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Vol 42, No 4 (2025)
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The potential for using the mechanism of hypoxic adaptation in lower eukaryotes
Abstract
The analytical review examines the key elements of mitochondrial adaptations that occur in eukaryotes forced to live in hypoxic conditions for extended periods. In these organisms, mitochondria retain their activity, including synthetic, signaling, and, albeit to a lesser extent, bioenergetic functions. These adaptations, primarily characterized by the reversal of the succinate dehydrogenase reaction and the presence of low-potential quinone, enable the organisms to sustain energy production via mitochondrial Complex I. A comprehensive analysis of all changes induced by prolonged hypoxia allows for the development of anti-hypoxic strategies aimed at mitigating the effects of undesirable factors associated with hypoxic remodeling. On the other hand, understanding all elements of hypoxia-induced adaptations makes it possible to exploit them in combating solid tumor cells, which inhabit in an oxygen-deprived microenvironment.
Membrane and Cell Biology. 2025;42(4):255-263
255-263
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Dominance of intracellular maturation of brain-derived neurotrophic factor provides its retrograde influence in newly formed mouse motor synapses
Abstract
Miniature endplate potentials (MEPPs) and multiquantal endplate potentials (EPPs) evoked by short rhythmic nerve stimulation were recorded in newly formed mouse neuromuscular junctions using intracellular microelectrode technique. The pathways of proteolysis (extra- or intracellular) of proBDNF to brain-derived neurotrophic factor (BDNF) in muscle fibers during their reinnervation were investigated. Matrix metalloprotease 3 (MMP-3) or intracellular proconvertase furin were selectively inhibited in combination with the release of endogenous neurotrophin from muscle fibers upon stimulation of protease-activated receptors (PAR1). It was confirmed that PAR1 stimulation leads to an increase of MEPP amplitude due to the release of endogenous BDNF from muscle fibers and its retrograde action which increases the size of acetylcholine (ACh) quanta. MMP-3 does not participate in BDNF maturation. Inhibition of furin resulted in a change in the synaptic effect upon PAR1 stimulation: an increase of MEPP amplitude was substituted by a decrease in MEPP frequency, which is characteristic of the action of proBDNF in newly formed synapses. Thus, it has been shown that by inhibiting furin activity, it is possible to stop the maturation of muscle BDNF at the proneurotrophin stage in weakened regenerating synapses and ultimately ensure the appearance of proBDNF in the synaptic cleft with its own spectrum of effects. This can change the balance of the retrograde influence of BDNF and its proneurotrophin on the signal transmission in newly formed motor synapses. Moreover, a change in such a balance can potentially influence not only the regulation of quantal ACh secretion, but also the rate and severity of reinnervation, since BDNF and proBDNF have opposite effects on the elimination of excessive synaptic contacts during embryogenesis or during post-traumatic muscle reinnervation.
Membrane and Cell Biology. 2025;42(4):264-276
264-276
Activation of pyruvate dehydrogenase complex and inhibition of Krebs cycle and mitochondrial respiration by excess pyruvate
Abstract
Experiments on isolated rat liver mitochondria showed that pyruvate (10–30 mM) in the presence of L-glutamate causes concentration-dependent inhibition of ADP-activated respiration. Respiration is reactivated by 3 mM L-malate. Both effects are reproduced in the presence of D,L-carnitine (AcCar), indicating the important role of acetylCoA (AcCoA) in the regulation of the Krebs cycle reactions. During pyruvate oxidation, the respiration rate decreases within a few hundred seconds. The effect is also reproduced in the presence of dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), and is not observed with excess AcCar, indicating dephosphorylation of pyruvate dehydrogenase (PDH) upon inhibition of PDK by pyruvate (+ADP) or DCA. The effects of pyruvate and AcCar depend on the duration of the preliminary incubation of mitochondria in state 2. Experiments on frozen/thawed mitochondria show that preincubation of mitochondria with pyruvate excess restores PDH activity and suppresses α-ketoglutarate dehydrogenase (α-KGDH) activity, as detected by NADH fluorescence. Thus, a possible mechanism of inhibition of respiration by pyruvate may be a combined mechanism that includes (1) allosteric inhibition of citrate synthase by excess AcCoA at low concentrations of oxaloacetate and α-KGDH with possible participation of acetoacetylCoA; (2) slow acetylation of α-KGDH and other enzymes of the cycle by excess AcCoA with slow reactivation of PDH by pyruvate.
Membrane and Cell Biology. 2025;42(4):277-288
277-288
The Effect of Extracellular Vesicles Produced by MMSC on the Growth Rate of Cultured Renal Epithelial Cells in Normal and Oxygen-Glucose Deprivation Conditions
Abstract
In this work, extracellular vesicles (EV) produced by multipotent mesenchymal stromal cells (MMSC) and released into the culture medium are characterized. The vesicles isolated from the culture medium by centrifugation contained proteins specific to exosomes and had their typical size. The effects of vesicles produced by MMSC (MMSC-EV) on renal cells under normal and pathological conditions were also analyzed. MMSC-EV increased the proliferation rate of renal cells after oxygen and glucose deprivation-induced injury. The microRNA expression profile in MMSC-EV showed that both damage-causing microRNAs and protective microRNAs were highly expressed in them. The effect of MMSC-EV on kidney cells may be determined by a complex network of target gene regulation.
Membrane and Cell Biology. 2025;42(4):289-300
289-300
Effect of the combination of TGFβ + MCSF + cholesterol on the percentage and functional activity of microglia in rat hippocampal cell cultures
Abstract
Microglial cells in the brain are considered resident macrophages possessing a number of functional and physiological characteristics typical of these immune cells. Microglia are involved in neuroinflammatory processes of various etiologies, during which they undergo phenotypic changes. In neuron–glia cultures, microglial cells typically have low proliferative capacity due to the absence of necessary growth factors. In this study, we evaluated the effect of a combination of compounds critical for microglial proliferation, such as transforming growth factor beta (TGFβ), macrophage colony-stimulating factor (MCSF), and cholesterol, on the number and functional activity of microglial cells in hippocampal cultures from newborn rats. We found that the combination TGFβ + MCSF + cholesterol increased the number of microglial cells in cultures by more than twofold. RT-PCR analysis showed that exposure to the pro-inflammatory agent lipopolysaccharide (LPS) in cultures grown using this combination of factors led to increased expression of genes encoding inflammation-associated proteins such as IL-1β, TNFα, STAT3, as well as the gene encoding vimentin protein, which acts as a situational marker of reactive microglia. Additionally, incubation with LPS led to increased cell death in the cultures. In the case of hypoxic episode exposure, suppression of gene expression encoding the mentioned pro-inflammatory proteins was observed, while the increase in cell death was insignificant. LPS, as well as chemotactic formylated peptide (fMLP, an immune cell activator), caused enhanced production of superoxide anion and increased intracellular Ca²+ concentration in microglial cells. Thus, the described effects of LPS may indicate that the combination of TGFβ + MCSF + cholesterol added to the culture medium promotes the preservation and proliferation of functionally active microglial cells in neuron–glia cultures.
Membrane and Cell Biology. 2025;42(4):301-314
301-314
Decompensated liver cirrhosis impairs the deformability of erythrocytes and their ability to pass through microchannels
Abstract
Erythrocytes are the most numerous cell population of blood, ensuring the necessary level of oxygenation of tissues and forming the orderly movement of cells through the vessels. Disorders of physiological deformability of erythrocytes exacerbate the degree of anemia in two ways: aberrant erythrocytes are rapidly eliminated from the bloodstream due to sequestration and destruction in the spleen and liver, and poorly deformable erythrocytes have a reduced potential for gas exchange in capillaries due to decreased membrane contact area. Regardless of the etiology of hepatosis, liver cirrhosis is accompanied by the development of persistent anemia, but erythrocyte deformability disorders in patients with decompensated liver cirrhosis (DLC) have been poorly studied. Using laser diffraction, flow cytometry, and microfluidic analysis, we showed that erythrocytes from patients with DLC have significant deformability disorders caused by stress-type erythropoiesis (release of immature reticulocytes into circulation; an increase in the proportion of phosphatidylserine-presenting erythrocytes, and a decrease in cytosolic esterase activity). In DLC, erythrocytes have strongly pronounced rigidity to hypoosmotic load: induced hemolysis is incomplete, and its rate is reduced, indicating impaired deformability. The revealed violations affected the ability of erythrocytes to pass through microchannels, the transit rate decreased, a high percentage of occlusions was observed, i.e., there are signs of micro-rheological disorders. A correlation was established between erythrocyte micro-rheological impairments and the degree of DLC progression.
Membrane and Cell Biology. 2025;42(4):315-331
315-331
Simultaneous Monitoring of Cytosolic and Reticular Ca2+ Indicates Heterogeneity of Ca2+ Store
Abstract
Transduction of many agonists involves mobilization of intracellular Ca2+. The dynamics and shape of intracellular Ca2+ signals are determined by Ca2+ fluxes across the plasma membrane and membranes of intracellular organelles, primarily the endoplasmic reticulum (ER). Although traditionally, intracellular Ca2+ signaling has been studied using chemical fluorescent Ca2+ probes, the advent of genetically encoded Ca2+ sensors with different intracellular localizations has significantly expanded the instrumental capabilities. In the present work, synchronous monitoring of cytosolic Ca2+ in HEK-293 cells using Fluo-8 and of reticular Ca2+ using the genetically encoded ER-localized Ca2+ sensor R-CEPIA1er was performed. Upon stepwise stimulation of cells with acetylcholine (ACh), a coordinated increase in cytosolic Ca2+ and a decrease in ER Ca2+ were observed in the front phase of the Ca2+ response, the relaxation of which was often accompanied by superimposed oscillations. A greater detailing of the correlated behavior of the cytosolic Ca2+ (C) and reticular Ca2+ (Cs) concentrations could be provided by representing cellular responses in the phase plane (Cs, C). Since Cs and C are not measurable directly but evaluated through Ca2+-dependent fluorescence of Ca2+ probes, the experimental data were presented in the (Fs, Fc) plane, where Fs and Fc are ΔF/F0 for R-CEPIA1er and Fluo-8, respectively. Qualitatively, this is equivalent to the presentation of data in the (Cs, C) plane. The phase trajectories indicated that Ca2+ responses to ACh could not be generated solely by Ca2+ exchange between the homogeneous Ca2+ store and the cytosol, since in approximately 30% of cases the phase trajectory contained a loop, wherein a simultaneous increase in Cs and C took place. The loop was observed in a calcium-free medium with negligible Ca2+ influx into the cell, indicating the involvement of an intracellular Ca2+ source that was not accessible to monitoring by R-CEPIA1er or did not significantly contribute to its fluorescence. The inhibitory analysis showed that the suggested source was not acidic endosomes and lysosomes containing two-pore channels, the Golgi apparatus, and vesicles loaded with Ca2+ by SPCA-type ATPase, organelles releasing Ca2+ through ryanodine receptors, and/or mitochondria releasing Ca2+ into the cytosol through the Na+/Ca2+ exchanger. The results obtained pointed out that a system mediating agonist-induced Ca2+ signals is more complex than one in the paradigm with one homogeneous pool of stored Ca2+.
Membrane and Cell Biology. 2025;42(4):332-344
332-344