Effects of Gasotransmitters on Membrane Elasticity and Microrheology of Erythrocytes Abstract Erythrocytes change their micromechanical properties associated with membrane viscoelasticity when they perform the transport function. There is some evidence that this is due to the influence of signaling molecules, including such gasotransmitters as nitrogen oxide (NO) and hydrogen sulfide (H2S). The aim of this study was to investigate the changes in microrheology of human erythrocytes under the influence of donors and stimulants of endogenous formation of gasotransmitters. The erythrocyte deformability, assessed by the elongation index of erythrocytes (EIE), and aggregation parameters, the indices of aggregation of erythrocytes (IAE), of washed cells were recorded after their incubation with sodium nitroprusside (SNP); sodium hydrosulfide (NaHS), the donor of H2S; 1H-[1,2,4]-oxadiazolo [4,3-a] quinoxalin-l-one (ODQ), an inhibitor of soluble guanylate cyclase; L-arginine and N-nitroarginine methyl ester (L-NAME), a substrate and an inhibitor of NO synthase, respectively; and glibenclamide (GBC ), the blocker of ATP-sensitive potassium channels. In addition, the resealed ghosts of erythrocytes were prepared, incubated with the above compounds, and the change in their deformability was evaluated. EIE increased by 8–11% (p < 0.01) and IAE decreased by 11–26% (p < 0.01) after the incubation of erythrocytes with SNP and NaHS, respectively. L-arginine and SNP exerted similar microrheological effects, which were eliminated by L-NAME or ODQ. GBC caused an increase in EIE by 8% (p < 0.05); the effects of GBC and NaHS were not additive. Incubation of the erythrocyte ghosts with SNP, L-arginine and NaHS was accompanied by a moderate but significant increase in membrane elasticity (p < 0.05). The obtained results indicate that the donors and stimulators of gasotransmitters NO and H2S moderately increase deformability of erythrocytes and markedly reduce their aggregability. The experiments on the erythrocyte ghosts suggest the existence of a direct effect of gasotransmitters on the viscoelastic properties of the membrane of these cells, which is independent of cGMP.

The Contribution of Changes of Intracelluar Potassium Ion Concentration to the Kinetics of Voltage-Dependent Potassium Current Abstract Voltage-dependent potassium channels (Kv channels) mediate the voltage-dependent potassium ionic current, contribute to the generation of the action potential and to the regulation of neuronal excitability. To date, a large number of studies of these ion channels have been carried out using the patch-clamp method in the whole-cell configuration. It is generally assumed that during the implementation of this method intracellular ion concentrations remain approximately constant due to the relatively rapid exchange of the content between the cytoplasm and the patch pipette. However, this assumption may be incorrect if the flow of ions through the membrane is large. It was demonstrated in this study that the large outward currents of potassium ions can lead to a decrease in their intracellular concentration even during the whole-cell patch-clamp recording. This phenomenon can accelerate the decay of the recorded voltage-dependent potassium currents and may consequently lead to the overestimation of the inactivation rate of voltage-dependent potassium channels.

Coagulation Activity of Membrane Microparticles Abstract Properties of membrane microparticles (MPs), as well as methods for their study are reviewed. Microparticles are vesicular fragments of a plasma membrane, which are detached from the surface of cells upon their activation and/or damage. An increase in intracellular calcium and subsequent remodeling of membrane cytoskeleton and redistribution of membrane phospholipids are key events leading to the MPs formation. Transfer of biologically active substances (proteins, lipids, and nucleic acids) from “parental” cell to other cells of the organism is the main function of MPs. MPs also have coagulation activity, that is, they are able to accelerate blood clotting. Procoagulant properties of MPs are determined by the expression on their surface of negatively charged phospholipids (first of all phosphatidylserine), which serve as substrates for assembling of coagulation complexes, and by the presence of tissue factor in some of them, the primary inducer of coagulation reactions. Methods of MPs counting and sizing are analyzed in this review with the indications of their limitations, advantages, and disadvantages. The main attention is focused on flow cytometry, the method that is most widely used in studies of MPs. The data on the coagulation activity of MPs originating from the blood cells (platelets, leukocytes, and erythrocytes) and endothelial cells are surveyed. Tissue factor-containing MPs derived from monocytes and endothelial cells have the highest capability to accelerate blood clotting. Information on the content of MPs of different cellular origin in the blood of healthy subjects and patients with thrombotic, inflammatory, and other diseases is presented.

Normal Fluctuations of Biological Membrane Shape as a Coupling Factor for Ordered Monolayer Domains Abstract It is believed that separation of lipid matrix of biological membranes into ordered and disordered phases plays an important role in the lateral distribution of proteins and transduction of cellular signals through the plasma membrane. In model lipid membranes with symmetric composition of monolayers, such phase separation always leads to formation of bilayer domains. However, the lipid composition of outer and inner monolayers of plasma membranes of cells is different, i.e., these membranes are asymmetric. The mechanism of coupling of monolayer domains into bilayer structures in cells still remains unexplained. The ordered lipid domain is thicker than the surrounding membrane; as a result, elastic deformations occur at their boundary. Minimization of the elastic part of the boundary energy contributes to the coupling of monolayer domains in opposite monolayers. However, this driving force is not enough to ensure that domains will come into register: at a certain fraction of the membrane area occupied by the domains, the elastic energy can reach the minimum in antisymmetric configurations (the ordered domain in the upper monolayer is opposed by disordered monolayer of the surrounding membrane). As an alternative factor of the coupling, we considered curvature thermal fluctuations of the membrane shape. Our theoretical analysis of elastic deformations in a lipid bilayer that is asymmetric in monolayer composition shows that more rigid lipid domains tend to distribute into the region with lower curvature of the monolayer; these regions naturally coincide in opposite monolayers. Thus, the coupling of ordered domains in different monolayers is provided by their greater bending stiffness as compared to the surrounding membrane. At the same time, greater ordering leads to lateral condensation, i.e., to the decrease of the average area per lipid molecule. The difference in area per lipid molecule in the coexisting membrane phases, on the contrary, tends to separate bilayer domains into their monolayer components. To quantify the coupling energy of ordered domains, it is necessary to take both of these effects into account.

cGMP-Dependent Protein Kinase Modulates the Sensitivity of Mesenchymal Stromal Cells to Purinergic Agonists Abstract Calcium signaling induced by ATP in mesenchymal stromal cells (MSCs) from the human adipose tissue was studied by using the Ca2+ dye Fluo-4 and Ca2+ imaging. Previously, purinergic agonists have been found to mobilize Ca2+ in the MSC cytoplasm by stimulating IP3 production and triggering Ca2+ release from Ca2+ stores via IP3 receptors. Here we demonstrated that the phosphodiesterase inhibitor IBMX increased a lag period of ATP-induced Ca2+ responses and also shifted the dose–response curve to higher concentrations. When stimulated by ATP at doses close to the threshold concentration, MSCs became unresponsive in the presence of 50 μM IBMX. PKA inhibitor H89 did not restore MSC responsiveness to ATP lost in the presence of IBMX, while the application of KT-5823, a PKG inhibitor, canceled the inhibitory effect of IBMX. Yet, the cell treatment with KT-5823 resulted in a marked decrease in the ATP response delay. Altogether, the data obtained suggest that PKG modulates the MSC sensitivity to ATP, presumably by phosphorylating IP3 receptors in a cGMP-dependent manner.

Analysis of Changes in Photochemical Reflectance Index (PRI) in Relation to the Acidification of the Lumen of the Chloroplasts of Pea and Geranium Leaves under a Short-Term Illumination Abstract— Measurement of the photochemical reflectance index (PRI) is a simple and non-invasive method for the evaluation of photosynthetic processes in higher plants. At the same time, the relationship between photosynthetic parameters and PRI can be significantly modified at the initial stages of illumination; thus, the study of the mechanisms of development of PRI after the beginning of illumination is an important task. The aim of this work was to analyze the relationship between the acidification of chloroplast lumen, which was assessed as an increase in the absorption of light by the leaf at a wavelength of 535 nm (light scattering, LS), and changes in PRI under short-term illumination in the leaves of pea and geranium. It was shown that the illumination caused an increase in LS and a decrease in PRI in both studied objects. Significant differences were found for the amplitude of the photochemical reflectance index decrease, while the differences in the absolute values of PRI at different stages of illumination were not reliable. Correlation analysis showed that the increase in LS during the first two minutes of illumination, reflecting light-induced acidification of chloroplast lumen, strongly correlated with the amplitude of the decrease in PRI; at the same time, at later stages of illumination, such a relationship was absent. Additional analysis carried out on geranium showed that the cessation of illumination caused the opposite dynamics: the decrease in LS was accompanied by an increase in PRI. The results show that the changes in PRI at the first minutes after the beginning of illumination, apparently, were due to the acidification of the chloroplast lumen.

Ways to Increase the Activity of Glutamate Dehydrogenase in Erythrocyte-Bioreactors for the Ammonium Removal Abstract— An increased blood ammonium concentration (hyperammonemia) is toxic to the central nervous system, so removing excess ammonium from the bloodstream is an important task. One of the solutions may be the use of erythrocyte-bioreactors (EBRs) with ammonium-processing enzymes loaded inside. Earlier attempts have been made to create such EBRs based on glutamate dehydrogenase (GDH) or glutamine synthetase; however, these EBRs were not effective enough. We have previously shown that the reasons for this were the low permeability of the erythrocyte membrane for the substrates of these reactions (α-ketoglutarate and glutamate) and the low activity of the included GDH (due to its high molecular mass and tendency towards aggregation at an increase in concentration above 0.1 mg/mL), and also that the low membrane permeability problem for α-ketoglutarate and glutamate could be overcome if GDH and alanine aminotransferase were included into EBR together, because these metabolites should be consumed and reproduced within such EBR cyclically. However, the problem of low GDH activity remains if GDH is the main ammonium processing enzyme. To improve the efficiency of GDH incorporation into erythrocytes and the quality of the EBRs (their erythrocyte indices and osmotic fragility), these parameters were compared for various hypoosmotic methods for loading GDH into cells. In addition, a comparison was made of previously used GDH from bovine liver and GDH from Proteus sp. It was shown that the method of flow dialysis was the most effective and allowed the inclusion of the GDH amount 3 times exceeding the inclusion achieved during the hypoosmotic dialysis. The properties of erythrocytes after exposure to this method changed slightly. GDH from Proteus sp. did not aggregate with an increase in its concentration and thereby allowed an approximately 18-fold increase in the specific activity of the enzyme in erythrocytes. Thus, this GDH is a promising enzyme for creating effective EBRs for ammonium removing.

Microdomain Organization of Internodal Myelin Abstract The orientation and ordering of the molecules of carotenoids and fatty acids of phospholipids in myelin of nerve fiber was investigated using Raman spectroscopy. A method for the quantitative description of the order of the molecules in myelin lipid bilayer has been developed. It was established that the difference in the distribution of the molecules of carotenoids and phospholipids is associated with the morphology of myelin and nerve fiber. The molecules of carotenoids are predominantly perpendicular to the surface of the lipid bilayer of myelin, while phospholipids are oriented at an angle of 45° to it. It is assumed that the microdomain organization of the internodal myelin is due to the presence of areas with high degree of saturation and order of the fatty acid chains of phospholipids.

Taxonomic Features of Specific Ca 2+ Transport Mechanisms in Mitochondria Abstract Mitochondria play an important role in the regulation of intracellular Ca2+ homeostasis in Eukaryotes. Progress in the development of molecular and genetic methods for the study of living systems made it possible to identify the structures that carry specific Ca2+transport in mitochondria, including Ca2+ uniporter (MCU), Na+/Ca2+ exchanger (NCLX) and Ca2+/H+ antiporter (Letm1). The study of the architecture and functioning of these systems at different levels of the organization of living organisms can provide insight into the origin and evolution of the systems of Ca2+ homeostasis and also reveal general mechanisms of regulation and control of these systems in normal and pathological conditions. This review is focused on the taxonomic features of the structure and functioning of specific calcium transport systems in eukaryotic mitochondria and provides evidence of the presence of homologous structures in prokaryotic organisms.

Adrenergic Modulation of Excitation Propagation in Peripheral Synapses Abstract A long history of studies of the effect of catecholamines on various physiological processes, a multidirectional and ambiguous interpretation of these effects, and a widespread use of adrenergic drugs in clinical practice raises a question of the mechanisms of action of these compounds on various functionally important elements of a living organism. The neuromuscular synapse plays a leading role in ensuring locomotor and respiratory functions, as well as in the posture maintenance. There is a number of conflicting reports on the multidirectional effects of adrenergic agonists on the muscle contraction, the release of acetylcholine from the motor nerve endings, and the state of the postsynaptic membrane of muscle fibers. The purpose of this review is to systematize the information concerning the effects of adrenergic compounds on different stages of the process of excitation propagation in peripheral synapses and to highlight recently revealed opportunities of the application of adrenergic compounds for the treatment of various diseases associated with neuromuscular pathology.