Chloride Gradient Is Involved in Ammonium Influx in Human Erythrocytes.

The ammonia/ammonium (NH3/NH4+, AM) concentration in human erythrocytes (RBCs) is significantly higher than in plasma. Two main possible mechanisms for AM transport, including simple and facilitated diffusion, are described; however, the driving force for AM transport is not yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a unique feature of RBCs to swell and lyse in isotonic NH4+ buffer. The kinetics of cell swelling and lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1 main ligand concentrations (Cl- and HCO3-). Additionally, the AM import kinetics was analyzed enzymatically and colorimetrically. In NH4+ buffer, RBCs concentration-dependently swelled and lysed when [NH4+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride concentration. The complete substitution of chloride with glutamate prevented NH4+-induced cell swelling and hemolysis, and the restoration of [Cl-] dose-dependently amplified the rates of RBC swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride anions in the buffer, significantly decreased NH4+ import. Our data indicate that the eAE1-mediated chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM transport in RBCs.

Testing Green Tea Extract and Ammonium Salts as Stimulants of Physical Performance in a Forced Swimming Rat Experimental Model.

The study of drugs of natural origin that increase endurance and/or accelerate recovery is an integral part of sports medicine and physiology. In this paper, decaffeinated green tea extract (GTE) and two ammonium salts-chloride (ACL) and carbonate (ACR)-were tested individually and in combination with GTE as stimulants of physical performance in a forced swimming rat experimental model. The determined parameters can be divided into seven blocks: functional (swimming duration); biochemistry of blood plasma; biochemistry of erythrocytes; hematology; immunology; gene expression of slow- and fast-twitch muscles (m. soleus, SOL, and m. extensor digitorum longus, EDL, respectively); and morphometric indicators of slow- and fast-twitch muscles. Regarding the negative control (intact animals), the maximum number of changes in all blocks of indicators was recorded in the GTE + ACR group, whose animals showed the maximum functional result and minimum lactate values on the last day of the experiment. Next, in terms of the number of changes, were the groups ACR, ACL, GTE + ACL, GTE and NaCl (positive control). In general, the number of identified adaptive changes was proportional to the functional state of the animals of the corresponding groups, in terms of the duration of the swimming load in the last four days of the experiment. However, not only the total number but also the qualitative composition of the identified changes is of interest. The results of a comparative analysis suggest that, in the model of forced swimming we developed, GTE promotes restoration of the body and moderate mobilization of the immune system, while small doses of ammonium salts, especially ammonium carbonate, contribute to an increase in physical performance, which is associated with satisfactory restoration of skeletal muscles and the entire body. The combined use of GTE with ammonium salts does not give a clearly positive effect.

ROS formation, mitochondrial potential and osmotic stability of the lamprey red blood cells: effect of adrenergic stimulation and hypoosmotic stress.

Semi-anadromous animals experience salinity fluctuations during their life-span period. Alterations of environmental conditions induce stress response where catecholamines (CA) play a central role. Physiological stress and changes in external and internal osmolarity are frequently associated with increased production of reactive oxygen species (ROS). In this work, we studied the involvement of the cAMP/PKA pathway in mediating catecholamine-dependent effects on osmoregulatory responses, intracellular production of ROS, and mitochondrial membrane potential of the river lamprey (Lampetra fluviatilis, Linnaeus, 1758) red blood cells (RBCs). We also investigated the role of hypoosmotic shock in the process of ROS production and mitochondrial respiration of RBCs. For this, osmotic stability and the dynamics of the regulatory volume decrease (RVD) following hypoosmotic swelling, intracellular ROS levels, and changes in mitochondrial membrane potential were assessed in RBCs treated with epinephrine (Epi, 25 µM) and forskolin (Forsk, 20 µM). Epi and Forsk markedly reduced the osmotic stability of the lamprey RBCs whereas did not affect the dynamics of the RVD response in a hypoosmotic environment. Activation of PKA with Epi and Forsk increased ROS levels and decreased mitochondrial membrane potential of the lamprey RBCs. In contrast, upon hypoosmotic shock enhanced ROS production in RBCs was accompanied by increased mitochondrial membrane potential. Overall, a decrease in RBC osmotic stability and the enhancement of ROS formation induced by ß-adrenergic stimulation raises concerns about stress-associated changes in RBC functions in agnathans. Increased ROS production in RBCs under hypoosmotic shock indicates that a decrease in blood osmolarity may be associated with oxidative damage of RBCs during lamprey migration.

Protein kinase A activity and NO are involved in the regulation of crucian carp (Carassius carassius) red blood cell osmotic fragility.

Activation of the cAMP pathway by ß-adrenergic stimulation and cGMP pathway by activation of guanylate cyclase substantially affects red blood cell (RBC) membrane properties in mammals. However, whether similar mechanisms are involved in RBC regulation of lower vertebrates, especially teleosts, is not elucidated yet. In this study, we evaluated the effects of adenylate cyclase activation by epinephrine and forskolin, guanylate cyclase activation by sodium nitroprusside, and the role of Na+/H+-exchanger in the changes of osmotic fragility and regulatory volume decrease (RVD) response in crucian carp RBCs. Western blot analysis of protein kinase A and protein kinase G substrate phosphorylation revealed that changes in osmotic fragility were regulated via the protein kinase A, but not protein kinase G signaling pathway. At the same time, the RVD response in crucian carp RBCs was not affected either by activation of adenylate or guanylate cyclase. Adenylate cyclase/protein kinase A activation significantly decreased RBC osmotic fragility, i.e., increased cell rigidity. Inhibition of Na+/H+-exchanger by amiloride had no effect on the epinephrine-mediated decrease of RBC osmotic fragility. NO donor SNP did not activate guanylate cyclase, however affected RBCs osmotic fragility by protein kinase G-independent mechanisms. Taken together, our data demonstrated that the cAMP/PKA signaling pathway and NO are involved in the regulation of crucian carp RBC osmotic fragility, but not in RVD response. The authors confirm that the study has no clinical trial.

Hypoxia inhibits the regulatory volume decrease in red blood cells of common frog (Rana temporaria).

Red blood cells of vertebrates can restore their cellular volume after hyposmotic swelling. The process strictly depends on oxygen availability in the environment. However, the role of hemoglobin in regulation of cell volume recovery is not clear yet. Little is known about the osmotic reactions and regulatory volume decrease of amphibian red blood cells. We investigated volume recovery process in oxygenated (oxyhemoglobin concentration 97 ±â€¯3% of total hemoglobin) deoxygenated (96 ±â€¯2% of deoxyhemolobin) and oxidized (47 ±â€¯2% of methemoglobin, 41 ±â€¯3% of deoxyhemoglobin) red blood cells of common frog (Rana temporaria) after hyposmotic swelling. Using the low-angle light scattering method we demonstrated the regulatory volume decrease in oxygenated cells and showed that the process was eliminated in hypoxic conditions. Reoxygenation of hypoxic cells restored the regulatory volume decrease. Oxidation of cellular hemoglobin to methemoglobin inhibited the volume recovery response in hyposmotically swollen oxygenated and reoxygenated hypoxic cells.

Hematin- and Hemin-Induced Spherization and Hemolysis of Human Erythrocytes Are Independent of Extracellular Calcium Concentration.

Pathologies such as malaria, hemorrhagic stroke, sickle cell disease, and thalassemia are characterized by the release of hemoglobin degradation products from damaged RBCs. Hematin (liganded with OH-) and hemin (liganded with Cl-)-are the oxidized forms of heme with toxic properties due to their hydrophobicity and the presence of redox-active Fe3. In the present study, using the original LaSca-TM laser particle analyzer, flow cytometry, and confocal microscopy, we showed that both hematin and hemin induce dose-dependent RBC spherization and hemolysis with ghost formation. Hematin and hemin at nanomolar concentrations increased [Ca2+]i in RBC; however, spherization and hemolysis occurred in the presence and absence of calcium, indicating that both processes are independent of [Ca2+]i. Both compounds triggered acute phosphatidylserine exposure on the membrane surface, reversible after 60 min of incubation. A comparison of hematin and hemin effects on RBCs revealed that hematin is a more reactive toxic metabolite than hemin towards human RBCs. The toxic effects of heme derivatives were reduced and even reversed in the presence of albumin, indicating the presence in RBCs of the own recovery system against the toxic effects of heme derivatives.

Assay variables and early clinical evaluation of low-angle light scattering for platelet function analysis.

INTRODUCTION: The recently developed platelet aggregation technique based on low-angle light scattering (LaSca) in diluted platelet-rich plasma (PRP) requires only a small sample volume and provides information about platelet aggregation and shape change. This study aimed to investigate the influence of preanalytical and analytical variables and to validate the method in a real-life pediatric hematology hospital setting. METHODS: Platelet aggregation was induced by ADP in diluted PRP in the presence of 2 mM calcium at 23 °C. The study included healthy adults (n = 30), healthy children (n = 20), and pediatric patients with suspected or diagnosed platelet function abnormalities (n = 25). RESULTS: The assay parameters were stable for at least 3 h after isolation of PRP and were sensitive to plasma dilution in the range of 2-8%. The initial aggregation velocity was significantly reduced in pediatric patients compared with healthy children (p < 0.05). ADP-induced light transmission amplitude was moderately correlated with LaSca amplitude of aggregation in healthy children (p = 0.52, p < 0.05) but not in pediatric patients. CONCLUSIONS: We standardized the protocol for platelet aggregation assessment by LaSca and characterized the influence of preanalytical and analytical variables on it.

Cellular osmoregulation of the ark clam (Anadara kagoshimensis) hemocytes to hyposmotic media.

Many bivalve species are considered to be euryhaline organisms due to effective adaptation to fluctuations of environmental salinity. Cellular mechanisms responsible for tolerance to salinity changes remain unclear for bivalves despite this question being critically important for commercially cultured species frequently introduced into regions differing from natural habitat by salinity regime. In the present work laser diffraction method was used for the analysis of volume changes in hemoglobin-containing ark clam (Anadara kagoshimensis) hemocytes following hyposmotic stimulation. Hemocytes responded to hyposmotic shock (decrease of media osmolarity from 461 to 216 mОsm/L) by a rapid swelling up to 171.5 ± 15.2% of control level. At normal osmotic conditions (osmolarity 461 mOsm/L), hemocyte mean cellular volume (MCV) was 354.0 ± 24.4 fl and maximum MCV of hyposmotically swollen cells prior lysis was 555.5 ± 57.4 fl (at the osmolarity 194 mOsm/L). Ark clam hemocytes demonstrated volume recovery response following hyposmotic swelling. Regulatory volume decrease (RVD) reaction did not depend on hemoglobin confirmation status. Final MCV of swollen hemocytes at the end of experimental period of RVD in oxygenated and deoxygenated suspensions did not significantly differ.

Persistent red blood cells retain their ability to move in microcapillaries under high levels of oxidative stress.

Oxidative stress is one of the key factors that leads to red blood cells (RBCs) aging, and impairs their biomechanics and oxygen delivery. It occurs during numerous pathological processes and causes anaemia, one of the most frequent side effects of cancer chemotherapy. Here, we used microfluidics to simulate the microcirculation of RBCs under oxidative stress induced by tert-Butyl hydroperoxide. Oxidative stress was expected to make RBCs more rigid, which would lead to decrease their transit velocity in microfluidic channels. However, single-cell tracking combined with cytological and AFM studies reveals cell heterogeneity, which increases with the level of oxidative stress. The data indicates that the built-in antioxidant defence system has a limit exceeding which haemoglobin oxidation, membrane, and cytoskeleton transformation occurs. It leads to cell swelling, increased stiffness and adhesion, resulting in a decrease in the transit velocity in microcapillaries. However, even at high levels of oxidative stress, there are persistent cells in the population with an undisturbed biophysical phenotype that retain the ability to move in microcapillaries. Developed microfluidic analysis can be used to determine RBCs' antioxidant capacity for the minimization of anaemia during cancer chemotherapy.

Microfluidic Characterization of Red Blood Cells Microcirculation under Oxidative Stress.

Microcirculation is one of the basic functional processes where the main gas exchange between red blood cells (RBCs) and surrounding tissues occurs. It is greatly influenced by the shape and deformability of RBCs, which can be affected by oxidative stress induced by different drugs and diseases leading to anemia. Here we investigated how in vitro microfluidic characterization of RBCs transit velocity in microcapillaries can indicate cells damage and its correlation with clinical hematological analysis. For this purpose, we compared an SU-8 mold with an Si-etched mold for fabrication of PDMS microfluidic devices and quantitatively figured out that oxidative stress induced by tert-Butyl hydroperoxide splits all RBCs into two subpopulations of normal and slow cells according to their transit velocity. Obtained results agree with the hematological analysis showing that such changes in RBCs velocities are due to violations of shape, volume, and increased heterogeneity of the cells. These data show that characterization of RBCs transport in microfluidic devices can directly reveal violations of microcirculation caused by oxidative stress. Therefore, it can be used for characterization of the ability of RBCs to move in microcapillaries, estimating possible side effects of cancer chemotherapy, and predicting the risk of anemia.