Search

—Using Raman spectroscopy (RS) approach in a spectral range of 1000–3000 cm–1 were used to study the conformational and structural changes that arise in the heme group and globin moiety of hemoglobin in human red blood cells at various temperatures and oxygen contents. In hypoxia, the hemoglobin conformation was shown to change as a result of the increasing contribution of hematoporphyrin pyrrole rings and vibrational motions of vinyl groups. Modifications were additionally detected in the contributions of symmetric and asymmetric vibrations of the CH2 and CH3 radicals of histidine (2850, 2860, and 2900 cm–1) and lysine (2880 and 2860 cm–1) residues. The mechanisms of oxygen binding are discussed for hemoglobin located in the submembrane region and cytoplasm of the cell.

Binding of dinitrosyl iron complex (DNIC) to albumin was studied using time-resolved fluorescence (TRF) and electron spin resonance (ESR) spectroscopy. It was found that the fluorescence lifetime of bovine serum albumin (BSA) and human serum albumin (HSA) decreases with binding and depends on DNIC concentration. The observed biexponential pattern of the BSA tryptophan (Trp) fluorescence decay is explained by the presence of two tryptophan residues in the protein molecule. We believe that DNIC forms stable complexes with the cysteine (Cys34) residue in the domain I of albumin. It was shown that the lifetime of albumin tryptophan fluorescence decreased during co-incubation of BSA with DNICs and glutathione. Effects of DNIC on the binding of specific spin-labeled fatty acids with albumin in human blood plasma were studied in vitro. The presence of DNIC in blood plasma does not change conformation of albumin domains II and III. We suggest that the most possible interaction between DNICs and albumin is the formation of a complex; and nitrosylation of the cysteine residue in the albumin domain I occurs without the changes in albumin conformation.

Recently redox-regulation of tip growth has been extensively studied, but differential sensitivity of growing cells to particular ROS and their subcellular localization is still unclear. Here we used specific dyes to provide mapping of H2O2 and O•2− in short and long pollen tubes. We found apical accumulation of H2O2 and H2O2–producing organelles in the shank that were not colocalized with O•2−-producing mitochondria. Differential modulation of ROS content of the germination medium affected both growth speed and pollen tube morphology. Oxygen radicals affected ionic zoning: membrane potential and pH gradients. OH• caused depolarization all along the tube while O•2− provoked hyperpolarization and cytoplasm alkalinization. O•2− accelerated growth and reduced tube diameter, indicating that this ROS can be considered as pollen tube growth stimulator along with H2O2. Serious structural disturbances were observed upon exposure to OH• and H2O2 and O•2− quencher MnTMPP: pollen tube growth slowed down and ballooned tips formed in both cases, but in the presence of OH• membrane transport and organelle distribution was affected as well. OH•, thus, can be considered as a negative influence on pollen tubes which, presumably, have mechanisms for leveling it. The assumption was confirmed by EPR spectroscopy: pollen tubes actively reduce OH• content in the incubation medium.

Differential modulation of ROS content of the microenvironment (O ¯/MnTMPP/OH·) affects growth speed and morphology in lily pollen tubes. Oxygen radicals influence ionic zoning: membrane potential and pH gradients. Recently, redox-regulation of tip growth has been extensively studied, but differential sensitivity of growing cells to particular ROS and their subcellular localization is still unclear. Here, we used specific dyes to provide mapping of H2O2 and O·2¯ in short and long pollen tubes. We found apical accumulation of H2O2 and H2O2-producing organelles in the shank that were not colocalized with O·2¯-producing mitochondria. Differential modulation of ROS content of the germination medium affected both growth speed and pollen tube morphology. Oxygen radicals affected ionic zoning: membrane potential and pH gradients. OH· caused depolarization all along the tube while O·2¯ provoked hyperpolarization and cytoplasm alkalinization. O·2¯accelerated growth and reduced tube diameter, indicating that this ROS can be considered as pollen tube growth stimulator. Serious structural disturbances were observed upon exposure to OH· and ROS quencher MnTMPP: pollen tube growth slowed down and ballooned tips formed in both cases, but OH· affected membrane transport and organelle distribution as well. OH·, thus, can be considered as a negative regulator of pollen tube growth. Pollen tubes, in turn, are able to reduce OH· concentration, which was assessed by electron paramagnetic resonance spectroscopy (EPR).

This study examines the action of hypoxia on integrity, fluidity and protein composition of red blood cell (RBC) membrane. Twenty-min exposure to oxygen-free environment decreases rat RBC integrity documented by 3-fold elevation of hemoglobin release without any action on the membrane fluidity estimated by electron magnetic resonance spectroscopy of spin-labeled stearic acid analogues. The proteomics technology in combination with relative label free quantification analysis revealed a dozen of membrane-bound proteins, including elevated content of hemoglobin, reproducibly affected by hypoxia. Mapping the identified proteins in the KEGG pathway database we found that the proteins of multi subunit Cullin-Rbx E3 ubiquitin ligase complex are presented in normoxic RBC ghosts but not in the hypoxic samples. Our results suggest that Cullin-Rbx E3 complex, associated with RBC membrane in normoxia, provides detection and deletion of membrane proteins damaged by reactive oxygen species. In hypoxic conditions, deoxy-Hb binds to band 3 protein, resulting in dissociation of Cullin-Rbx E3 complex from RBC membrane and impaired clearance of damaged cytoskeleton proteins. These rearrangements of membrane proteins might be involved in attenuated membrane integrity revealed in hypoxic RBC. Significance This study demonstrate that sustained deoxygenation of rat erythrocytes alters the composition of membrane-bound proteins including elevation of the content of hemoglobin without any changes in the viscosity of erythrocyte membrane lipid bilayer. These results suggest that the changes the composition of membrane proteins result in attenuated membrane integrity and contribute to augment release of hemoglobin seen in hypoxic conditions.

Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat erythrocytes. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5- and 2.5-fold, respectively. In rat erythrocytes a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL 67156. In these conditions we observed a positive correlation (R2 = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia-induced ATP release from rat erythrocytes. The obtained results indicate a key role of hemolysis in the ATP release from red blood cells.

We studied microfluidity and selective ion permeability of plasma membranes and O2-binding properties of erythrocytes of cosmonauts during early rehabilitation after a long-term space flight (LTSF). Microfluidity of plasma membranes in surface regions was found to undergo a reversible decrease during 13–15 days following LTSF, which was accompanied by a reversible increase in relative cholesterol content. Cosmonauts’ erythrocytes revealed an increased activity of Na/H-exchanger and KCa-channel as well as a decrease in number of discocytes and increase in number of echinocytes, stomatocytes and knizocytes. Total hemoglobin content as well as oxyhemoglobin content were lowered after the LTSF, while the affinity of hemoglobin to O2 was advanced. It is suggested that the changes in Hb properties, microfluidity and selective permeability of plasma membranes following the elevated cholesterol content in the membranes can decrease tissue supply with O2.

The article presents a noninvasive approach to the study of erythrocyte properties by means of a comparative analysis of signals obtained by surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RS). We report step-by-step the procedure for preparing experimental samples containing erythrocytes in their normal physiological environment in a mixture of colloid solution with silver nanoparticles and the procedure for the optimization of SERS conditions to achieve high signal enhancement without affecting the properties of living erythrocytes. By means of three independent techniques, we demonstrate that under the proposed conditions a colloid solution of silver nanoparticles does not affect the properties of erythrocytes. For the first time to our knowledge, we describe how to use the SERS-RS approach to study two populations of hemoglobin molecules inside an intact living erythrocyte: submembrane and cytosolic hemoglobin (Hb(sm) and Hb(c)). We show that the conformation of Hb(sm) differs from the conformation of Hb(c). This finding has an important application, as the comparative study of Hb(sm) and Hb(c) could be successfully used in biomedical research and diagnostic tests.

In smooth muscles, inhibition of Na(+),K(+),2Cl(-) cotransport (NKCC) by bumetanide decreased intracellular Cl(-) content ([Cl(-)](i)) and suppressed the contractions triggered by diverse stimuli. This study examines whether or not bicarbonate, a regulator of several Cl(-) transporters, affects the impact of NKCC in excitation-contraction coupling. Addition of 25 mM NaHCO(3) attenuated the inhibitory action of bumetanide on mesenteric artery contractions evoked by 30 mM KCl and phenylephrine (PE) by 5 and 3-fold, respectively. In cultured vascular smooth muscle cells, NaHCO(3) almost completely abolished inhibitory actions of bumetanide on transient depolarization and [Ca(2+)](i) elevation triggered by PE. In bicarbonate-free medium, bumetanide decreased [Cl(-)](i) by approximately 15%; this effect was almost totally abrogated by NaHCO(3). The addition of NaHCO(3) resulted in 2-fold inhibition of NKCC activity and 3-fold attenuation of [Cl(-)](i). These data strongly suggest that extracellular HCO(3)(-) diminishes the NKCC-sensitive component of excitation-contraction coupling via suppression of this carrier.

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.

Tissue hypoxia, which plays a key role in the development of renal and vascular complications of cardiovascular diseases (CVD), might be considered a consequence of vascular remodeling and/or attenuated oxygen (O(2)) delivery by erythrocytes. Using Raman spectroscopy (RS), we observed that erythrocytes from patients with CVD exhibit changes in the conformation of haemoglobin (Hb) haemoporphyrin (HP), reflecting its lower O(2) transport capacity. Hypertriglyceridemia and hypercholesterolemia are well-known hallmarks of CVD. This study examined the role of plasma lipids in the regulation of erythrocyte membrane viscosity, oxy-Hb content as well as Na(+)/H(+) exchange and Ca(2+)-ATPase, whose activities are altered in patients with CVD. HP conformation was assessed by RS of blood samples. Membrane fluidity was estimated at depths of 0.6-0.8 and 2.2nm by electron-paramagnetic resonance spectroscopy of erythrocytes loaded with spin-labeled 5-doxylstearic acid and 16-doxylstearic acid, respectively. Ion-selective electrodes were employed for the study of H(+) and Ca(2+) fluxes. Both oxy-Hb content and erythrocyte membrane fluidity were decreased in essential hypertension and coronary artery disease patients and negatively correlated with plasma cholesterol but not triglyceride content. This observation allows us to assume that decreased oxy-Hb content in patients with CVD is caused by high plasma cholesterol via attenuation of erythrocyte membrane fluidity and its permeability to O(2). Plasma cholesterol level correlated positively and negatively with erythrocyte Na(+)/H(+) exchange and Ca(2+)-ATPase, respectively. However, in contrast to membrane fluidity, the impact of these ion transporters in oxy-Hb regulation under baseline conditions seems to be negligible. We propose that decreased oxy-Hb content contributes to the reduced O(2) tissue supply seen in patients with CVD.

Morphology of erythrocytes and conformation of hemoglobin-derived hematoporphyrin were studied in patients with coronary heart disease (CHD) and patients with circulatory failure using laser interference microscopy and Raman spectroscopy. Correlation was revealed (r=0.81) between hemoglobin oxygen saturation and oxyhemoglobin fraction in erythrocytes evaluated by Raman spectroscopy. Patients with CHD and patients with circulatory failure showed reduced oxygen-releasing capacity of hemoglobin and hemoglobin content and increased oxygen-binding capacity of hemoglobin, and hemoglobin affinity for oxygen. Significant differences from the control were observed only in patients with circulatory failure. It was found that hemoglobin content, hematocrit, and the shape of erythrocytes during CHD and circulatory failure did not differ from the control, whereas the area of erythrocytes was increased.

A drop in oxygen partial pressure results in elevation of blood vessel diameter. It has been demonstrated that isolated vessels exhibit this unique feature only when they are perfused in the presence of erythrocytes. More recently, it was shown that haemoglobin plays a key role in oxygen sensing. Its deoxygenated form interacts with band 3 protein, triggering the cascade of non-identified intracellular signals involved in nitric oxide production and release of ATP interacting with P2Y purinergic receptors in endothelial cells. In this review, we summarize the data on mechanisms of ATP release from erythrocytes, as well as on its physiological and pathophysiological implications.

Changes in viscosity and permeability of the plasma membrane and conformation of erythrocyte hemoglobin hematoporphyrin were found in patients with diabetes mellitus. The decrease in oxygen binding and increase in deoxyhemoglobin concentration during diabetes mellitus were accompanied by changes in viscosity and permeability of the membrane for Na+, H+, Ca2+, and K+. Our results suggest that oxygen-binding properties of hemoglobin depend on viscosity and permeability of the erythrocyte plasma membrane.

Hypoxia of various origin and localization is accompanied by changes in some physical and chemical properties of erythrocytes: deformability, plasma membrane viscosity, and the oxygen-binding capacity of hemoglobin [1, 4‐6]. Under the conditions of brain ischemia, these properties are studied insufficiently. After postischemic reperfusion (PIR) that restores blood circulation, the oxygen partial pressure in plasma increases, which may stimulate the generation of reactive oxygen species (ROS) and affect the erythrocyte functions. In blood plasma, Cu,Zn-superoxide dismutase (SOD) and ceruloplasmin (CP) are involved in utilization of superoxide anion radical ( ) that triggers ROS formation. In this study, changes in the viscosity of erythrocyte plasma membrane and the e 2 -binding ability of hemoporphyrin of deoxyhemoglobin were studied. SOD activity and CP level were also measured in blood plasma of rats with brain ischemia before and after brain PIR. White outbred male rats weighing 272 ± 11 g were used in experiments. The animals were divided into three groups: the sham-operated rats (the control) ( n = 10), the rats with brain ischemia ( n = 10), and the rats with postischemic brain reperfusion ( n = 7). One day before the experiment, both carotids of the anesthetized animals were underpinned with a fishing line (0.3 mm) that was later withdrawn under skin through the polyethylene tubes into the interscapular region. After one day, a one-stage complete occlusion of both carotids was induced by carotid retraction into the tubes by means of the fishing line; subsequent release of carotids led to PIR. Blood samples (3 ml of blood mixed with heparin, 10 U/ml) were taken from the jugular veins of

This study examined the possible involvement of abnormal erythrocyte oxygen (O 2 ) transport in the pathogenesis of heart failure. Haemoglobin (Hb) haemoporphyrin conformation was assessed by Raman spectroscopy (RS) of blood samples, whereas membrane fluidity was estimated at depths of 0.6–0.8 and 2.2nm by electron-paramagnetic resonance spectroscopy of erythrocytes loaded with spin-labeled 5-doxylstearic acid and 16-doxylstearic acid, respectively. The fluidity of erythrocyte membranes from patients with heart failure was decreased in the area near the membrane surface and remained unchanged in the deeper hydrophobic membrane regions. The same differences were also detected in healthy controls subjected to chronic high-altitude hypoxia. RS demonstrated that in heart failure the total content of Hb–ligand complexes and the relative content of Hb–nitric oxide (NO) complexes with cleaved Fe 2+ -globin bond was decreased, whereas content of Hb–NO complexes with preserved Fe 2+ -globin bond was increased. We propose that this phenomenon contributes to the reduced O 2 tissue supply seen in patients with heart failure.