Hypotonic Environment Research Articles

Osteopontin Regulates AQP4 Expression by TRPV4 Activation in Müller Cells: Implications for Retinal Homeostasis.

During the intense neuronal activity in the retina, Müller cells are exposed to a hypotonic environment and activate a regulatory volume decrease (RVD) response, which depends on Aquaporin-4 (AQP4) and the calcium channel Transient Receptor Potential Vanilloid 4 (TRPV4). It was reported that Osteopontin (OPN), a cytokine and component of the extracellular matrix (ECM), may modulate the RVD of Müller cells. In other cell types, OPN participates in cell survival and migration, which Müller cells undergo to maintain retinal homeostasis. Therefore, the aim of this work was to study the putative crosstalk of OPN with AQP4 and/or TRPV4 in the main functions of Müller cells: RVD, morphology maintenance and migration. We used a human Müller cell line (MIO-M1) exposed to OPN and evaluated cell volume and osmotic permeability (Pf) during an osmotic swelling, AQP4 expression, cell morphology and migration. We observed that OPN induced a reduced Pf and RVD by downregulating AQP4 expression, which was prevented by TRPV4 inhibition. OPN also induced significant changes in cell morphology with an increased number of cytoplasmic projections. Finally, OPN reduced the migration of Müller cells, being this effect dependent on TRPV4. We propose that OPN affects water permeability and cell volume regulation of Müller cells by activating TRPV4 to reduce AQP4 expression. This represents a novel mechanism of regulation of water permeability by the ECM in Müller cells. Additionally, OPN-induced changes in morphology and migration of Müller cells may have an impact on retinal physiology.

Structural characteristics of the epidermis in marine and freshwater finless porpoises adapted to distinct osmotic environments

Abstract The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, Pilleri & Gihr, 1972; YFP) is an exclusively freshwater cetacean species inhabiting the Yangtze River and its connecting large lakes. As the primary line of defense in maintaining physiological equilibrium, the epidermis of the porpoise is expected to have undergone structural adaptations due to the shift from the marine to the freshwater environment. This study compared the microstructural and ultrastructural features of the epidermis of YFP and its marine counterpart, the East Asian finless porpoise (Neophocaena asiaeorientalis sunameri, Pilleri & Gihr, 1975; EAFP). Microscopic structural observations and statistical analyses of the epidermal thickness demonstrated no significant differences in the cell structure or distribution between the two porpoise species. However, the epidermis of the YFP contained more abundant stratum basale cells. The outermost lipid stratum corneum exhibited a thinner cell layer with wider neutral lipid droplets to resist the passive entry of water molecules in the hypotonic environment. In contrast, for the EAFP, a more uniformly arranged stratum basale in the epidermis led to denser keratin fibers and robust desmosomes within each epidermal layer at the ultrastructural level. This tight arrangement of cells can reduce transepidermal water loss (TEWL) in an environment with higher osmotic pressure. In conclusion, the 2 finless porpoise species appear to employ different epidermal mechanisms to adapt to their distinct osmotic environments. The YFP appears to possess a “lipid waterproofing” epidermal structure, while the EAFP possesses a “thick and compact water-retaining” epidermal structure to cope with potential water loss.

Towards phenotyping adaptive traits in camels: A study of the influence of hypotonic saline solutions on blood cell area.

Single-humped camels are livestock of physical, physiological, and biochemical adaptations to hot desert environments and to water scarcity. The tolerance of camels to water deprivation and their exceptional capacity for rapid rehydration requires blood cells with membranes of specialized organization and chemical composition. The objectives of this study are to examine the changes in the area (a proxy for volume) of camel blood cells in solutions with decreasing concentrations of NaCl and consequently identify the conditions under which blood cells can be phenotyped in a large population. Whole-blood samples from three healthy adult female camels were treated with four different concentrations of NaCl and examined at six incubation-periods. Observationally, red blood cells in all treatments remained intact and maintained their elliptical shape while white blood cells experienced some damage, lysing at concentrations below 0.90%. Average basal (in 0.90% NaCl) RBC area was ~15 μm² and swelled in the various treatments, in some cases reaching twice its original size. Excluding the damaged cells, the average area of combined WBCs, ~32.7 μm², expanded approximately three times its original size. We find that camel WBCs, like their RBCs, are adapted to hypotonic environments, and are capable of expanding while maintaining their structural integrity.

Docosahexaenoic Acid Promotes Eryptosis and Haemolysis through Oxidative Stress/Calcium/Rac1 GTPase Signalling.

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid with promising anticancer potential. Anaemia is a frequent adverse effect of anticancer treatment caused in part by eryptosis and haemolysis. Thus, it is important to investigate the role of DHA in red blood cell (RBC) death. RBCs were treated with anticancer concentrations (10-100 μM) of DHA under different physiological conditions, and fluorescence-assisted cell sorting was employed to measure eryptotic markers. Cell membrane scrambling was detected by annexin-V-FITC labelling, cytoplasmic Ca2+ by Fluo4/AM, cell size by forward scatter (FSC), and oxidative stress by H2DCFDA. Haemolytic markers were also assayed by photometric methods. DHA caused significant phospholipid scrambling with Ca2+ accumulation, loss of cellular volume, and oxidative stress. These changes were associated with dacrocyte formation, as revealed by electron microscopy. Moreover, DHA exhibited a dual effect on membrane integrity: it was haemolytic under isotonic conditions and anti-haemolytic in hypotonic environments. Importantly, inhibition of Rac1 GTPase activity with NSC23766 significantly reduced DHA-mediated haemolysis, as did co-administration of either sucrose or polyethylene glycol 8,000. Conversely, the presence of 125 mM KCl and urea without extracellular Ca2+ significantly exacerbated DHA toxicity. In conclusion, this is the first report that identifies key biochemical mechanisms underlying the cytotoxic effects of DHA in RBCs, promoting further development and validation of DHA in anticancer therapy.

Genetically Encoded Fluorescence Resonance Energy Transfer Biosensor for Live-Cell Visualization of Lamin A Phosphorylation at Serine 22.

Extensive phosphorylation at serine 22 (pSer22) on lamin A is the hallmark of cell mitosis, which contributes to the breakdown of nuclear envelope. In the interphase, pSer22 lamin A exists in low abundance and is involved in mechanotransduction, virus infection, and gene expression. Numerous evidences emerge to support lamin A regulation on cell function and fate by phosphorylation. However, live-cell imaging tools for visualizing the dynamics of pSer22 lamin A are yet to be established. Herein, we developed a novel lamin A phosphorylation sensor (LAPS) based on fluorescence resonance energy transfer (FRET) with high sensitivity and specificity. We observed the dynamic lamin A phosphorylation during the cell cycle progression in single living cells: the increase of pSer22 modification when cells entered the mitosis and recovered upon the mitosis exit. Our biosensor also showed the gradual reduction of pSer22 modification during cell adhesion and in response to hypotonic environment. By applying LAPS, we captured the propagation of pSer22 modification from inside to outside of the inner nuclear membrane, which further led to the breakdown of nuclear envelope. Meanwhile, we found the synchronous phosphorylation of pSer22 lamin A and H3S10ph at mitosis entry. Inhibition of Aurora B, the responsible kinase for H3S10ph, could shorten the mitotic period without obvious effect on the pSer22 modification level of lamin A. Thus, LAPS allows the spatiotemporal visualization of the lamin A pSer22, which will be useful for elucidating the molecular mechanisms underlying cell mitosis and mechanoresponsive processes.

Tamoxifen induces eryptosis through calcium accumulation and oxidative stress.

Chemotherapy-related anemia is a major obstacle in anticancer therapy. Tamoxifen (TAM) is an antiestrogen prescribed for breast cancer patients with hemolytic potential and apoptotic properties in nucleated cells. However, the eryptotic activity of TAM has hitherto escaped the efforts of investigators. RBCs from apparently healthy volunteers were treated with 1-50μM of TAM for 24h at 37°C. Hemoglobin leakage and LDH, AST, and AChE activities were photometrically determined while K+, Na+, and Mg2+ were detected by ion-selective electrode. Flow cytometry was used to identify eryptotic cells by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, sell size and morphology by FSC and SSC signals, respectively, and oxidative stress by H2DCFDA. Whole blood was also exposed to 30μM of TAM for 24h at 37°C to examine the toxicity of TAM to WBCs and platelets. TAM caused Ca2+-independent, dose-responsive hemolysis accompanied by K+, LDH, and AST leakage without improving the mechanical stability of RBCs in hypotonic environments. TAM treatment also increased the proportion of cells positive for annexin-V-FITC, Fluo4, and DCF, along with diminished FSC and SSC signals and AChE activity. Notably, TAM toxicity was aggravated by sucrose but abrogated by vitamin C, PEG 8000, and urea. Moreover, TAM exhibited distinct cytotoxic profiles against leukocytes and platelets. TAM-induced eryptosis is characterized by breakdown of membrane asymmetry, inhibition of AChE activity, Ca2+ accumulation, cell shrinkage, and oxidative stress. Vitamin C, PEG 8000, and urea may hold promise to subvert the undesirable toxic effects of TAM on RBCs.

Respirometry reveals major lineage-based differences in the energetics of osmoregulation in aquatic invertebrates.

All freshwater organisms are challenged to control their internal balance of water and ions in strongly hypotonic environments. We compared the influence of external salinity on the oxygen consumption rates (ṀO2) of three species of freshwater insects, one snail and two crustaceans. Consistent with available literature, we found a clear decrease in ṀO2with increasing salinity in the snail Elimia sp. and crustaceans Hyalella azteca and Gammarus pulex (r5=-0.90, P=0.03). However, we show here for the first time that metabolic rate was unchanged by salinity in the aquatic insects, whereas ion transport rates were positively correlated with higher salinities. In contrast, when we examined the ionic influx rates in the freshwater snail and crustaceans, we found that Ca uptake rates were highest under the most dilute conditions, while Na uptake rates increased with salinity. In G. pulex exposed to a serially diluted ion matrix, Ca uptake rates were positively associated with ṀO2(r5=-0.93, P=0.02). This positive association between Ca uptake rate and ṀO2was also observed when conductivity was held constant but Ca concentration was manipulated (1.7-17.3 mg Cal-1) (r5=0.94, P=0.05). This finding potentially implicates the cost of calcium uptake as a driver of increased metabolic rate under dilute conditions in organisms with calcified exoskeletons and suggests major phyletic differences in osmoregulatory physiology. Freshwater insects may be energetically challenged by higher salinities, while lower salinities may be more challenging for other freshwater taxa.

Membrane stretch due to hypoosmolality leads to the development of various pulmonary and cardiovascular diseases

High blood pressures over a long period of time can lead to the development of diseases such as congestive heart failure(CHF). Congestive heart failure also commonly leads to the development of conditions such as hyponatremia, a common electrolyte disorder which increases the body’s water levels, creating hypoosmotic conditions for cells; these hypoosmotic conditions cause cell swelling, which subsequently causes mechanical stress on the cells. In these hypoosmotic environments, specific mechanosensitive ion channels which are activated by these mechanical stresses on cells may play a critical role in the progression of conditions resulting from these hypoosmotic environments, such as pulmonary arterial hypertension (PAH) and CHF. This study aims to look at the connection between the effects of hypoosmotic conditions in cells on the pathogenesis of the development of chronic diseases such as PAH and CHF. Mechanical actions on specific mechanosensitive ion channels such as Piezo1 have already been shown to play a critical role in regulating intracellular Ca2+homeostasis, which play an important part in the regulation of the vascular tone in places such as the pulmonary arteries; mechanical stimulation from hypotonic environments such as those seen in hyponatremia lead to Piezo1-mediated increases of intracellular Ca2+, which are subsequently linked to the increased contraction and proliferation of pulmonary arterial endothelial cells(PAECs). The proliferation of PAECs contribute to the pathogenesis of diseases, such as pulmonary arterial hypertension(PAH) or CHF, which there is currently no cure for. Patch-clamp experiments were conducted to examine the activity of mechanosensitive cation currents; graphed results showed that the activity of mechanosensitive cation currents were significantly increased (p<0.05) in PAECs treated with a hypoosmotic solution when compared with PAECs treated in an isosmotic control solution. Digital fluorescence microscopy using fura-2 AM, a membrane-permeable Ca2+ sensitive fluorescent indicator, and a Nikon digital imaging fluorescent microscopy system was also used to measure intracellular Ca2+ concentration in PAECs; graphed data indicated that the change in intracellular calcium concentration in PAECs was significantly increased(p<0.05) when placed in a hypoosmotic solution, than when placed in an isosmotic solution. The collective data indicates that hypoosmotic conditions increase the activity of mechanosensitive ion channels, as well as intracellular Ca2+ levels, which may prove the role of mechanosensitive ion channels like Piezo1 and TRPC6 in the development of diseases such as PAH and CHF. This work is supported in part by the National Heart, Lung, and Blood Institute of the National Institutes of Health Grants HL135807 and HL146764. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Observing temporal variation in hemolysis through photoacoustics with a low cost LASER diode based system

Patients under hemolytic condition need continuous monitoring of lysis as depletion of Red Blood Cells (RBC) and the presence of antioxidant free hemoglobin (Hb) in excess amount due to hemolysis lead to severe deterioration of their health. Out of many modalities, Photoacoustics (PA) offers real time information noninvasively from deep lying blood vessels since Hb is the strongest chromophore in mammalian blood and the PA response of blood varies with the amount of Hb present. During hemolysis, total Hb content in blood however remains unchanged, thus, questions the use of PA in hemolysis detection. In this report, a hypothesis that the amplitude of the PA signal would not change with the amount of lysis is framed and tested by applying osmotic shock to the RBCs in hypotonic environment and the PA response is recorded over time using a low cost NIR based PA system. The experimental outcome indicates that PA amplitude falls off as lysis progresses in course of time consequently rejecting the hypothesis. The decaying PA response also carries the signature of RBC swelling during the early phase of lysis. The PA measurement can detect hemolysis as low as 1.7%. These findings further advocate transforming this NIR-PA system into a portable, noninvasive patient care device to monitor hemolysis in-vivo.

Freshwater transfer affected intestinal microbiota with correlation to cytokine gene expression in Asian sea bass.

As a catadromous fish, Asian sea bass (Lates calcarifer) juveniles migrate from seawater (SW) to freshwater (FW) for growth and development. During migration, they undergo physiological changes to acclimate to environmental salinity. Thus, it is crucial to understand how SW-to-FW migration affects the gut microbiota of catadromous fish. To the best of our knowledge, no study has revealed the effects of transfer to hypotonic environments on a catadromous fish microbiota. In this study, we aimed to determine the effects of FW transfer on the microbiota and cytokine gene expression in the intestines of juvenile catadromous Asian sea bass. The relationship between the water and the gut microbiota of this euryhaline species was also examined. We found that FW transfer affected both mucosa- and digesta-associated microbiota of Asian sea bass. Plesiomonas and Cetobacterium were dominant in both the mucosa- and digesta-associated microbiota of FW-acclimated sea bass. The pathogenic genera Vibrio, Staphylococcus, and Acinetobacter were dominant in the SW group. Although dominant fish microbes were present in the water, fish had their own unique microbes. Vitamin B6 metabolism was highly expressed in the FW fish microbiota, whereas arginine, proline, and lipid metabolism were highly expressed in the SW fish microbiota. Additionally, the correlation between cytokine gene expression and microbiota was found to be affected by FW transfer. Taken together, our results demonstrated that FW transfer altered the composition and functions of mucosa- and digesta-associated microbiota of catadromous Asian sea bass intestines, which correlated with cytokine gene expression.

Spatiotemporal tracking of small extracellular vesicle nanotopology in response to physicochemical stresses revealed by HS-AFM.

Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high-speed atomic force microscopy (HS-AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high-temperature, high-pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real-time observation by HS-AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS-AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles.

Effect of mechanical properties of Jurkat cell on adhesion properties of Jurkat integrin and VCAM-1: An AFM study

Mechanical properties play key roles in the immune system, especially the activation, transformation and subsequent effector responses of immune cells. As transmembrane adhesion receptors, integrins mediate the adhesion events of both cells and cell-extracellular matrix (ECM). Integrin affinity would influence the crosslinking of cytoskeleton, leading to the change of elastic properties of cells. In this study, the cells were treated with F-actin destabilizing agent Cytochalasin-D (Cyt-D), fixed by Glutaraldehyde, and cultivated in hypotonic solution respectively. We used Atomic force microscopy (AFM) to quantitatively measure the elasticity of Jurkat cells and adhesion properties between integrins and vascular cell adhesion molecule-1 (VCAM-1), and immunofluorescence to study the alteration of cytoskeleton. Glutaraldehyde had a positive effect on the adhesion force and Young’s modulus. However, these mechanical properties decreased in a hypotonic environment, confirming the findings of cellular physiological structure. There was no significant difference in the bond strength and elasticity of Jurkat cells treated with Cytochalasin-D, probably because of lower importance of actin in suspension cells. All the treatments in this study pose a negative effect on the adhesion probability between integrins and VCAM-1, which demonstrates the effect of structural alteration of the cytoskeleton on the conformation of integrin. Clear consistency between adhesion force of integrin/VCAM-1 bond and Young's modulus of Jurkat cells was shown. Our results further demonstrated the relationship between cytoskeleton and integrin-ligand by mechanical characteristics.

Roles of Bacterial Mechanosensitive Channels in Infection and Antibiotic Susceptibility

Bacteria accumulate osmolytes to prevent cell dehydration during hyperosmotic stress. A sudden change to a hypotonic environment leads to a rapid water influx, causing swelling of the protoplast. To prevent cell lysis through osmotic bursting, mechanosensitive channels detect changes in turgor pressure and act as emergency-release valves for the ions and osmolytes, restoring the osmotic balance. This adaptation mechanism is well-characterized with respect to the osmotic challenges bacteria face in environments such as soil or an aquatic habitat. However, mechanosensitive channels also play a role during infection, e.g., during host colonization or release into environmental reservoirs. Moreover, recent studies have proposed roles for mechanosensitive channels as determinants of antibiotic susceptibility. Interestingly, some studies suggest that they serve as entry gates for antimicrobials into cells, enhancing antibiotic efficiency, while others propose that they play a role in antibiotic-stress adaptation, reducing susceptibility to certain antimicrobials. These findings suggest different facets regarding the relevance of mechanosensitive channels during infection and antibiotic exposure as well as illustrate that they may be interesting targets for antibacterial chemotherapy. Here, we summarize the recent findings on the relevance of mechanosensitive channels for bacterial infections, including transitioning between host and environment, virulence, and susceptibility to antimicrobials, and discuss their potential as antibacterial drug targets.

RNA-seq analyses of Marine Medaka (Oryzias melastigma) reveals salinity responsive transcriptomes in the gills and livers.

Increasing salinity levels in marine and estuarine ecosystems greatly influence developmental, physiological and molecular activities of inhabiting fauna. Marine medaka (Oryzias melastigma), a euryhaline research model, has extraordinary abilities to survive in a wide range of aquatic salinity. To elucidate how marine medaka copes with salinity differences, the responses of Oryzias melastigma after being transferred to different salt concentrations [0 practical salinity units (psu), 15 psu, 30 psu (control), 45 psu] were studied at developmental, histochemical and transcriptome levels in the gill and liver tissues. A greater number of gills differentially expressed genes (DEG) under 0 psu (609) than 15 psu (157) and 45 psu (312), indicating transcriptomic adjustments in gills were more sensitive to the extreme hypotonic environment. A greater number of livers DEGs were observed in 45 psu (1,664) than 0 psu (87) and L15 psu (512), suggesting that liver was more susceptible to hypertonic environment. Further functional analyses of DEGs showed that gills have a more immediate response, mainly in adjusting ion balance, immune and signal transduction. In contrast, DEGs in livers were involved in protein synthesis and processing. We also identified common DEGs in both gill and liver and found they were mostly involved in osmotic regulation of amino sugar and nucleotide sugar metabolism and steroid biosynthesis. Additionally, salinity stresses showed no significant effects on most developmental and histochemical parameters except increased heartbeat with increasing salinity and decreased glycogen after transferred from stable conditions (30 psu) to other salinity environments. These findings suggested that salinity-stress induced changes in gene expressions could reduce the effects on developmental and histochemical parameters. Overall, this study provides a useful resource for understanding the molecular mechanisms of fish responses to salinity stresses.

102 Loss of volume-regulated anion channel LRRC8 interferes with cell volume regulation and epidermal homeostasis

The volume-regulated anion channel LRRC8 contributes to cell volume regulation upon hypoosmotic stress in keratinocytes. In a hypotonic environment water enters the cell and leads to an increase in cell volume, which is counteracted by a chloride efflux mediated by LRRC8. We found that the essential LRRC8 subunit namely LRRC8A is mainly expressed in the basal epidermal layers with highest expression in transient amplifying cells. Thus, we hypothesize that it potentially plays an important role during the switch between proliferation and differentiation in these cells. To analyze the function of LRRC8A in human keratinocytes, we established a siRNA-mediated knockdown in primary keratinocytes. These cells displayed aberrant expression of involucrin and keratin 10 in differentiating cells, while filaggrin and keratin 14 were unchanged. To overcome experimental variations and get a better understanding of the underlying processes, we generated an LRRC8A- knockout cell line from immortalized keratinocytes using CRISPR/Cas9. The knockout was validated on DNA and protein levels and further verified by manual patch clamping, which showed complete disappearance of VRAC-mediated ionic currents. LRRC8A-/- cells showed a reduced proliferation rate and disturbed epidermal maturation in 2D differentiation assays. Interestingly, LRRC8A-/- cells were unable to reconstitute a 3D epidermis in vitro, indicating that LRRC8 is potentially involved in differentiation initiation. Taken together, our findings suggest that LRRC8 ion channels control cell volume changes in the epidermis, which could represent an essential mechanism, when keratinocytes leave the proliferative, basal compartment and initiate differentiation. Further work will indicate via which molecular processes LRRC8 regulates epidermal renewal and contributes to skin homeostasis.

Optimisation of phagocytosis assay in rainbow trout (Oncorhynchus mykiss)

A phagocytosis assay is one of the most commonly used functional immunological methods. There are many possible ways of assessing leucocytes and their ability to ingest different particles. The aim of this study was to optimise the phagocytosis assay in rainbow trout (Oncorhynchus mykiss) using labelled zymosan particles (Alexa Fluor 488 and Texas Red conjugate). Whole blood was incubated with the particles under different conditions and leucocytes were subsequently isolated by haemolysis in a hypotonic environment. The effect of the different incubation time, temperature, blood volume and dilution on the phagocytic activity was evaluated by flow cytometry. Our experiments showed that the incubation for at least 2 h at 15 °C provided optimal results, while the blood volume and dilution had no significant effect. The optimised assay will be used for the examination of fish health and in further experimental studies.

Micromechanical properties and functional activity of granulocytes when simulating exogenous loading with ATP in vitro

The micromechanical properties of leukocytes make a certain contribution to the blood flow velocity in the microcirculatory bed, while the micromechanical properties themselves change under the influence of a complex network of purinergic signals.The aim of the work was to study the micromechanical properties and functional activity of granulocytes in normal conditions and in patients with acute lymphoblastic leukemia when simulating exogenous loading with ATP in vitro.Materials and methods. Leukocytes were isolated from the blood of patients with acute lymphoblastic leukemia and healthy people. Each sample was divided into a test sample and a control sample. In the test samples, the loading with ATP in vitro was simulated. Leukocytes of the control samples were incubated in the culture medium without the addition of ATP. Young’s modulus and adhesion force were measured using an atomic force microscope in the force spectroscopy mode. The cell surface potential was measured in an atomic force microscope in the Kelvin mode. To assess the functional activity of granulocytes, hypoosmotic tests in vitro and determination of migration activity were used.Results. In tests with exogenous ATP, both in samples from healthy people and from patients with acute lymphoblastic leukemia, a decrease in the rigidity and potential of the plasma membrane surface, an increase in the adhesive properties of leukocytes and migration activity were found. At the same time, the responses of granulocytes to the osmotic loading were different: for example, in the group of healthy people, the loading with ATP caused cell contraction and a decrease in the use of the membrane reserve by the cell in a hypotonic environment, and in patients with acute lymphoblastic leukemia, it caused an increase in the volume and more intensive use of the membrane reserve in volume regulation.Conclusion. The revealed effects indicate the leading role of the ATP molecule in the signal transduction mechanisms between blood cells in the microvasculature. The increase in the adhesive properties of the cell surface of granulocytes revealed in the study, in parallel with the increase in their migration activity under the influence of the ATP molecule, can contribute to the development of inflammation in the vessel wall.

Interleukin 1 beta-induced chloride currents are important in osteoarthritis onset: an in vitro study

Persistent hypotonic and inflammatory conditions in the joint cavity can lead to the loss of cartilage matrix and cell death, which are the important mechanisms of osteoarthritis (OA) onset. Previous studies have confirmed that the existence of a hypotonic environment is a red flag for inflammation, as hypotonic environment induces the opening of the chloride channel of the cell and promotes chloride ion efflux, which prompts the cell volume to increase. Chloride channels play an important role in the regulation of mineralization and chondrocyte death. Here, we reported that OA chondrocytes showed a significant increase of cell death rate and the imbalance of cartilage matrix catabolism. We found that the distribution of skeleton protein F-actin was disordered. In addition, the volume-sensitive chloride current of OA chondrocytes decreased significantly with the increase of the expression levels of inflammation-related proteins caspase-1, caspase-3, and NLRP3. Moreover, interleukin-1β (IL-1β) showed a potential to activate the chloride current of normal chondrocytes. These results indicate that IL-1β-induced chloride channel opening in chondrocytes may be closely related to the occurrence of OA. This chloride channel opening process may therefore be a potential target for the treatment of OA.

PROFIL OSMOTIK GELONDONGAN IKAN BANDENG (Chanos chanos Forsskal) SELAMA PROSES KULTIVASI DI TAMBAK BANDENG DESA WONOREJO KABUPATEN KENDAL

Milkfish is euryhaline fish that can live in wide range salinity, so it will affect with osmoregulation processes. This research aimed to examine the osmotic response of milkfish fingerling during cultivation processes in brackishwater ponds at Wonorejo Village district of Kendal. The research was conducted on July-October 2016. The cultivation process of milkfish fingerling has been cultured for 60 days in 2000 m2 brackishwater pond. The initial density of milkfish larvae was 100 individuals /m2 with a lenght size of 10 mm on average and 15 days old. Samples of milkfish fingerling were taken on the 20th, 40th and 60th days, the number of samples was 3 individuals every size. The measurement result of blood osmolarity was 460, 05 to 490.10 mOsm / l H2O higher than media osmolarity 38,98 – 194,5 mOsm/l H2O. The osmotic response of milkfish fingerling were hyperosmotic to the hypotonic environment and osmoregulator fish.

Capacity for freshwater acclimation and differences in the transcription of ion transporter genes underlying different migratory life histories of Takifugu fish.

The genus Takifugu is a group of approximately 20 species of puffer fishes living in a wide range of salinity environments around East Asian countries. This group presents a broad spectrum of evolutionary stages adapted to anadromy as a result of speciation that occurred a short time (2-5 million years) ago on an evolutionary timescale. This group thus can be considered as a model for studying the evolutionary mechanisms of anadromy. We firstly conducted a transfer experiment from seawater to low-salinity waters on five Takifugu species: two anadromous species T. obscurus and T. ocellatus, two euryhaline wanderer marine species T. rubripes and T. niphobles, and a strictly marine species T. snyderi, and confirmed that the capacity for acclimation to hypotonic environments was associated with their life history strategies. Next, transcriptomes of the gill and intestine of these species in hypotonic condition were compared to those under hypertonic condition for each species using RNA-Sequencing so as to determine possible candidate transporters playing an important role on freshwater adaptation. As this analysis suggested that cftr, encoding an important ion transporter for seawater acclimation in the gill, and ncc, encoding a transporter that is suggested to play important osmoregulatory roles in the intestine, are important candidates, their expression was validated by quantitative real-time PCR analysis. Expression of cftr was downregulated in the gills of the four euryhaline species under the hypotonic condition, but no change was detected in the gill of stenohaline T. snyderi, which may be one reason for the poor hypotonic acclimation capacity of T. snyderi. Expression of ncc was clearly upregulated in the intestines of the two anadromous species under the hypotonic condition, but not in other three species. Different ion transporter expression patterns between the five species indicate that the transcriptional regulation of cftr in the gill and ncc in the intestine may be important for the improvement of hypotonic acclimation capacity and evolution of anadromy in the Takifugu species.