O. G. Luneva

New Insight into Erythrocyte through In Vivo Surface-Enhanced Raman Spectroscopy

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.

HCO3-dependent impact of Na+,K+,2Cl- cotransport in vascular smooth muscle excitation-contraction coupling.

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 NaHCO₃ 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, NaHCO₃ almost completely abolished inhibitory actions of bumetanide on transient depolarization and [Ca²⁺]_i elevation triggered by PE. In bicarbonate-free medium, bumetanide decreased [Cl^-]_i by ∼15%; this effect was almost totally abrogated by NaHCO₃. The addition of NaHCO₃ resulted in 2-fold inhibition of NKCC activity and 3-fold attenuation of [Cl^-]_i. These data strongly suggest that extracellular HCO₃^- diminishes the NKCC-sensitive component of excitation-contraction coupling via suppression of this carrier.

Changes in the State of Hemoglobin in Patients with Coronary Heart Disease and Patients with Circulatory Failure

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.

Changes in plasma membrane viscosity and hemoporphyrin conformation in erythrocyte hemoglobin under the conditions of ischemia and reperfusion of rat brain.

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

Role of viscosity and permeability of the erythrocyte plasma membrane in changes in oxygen-binding properties of hemoglobin during diabetes mellitus.

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.

Erythrocytes as regulators of blood vessel tone

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.

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.

Modifying effect of nitric oxide on rat blood plasma proteins and hemoglobin.

222 It is known that nitric oxide (NO), as a potent hemostatic factor, vasodilator, and neurotransmitter is involved in regulation of various physiological and biochemical processes [9, 13]. An increase in NO concentration and accumulation of reactive oxygen species (ROS) in tissues in pathologies may lead to formation of peroxynitrite, a modifier of activity of many enzymes [12]. The main antioxidant enzymes in blood plasma, which are involved in superoxide anion radical

Proteomics-based identification of hypoxia-sensitive membrane-bound proteins in rat erythrocytes

Abstract 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.

Hemolysis and ATP Release from Human and Rat Erythrocytes under Conditions of Hypoxia: A Comparative Study

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.