after retransfusion to detect the phagocytosis rate of salvaged erythrocytes by monocytes. Results There was no significant difference in ATP content before and after blood salvage between the two groups (P>0.05). Compared with NS group, the PS exposure rate of the salvaged erythrocytes at each time point after retransfusion and phagocytosis rate of salvaged erythrocytes by monocytes were significantly decreased in MEI group (P<0.05). Conclusion Compound electrolyte injection as a washing solution for intraoperative blood salvage can reduce the PS exposure in salvaged erythrocytes and is helpful in prolonging the lifespan of erythrocytes after retransfusion in dogs. Key words: Electrolytes; Erythrocytes; Operative blood salvage; Phosphatidylserines; Monocytes

Hypoxia enhances blood O2 affinity and depresses skeletal muscle O2 consumption in zebrafish (Danio rerio).

Zebrafish (Danio rerio) are widely used animal models. Nevertheless, the mechanisms underlying hypoxia tolerance in this species have remained poorly understood. In the present study, we have determined the effects of hypoxia on blood-O2 transport properties and mitochondrial respiration rate in permeabilized muscle fibres of adult zebrafish exposed to either 1) a gradual decrease in O2 levels until fish lost equilibrium (~1 h, acute hypoxia), or 2) severe hypoxia (PO2 ∼ 15 Torr) for 48 h (prolonged hypoxia). Acute, short-term hypoxia caused an increase in hemoglobin (Hb) O2 affinity (decrease in P50), due to a decrease in erythrocyte ATP after erythrocyte swelling. No changes in isoHb expression patterns were observed between hypoxic and normoxic treatments. Prolonged hypoxia elicited additional reponses on O2 consumption: lactate accumulated in the blood, indicating that zebrafish relied on glycolysis for ATP production, and mitochondrial respiration of skeletal muscle was overall significantly inhibited. In addition, male zebrafish had higher hypoxia tolerance (measured as time to loss of equilibrium) than females. The present study contributes to our understanding of the adaptive mechanisms that allow zebrafish, and by inference other fish species, to cope with low O2 levels.

Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis.We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry.Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3–36-fold) and hemolysis (1–44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43–67%).The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis.For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure.HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60–80%. HlyA-RBCs showed an acute 1.3–2.2-fold increase of Ca2+i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media.Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.

Interrelations of Hemorheological Parameters and Microcirculation in Subjects with an Increased Blood Pressure

Vascular resistance and blood pressure (BP) have known effects on blood viscosity. Therefore, the purpose of this study was to investigate the interrelation between the set of rheological characteristics and the parameters of microcirculation in patients with moderately increased blood pressure. We measured the hemorheological characteristics, including blood viscosity (BV) at high and low shear rates, plasma viscosity (PV), the viscosity of erythrocyte suspension, hematocrit (Hct), and erythrocyte aggregation and deformability. We also evaluated the efficacy of the transport function of blood. We determined the functional capillary density (FCD) using the method of microscopy and recorded the value of microvascular perfusion in patients with elevated blood pressure (mean blood pressure above 97 mmHg) and a group of subjects with normal blood pressure (control group, mean blood pressure of 87 mmHg) using laser Doppler imaging (LDI). We studied the changes in microrheological characteristics of red blood cells during their incubation in the presence of compounds with vasoactive properties: spermine (a donor of nitric oxide), ADP, and adenosine. Subjects with high blood pressure had a higher BP than the controls. This was due to an increase in PV, Hct, and erythrocyte aggregation with a decrease in their deformability. The ATP content of red blood cells was measured in patients with elevated blood pressure; it was found to be lower by 25% (p < 0.01) than in the control group. It was found that the content of ATP in erythrocytes was significantly negatively correlated with the values of blood pressure (systolic and diastolic blood pressures); the correlation was stronger in patients with a high blood pressure. These findings suggest that the characteristics of the hemorheological profile and the parameters of microcirculation in patients with moderately elevated blood pressure were significantly different from those in subjects with normal blood pressure. This indicates a reduction of the effectiveness of microvascular tissue perfusion.

In vitro enhancement of ATP in human erythrocytes from a healthy subject and two patients with thalassemia and hemoglobinopathy.

<b>In vitro enhancement of ATP in human erythrocytes from a healthy subject and two patients with thalassemia and hemoglobinopathy.</b>

Electrokinetic and Biochemical Changes in Erythrocytes under the Action of Terahertz Range Electromagnetic Waves

The effects of electromagnetic radiation of a gyrotron with an operating frequency of 263 GHz on rat erythrocyte electrophoretic mobility, malonic dialdehyde, ATP, and 2,3-diphosphoglycerate concentration have been studied in vitro. The blood was irradiated under continuous and pulsed gyrotron emission modes with the estimated incident energy flux on an object from 0.1 to 20 mW/cm2. It has been established that the studied erythrocyte characteristics changed in different directions depending on the mode and the incident energy flux: the erythrocyte electrophoretic mobility rate and ATP concentration decreased, while the malonic dialdehyde and 2,3-diphosphoglycerate concentration increased at a low incident energy flux and continuous exposure mode; all studied parameters increased under the pulsed exposure mode. An incident energy flux increase caused a reduction of these effects.

New insights on the regulation of the adenine nucleotide pool of erythrocytes in mouse models.

The observation that induced torpor in non-hibernating mammals could result from an increased AMP concentration in circulation led our investigation to reveal that the added AMP altered oxygen transport of erythrocytes. To further study the effect of AMP in regulation of erythrocyte function and systemic metabolism, we generated mouse models deficient in key erythrocyte enzymes in AMP metabolism. We have previously reported altered erythrocyte adenine nucleotide levels corresponding to altered oxygen saturation in mice deficient in both CD73 and AMPD3. Here we further investigate how these Ampd3-/-/Cd73-/- mice respond to the administered dose of AMP in comparison with the control models of single enzyme deficiency and wild type. We found that Ampd3-/-/Cd73-/- mice are more sensitive to AMP-induced hypometabolism than mice with a single enzyme deficiency, which are more sensitive than wild type. A dose-dependent rightward shift of erythrocyte p50 values in response to increasing amounts of extracellular AMP was observed. We provide further evidence for the direct uptake of AMP by erythrocytes that is insensitive to dipyridamole, a blocker for ENT1. The uptake of AMP by the erythrocytes remained linear at the highest concentration tested, 10mM. We also observed competitive inhibition of AMP uptake by ATP and ADP but not by the other nucleotides and metabolites tested. Importantly, our studies suggest that AMP uptake is associated with an erythrocyte ATP release that is partially sensitive to inhibition by TRO19622 and Ca++ ion. Taken together, our study suggests a novel mechanism by which erythrocytes recycle and maintain their adenine nucleotide pool through AMP uptake and ATP release.

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature-dependent ATP release from human blood and endothelial cells.

New Findings What is the central question of this study? Skin and muscle blood flow increases with heating and decreases with cooling, but the temperature‐sensitive mechanisms underlying these responses are not fully elucidated. What is the main finding and its importance? We found that local tissue hyperaemia was related to elevations in ATP release from erythrocytes. Increasing intravascular ATP augmented skin and tissue perfusion to levels equal or above thermal hyperaemia. ATP release from isolated erythrocytes was altered by heating and cooling. Our findings suggest that erythrocytes are involved in thermal regulation of blood flow via modulation of ATP release. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here, we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature, brachial artery blood flow and plasma [ATP] during passive arm heating and cooling in healthy men and found that they increased by 3.0 ± 1.2°C, 105 ± 25 ml min−1 °C−1 and twofold, respectively, (all P < 0.05) with heating, but decreased or remained unchanged with cooling. In additional men, infusion of ATP into the brachial artery increased skin and deep tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9‐fold from 33 to 39°C (P < 0.05) and declined by ∼50% at 20°C (P < 0.05), but no changes were observed in cultured human endothelial cells, plasma or serum samples. In conclusion, increases in plasma [ATP] and skin and deep tissue perfusion with limb heating are associated with elevations in ATP release from erythrocytes, but not from endothelial cells or other blood constituents. Erythrocyte ATP release is also sensitive to temperature reductions, suggesting that erythrocytes may function as thermal sensors and ATP signalling generators for control of tissue perfusion during thermal interventions.

Interspecific variation and plasticity in hemoglobin nitrite reductase activity and its correlation with oxygen affinity in vertebrates

Deoxygenated hemoglobin (Hb) is a nitrite reductase that reduces naturally occurring nitrite to nitric oxide (NO), supplying physiological relevant NO under hypoxic conditions. The nitrite reductase activity is modulated by the allosteric equilibrium between the R and T structures of Hb that also determines oxygen affinity. In the present study we investigated nitrite reductase activity and O2 affinity in Hbs from ten different vertebrate species under identical conditions to disclose interspecific variations and allow an extended test for a correlation between the rate constant for nitrite reduction and O2 affinity. We also tested plastic changes in Hb properties via addition of T-structure-stabilizing organic phosphates (ATP and GTP). The decay in deoxyHb during its reaction with nitrite was exponential-like in ectotherms (Atlantic hagfish, carp, crucian carp, brown trout, rainbow trout, cane toad, Indian python and red-eared slider turtle), while it was sigmoid in mammals (harbor porpoise and rabbit). Typically, hypoxia-tolerant species showed a faster reaction than intolerant species. Addition of ATP and GTP decreased O2 affinity and slowed the rate of nitrite reduction in a concentration-dependent manner. The initial second order rate constant of the deoxyHb-mediated nitrite reduction showed a strong curvilinear correlation with oxygen affinity among all ectothermic vertebrates, and the relationship also applied to plastic variations of Hb properties via organic phosphates. The relationship predicts high nitrite reductase activity in hypoxic tolerant species with high Hb-O2 affinity and reveals that the decrease in erythrocyte ATP and/or GTP during acclimation to hypoxia in ectotherms increases the erythrocyte NO generating capacity.

Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes.

IntroductionThe peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe.Methods and TreatmentsWe made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors.ResultsIn rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40–50% and swelling by 40–60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%.Analysis and DiscussionResults were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.

Deoxygenation Affects Composition of Membrane-Bound Proteins in Human Erythrocytes

Background/Aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions.