Hyperosmotic Medium Research Articles

The intracellular water volume modulates the accumulation of cadmium in Euglena gracilis

In order to describe a relatively novel mechanism associated to heavy metal resistance in Euglena gracilis, the osmotic responses during cadmium accumulation were analyzed. Several physiological/biochemical parameters were assessed in E. gracilis exposed to 50 and 200 μM Cd2+, concentrations well within the range found in polluted soils and aquatic environments. The Cd2+ accumulation attained after only 24 h exposure induced marked increases in both the intracellular water volume and cellular size, which were maintained for several days; these changes correlated with (i) an increased intracellular osmolarity (i.e. osmotic pressure) driven by a generalized increase in the osmo-metabolites (trehalose, phosphate-molecules, amino acids, thiol-molecules, betaines and polyamines) content; and (ii) the triggering of antioxidant defenses (increased GSH/GSSG ratios and GPx, GR and APx activities). In contrast, no changes in intracellular volume, cellular size and antioxidant status were observed under hyperaccumulation of Zn2+. Furthermore, E. gracilis cultured in a hypoosmotic medium promoted greater Cd2+ accumulation and water volume, compared to a hyperosmotic medium. These changes were significantly decreased by the aquaporin inhibitors Hg2+ and pentamidine. The results suggested that the cell responses to Cd2+ and osmotic stress share biochemical mechanisms in this algae-like protist, in which Cd2+ accumulation closely correlates with the intracellular water volume changes in an oxidative stress-mediated process.

Effect of Diquafosol on Hyperosmotic Stress-induced Tumor Necrosis Factor-α and Interleukin-6 Expression in Human Corneal Epithelial Cells.

PurposeDiquafosol is a pharmaceutical drug used for dry eye treatment with a novel mechanism of action. It is a purinergic P2Y2 receptor agonist that promotes the secretion of tears and healing of corneal epithelial wounds. However, its inhibitory effect on hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs) remains unclear.MethodsA hyperosmotic stress model was established by transferring HCECs from isosmotic (312 mOsm/kg to hyperosmotic medium (500 mOsm/kg). HCECs were incubated with 500 mOsm/kg hyperosmotic medium for 30 minutes, and then treated with diquafosol (0.6–6 mg/mL) for 4 or 24 hours. Cells were then harvested and analyzed by western blot, immunocytochemistry, and real-time polymerase chain reaction to evaluate the expression of interleukin-6, tumor necrosis factor-alpha, and the phosphorylation status of nuclear factor-kappa B.ResultsDiquafosol significantly decreased the mRNA and protein expression of hyperosmotic stress-induced tumor necrosis factor-alpha and interleukin-6. These results were supported by immunofluorescence staining and quantitative real-time polymerase chain reaction analysis. Furthermore, diquafosol inhibits nuclear factor-kappa B activation by suppressing the phosphorylation and degradation of the inhibitor of кB.ConclusionsThis study shows that diquafosol inhibits nuclear factor-kappa B signaling and inflammatory factors induced by hyperosmotic stress in HCECs. This suggests that using diquafosol for the improvement of dry eye syndrome could be effective in the treatment of inflammation-related corneal and conjunctival diseases.

Reaction of osmoregulatory system of sterlet Acipenser ruthenus Linnaeus, 1758 (Acipenseridae) on influence of hyperosmotic medium

Some features of response to hyperosmotic environment (artificial sea water with a salinity of 12.5–12.7 ‰ (403–409 mosm/l) were studied in the osmoregulatory system of freshwater sturgeon species from the Volga river, starlet Acipenser ruthenus Linnaeus, 1758. Morphofunctional changes were traced in certain elements of the organ complex responsible for the osmoregulatory process. 72 hours after the immature sterlet individuals (age 2+) were transferred from fresh water to the hyperosmotic medium, blood serum osmolarity becomes iso-osmotic respective to the external environment. The interrenal gland responds to saline exposure by increasing cortisol concentration in the blood serum. A high cortisol level (75.13 ± 12.96 vs. 19.29 ± 6.36 ng/ml in the control group) persists throughout the entire experimental period (7 days), which indicates that fishes are under stress. The cortisol excretion into the bloodstream is not followed by an increased activity of the transport enzyme, Na+/K+ ATPase, in homogenate of the gills and an increased Na+ concentration in chloride cells, was identified by A-gold technique. The ultrastructure of chloride cells, being the main site for the removal and sorption of monovalent ions (Na+, K+, Cl-) in fishes, does not display the characteristic of an active excretion in sterlet. Thus, cortisol does not provide a stimulating effect on increasing Na+/K+ ATPase activity, an enzyme needed to transport excess ions from the body. The thyroid gland responds by increasing the thyroxin (T4) concentration during 3–6 hours of saline exposure. In the next 114 hours, the concentration of this hormone decreases to its initial level (in the control group). The thyroxin dynamics in serum does not correlate with the dynamics of serum osmolarity. So it seems possible to conclude that no functional relationship exists between the effector “organ” (the set of numerous chloride cells of the gills epithelium) and the endocrine glands (interrenal and thyroid) controlled by the hormones of tropic pituitary cells. In sterlet dwelling in hyperosmotic medium the kidney keeps a higher Na+/K+ ATPase activity, as compared with the gills. High Na+ concentration in the urine (163.2 ± 5.3 meq/l), close to its concentration in the hyperosmotic environment, high Na+ proportion (87.1 ± 0.1%) in the total concentration of major urinary cations, low level of water sorption (50.8 ± 4.0%) in the renal tubules, high diuresis (0.58 ± 0.09, ml/hr/100 g body weight) determine high level of Na+ excretion in the urine (100.95 ± 13.21 meq/ml/hr /100 g body weight). The important role of the kidney in removing Na+ excess under hyperosmotic environment underlies the osmoconformity strategy in sterlet.

Hypertonic external medium represses cellular respiration and promotes Warburg/Crabtree effect.

Hyperosmotic conditions are associated to several pathological states. In this article, we evaluate the consequence of hyperosmotic medium on cellular energy metabolism. We demonstrate that exposure of cells to hyperosmotic conditions immediately reduces the mitochondrial oxidative phosphorylation rate. This causes an increase in glycolysis, which represses further respiration. This is known as the Warburg or Crabtree effect. In addition to aerobic glycolysis, we observed two other cellular responses that would help to preserve cellular ATP level and viability: A reduction in the cellular ATP turnover rate and a partial mitochondrial uncoupling which is expected to enhance ATP production by Krebs cycle. The latter is likely to constitute another metabolic adaptation to compensate for deficient oxidative phosphorylation that, importantly, is not dependent on glucose.

Colon cancer cells cultured under hyperosmotic conditions as in vitro model to investigate dehydration effects on cancer drug susceptibility

In most clinical studies older people are underrepresented compared to the demographic reality. However, risk for some severe diseases like cancer typically increase with age. Most insight into cancer treatment comes from mixed-age patient cohorts, leading to a lack of detailed understanding of cancer drug effects in the elderly population. There is growing evidence that cancer drug effects can be influenced by dehydration conditions often found in older people. Colon cancer remains the second leading cause of death by cancer in Europe. Inter- and intra-heterogeneity of tumors contribute to why some individuals do not respond to specific cancer therapies or may often suffer a relapse. Our study applies an in vitro drug test system for simulating treatment with cytostatics of colorectal cancer in elderly patients with dehydration condition. Two well-known colon cancer cell lines, Caco-2 and RKO, harboring defined cancer-related mutations, were step-wisely adapted from routine culture medium to a severe hyperosmotic condition (397 mOmol/kg) by adding sodium chloride to the medium. We investigated the effects of these cell culture conditions, which should mimic cellular dehydration in elderly people, on the growth characteristics of the cells. Therefore, cell proliferation was investigated by measuring population doubling times. Furthermore, we investigated how the metabolic activity of the cells was influenced by treatment with different concentrations of cyclophosphamide (CPA) under normal and hyperosmotic conditions. We found that Caco-2 and RKO cell lines have an identical cell doubling time of 23 hours in normosmotic medium. However, hyperosmotic medium lifted the doubling time of Caco-2 cells to 31 hours while that of RKO cells did not change. Despite reduced cell proliferation rates, hyperosmotic medium sensitized Caco-2 cells to treatment with 10 mM CPA for 48 hours as measured by metabolic activity assays on ATP levels. The two investigated colon cancer cells lines reacted differently to hyperosmotic conditions. Only the growth of Caco-2 cells was reduced by increased osmolality. Despite this reduced growth their sensitivity to an alkylating cytostatic agent was even slightly increased. We are now in line to examine these effects in more detail and with more tumor cell lines.

Elevation of Intracellular Na+ Contributes to Expression of Early Response Genes Triggered by Endothelial Cell Shrinkage.

Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC. Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR. Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition. Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.

Acute and chronic response to a change in salinity of the euryhaline polychaete Pygospio elegans (Claparède)

Estuaries are thought to be very harsh environments because their physico-chemical parameters, such as salinity, temperature and oxygen, can fluctuate substantially. The distribution of species living in an estuary is largely determined by how well they can cope with such fluctuations. The spionid polychaete Pygospio elegans is common in boreal estuarine habitats that show strong salinity fluctuations, living in habitats ranging from fully marine salinities to brackish environments with salinities as low as 5. In this study we investigated the abilities of P. elegans to cope with an acute as well as a long-term change in salinity. Specimens originating from a salinity of about 15 were exposed to salinity 15 as control and 5 and 30 as low and high salinity treatments. In acute exposure experiments, we measured body volume and tissue water content as well as gene expression of seven genes related to volume- and osmoregulation within 4 h of exposure. In a long-term experiment, we monitored survival, growth and reproduction as well as potential changes in DNA methylation within 6 weeks after a gradual salinity change. In response to abrupt exposure to a hyposmotic medium (salinity 5) we observed increased body volume that could not be fully restored, increased mortality, and no clear change in gene expression. Hence, P. elegans might be a weak cell-volume regulator that cannot cope well with sudden drops in salinity. In contrast, a gradual, long-term change in salinity seemed to be less stressful, although worms at salinity 5 showed slightly increased mortality and reduced or delayed maturity. Overall, there were fewer detrimental effects of exposure to a higher salinity. While the tested salinities fall within the tolerance range of P. elegans from this population, decrease in salinity seems to be a stronger stress.

Osmolarity and calcium regulate connective tissue growth factor (CTGF/CCN2) expression in nucleus pulposus cells

ObjectiveThe objective of our study was to verify the hypothesis that the expression of connective tissue growth factor (CTGF/CCN2), a key molecule essential for the maintenance of nucleus pulposus (NP) matrix homeostasis, is regulated by osmolarity and intracellular calcium in NP cells. MethodsGene and protein expression levels of CCN2 were assessed using quantitative real-time PCR and western blot. Transfections and dual luciferase assays were performed to measure the effect of hyperosmolarity, tonicity enhancer binding protein (TonEBP) and Ca2+-calcineurin (Cn)-NFAT signaling on CCN2 promoter activity. ResultsCultured in hyperosmotic media, there was a significant decrease in the levels of CCN2 promoter activity, gene and protein expression in NP cells. The JASPAR database was used to analyze the construction of human CCN2 promoter, we found conserved TonE and NFAT binding sites. We then investigated whether TonEBP controlled CCN2 expression. Forced expression of TonEBP in NP cells showed that TonEBP negatively regulated CCN2 promoter activity, while suppression of TonEBP induced CCN2 promoter activity and expression. We then examined if Ca2+-Cn-NFAT signaling participated in the regulation of CCN2 expression. Co-expression of CCN2 reporter with individual NFAT1–4 expression plasmids and/or calcineurin A/B constructs suggested this signaling pathway played a role in the regulation of CCN2expression in NP cells. ConclusionsResults of these studies illustrated that the expression of CCN2 in NP cells was regulated by the NFAT family through a signaling pathway network involving both activator (Ca2+-Cn-NFAT signaling) and suppressor (Hyperosmolarity-TonEBP) molecules.

Calcitriol inhibits ROS-NLRP3-IL-1β signaling axis via activation of Nrf2-antioxidant signaling in hyperosmotic stress stimulated human corneal epithelial cells

PurposeThe activation of ROS-NLRP3-IL-1β signaling axis induced by hyperosmotic stress (HS) has been recognized as a key priming stage of epithelial inflammation in dry eye pathogenesis. The current study aims to investigate whether calcitriol, the active metabolite of vitamin D3, could protect cells against HS-induced inflammation through modulating this critical step. MethodsHuman corneal epithelial cells (iHCECs) were cultured in hyperosmotic medium (450 mOsM) with various concentrations of calcitriol. Small interfering RNA (siRNA) was used to knock down the expression of vitamin D receptor (VDR) in iHCECs. NLRP3 activation and IL-1β generation were detected by RT-qPCR or ELISA, respectively. Oxidative stress markers including ROS and 8-OHdG were examined by fluorometric analysis. The nuclear translocation of NRF2 was assessed by western blotting. ResultsCalcitriol could protect cells against HS-induced injury through inhibiting ROS-NLRP3-IL-1β signaling axis. Calcitriol remarkably suppressed the expression of NLRP3 inflammasome related genes and the production of IL-1β in cells that were exposed to HS. It could also significantly attenuate HS-induced oxidative stress, shown as the reduced intracellular ROS generation and 8-OHdG staining cells after calcitriol treatment. Calcitriol induced the translocation of NRF2 to the nucleus, and thereby triggered the expression of several antioxidant enzymes. ConclusionThe current study indicated that calcitriol could inhibit the priming stage of HS-induced cellular inflammation, highlighting its potential capacity to prevent and mitigate dry eye related corneal inflammation at an earlier stage.

Osteogenic protein-1 attenuates nucleus pulposus cell apoptosis through activating the PI3K/Akt/mTOR pathway in a hyperosmotic culture.

Background: Previous studies have indicated that osteogenic protein-1 has protective effects on the biological functions of intervertebral disc cells. Hyperosmolarity is an important physicochemical factor within the disc nucleus pulposus (NP) region, which obviously promotes NP cell apoptosis.Objective: To study the effects of osteogenic protein-1 (OP-1) on NP cell apoptosis induced by hyperosmolarity and the potential signaling transduction pathway.Methods: Rat NP cells were cultured in a hyperosmotic medium with or without OP-1 addition for 7 days. Inhibitor 294002 and inhibitor FK-506 were used to investigate the role of the PI3K/Akt/mTOR pathway in this process. NP cell apoptosis were evaluated by cell apoptosis ratio, activity of caspase-3/9 and gene/protein expression of apoptosis-related molecules (Bax, Bcl-2, caspase-3/cleaved caspase-3 and cleaved PARP).Results: OP-1 addition obviously decreased cell apoptosis ratio and caspase-3/9 activity, down-regulated gene/protein expression of pro-apoptosis molecules (Bax, caspase-3/cleaved casepase-3 and cleaved PARP), up-regulated gene/protein expression of anti-apoptosis molecule (Bcl-2) in a hyperosmotic culture. Moreover, OP-1 addition significantly increased protein expression of p-Akt and p-mTOR. Further analysis showed that addition of LY294002 and FK-506 partly attenuated these protective effects of OP-1 against NP cell apoptosis and activation of the PI3K/Akt/mTOR pathway in a hyperosmotic culture.Conclusion: OP-1 can attenuate NP cell apoptosis through activating the PI3K/Akt/mTOR pathway in a hyperosmotic culture. The present study sheds a new light on the protective role of OP-1 in regulating disc cell biology and provides some theoretical basis for the application of OP-1 in retarding/regenerating disc degeneration.

Poly(ethylene glycol) induces cell toxicity in melanoma cells by producing a hyperosmotic extracellular medium.

Poly(ethylene glycol) is a polymer that is widely used as a biomaterial and has been approved in a host of applications. While generally viewed as inert, recent studies with poly(ethylene glycol) suggest that it may have some effects on cells and tissues, making it potentially attractive as a therapeutic agent. In this study, the effect of poly(ethylene glycol) on the cell viability, membrane transport and apoptotic markers of metastatic melanoma cells was examined. The data were combined with observed effects of the polymer on the cell media, including osmolality and viscosity, in order to elucidate any structure-function relationship between the polymer and cells. It was observed that poly(ethylene glycol) reduced the cellular viability of A375 cells, and that the effect was dependent on poly(ethylene glycol) molecular weight and concentration. The mechanism was highly correlated with changes in the osmolality of the cell medium, which is determined by the inherent structure of poly(ethylene glycol), and in particular the ethylene oxide units. This mechanism was specific to poly(ethylene glycol) and was not observed with the similar linear, hydrophilic polymer poly(vinyl pyrrolidone). Overall, the data suggest that poly(ethylene glycol) and poly(ethylene glycol)-like compounds have a distinct effect on cellular activity, presumably mediated in part by their osmotic effects, supporting the further investigation of these polymers as pharmaceutically active compounds.

The Protective Effect of Polygonum cuspidatum (PCE) Aqueous Extract in a Dry Eye Model.

Dry eyes are caused by highly increased osmolarity of tear film, inflammation, and apoptosis of the ocular surface. In this study, we investigated the effect of Polygonum cuspidatum (PCE) aqueous extract in in vivo and in vitro dry eye models. Dry eye was induced by excision of the lacrimal gland and hyperosmotic media. In vivo, oral administration of PCE in exorbital lacrimal gland-excised rats recovered tear volume and Mucin4 (MUC4) expression by inhibiting corneal irregularity and expression of inflammatory cytokines. In vitro, hyperosmotic media induced human corneal epithelial cell (HCEC) cytotoxicity though increased inflammation, apoptosis, and oxidative stress. PCE treatment significantly inhibited expression of cyclooxygenase-2 and inflammatory cytokines (interleukin-6 and tumor necrosis factor-α), and activation of NF-κB p65 in hyperosmolar stress-induced HCECs. Hyperosmolarity-induced increase in Bcl-2-associated X protein (BAX) expression and activation of cleaved poly (ADP-ribose) polymerase and caspase 3 were attenuated in a concentration-dependent manner by PCE. PCE treatment restored anti-oxidative proteins such as heme oxygenase-1 (HO-1), superoxide dismutase-1 (SOD-1), and glutathione peroxidase (GPx) in hyperosmolar stress-induced HCECs. These data demonstrate that PCE prevents adverse changes in the ocular surface and tear fluid through inhibition of hyperosmolar stress-induced inflammation, apoptosis, and oxidation, suggesting that PCE may have the potential to preserve eye health.

In-Cell Titration of Small Solutes Controls Protein Stability and Aggregation.

The components of the intracellular environment vary widely in size: from large multiprotein complexes to atomic ions. Besides water, low-molecular-weight solutes (<1 kDa) such as electrolytes, metabolites, and carbohydrates are by far the most abundant of these components. Small solutes are thus key contributors to the solvation environment in the cell. Small solutes have been known for decades to alter protein structure or activity in vitro, through their interactions with protein surfaces or hydration shells. Here we use the cell itself as our test tube, by titrating its hydration, ion, or carbohydrate composition systematically. We trigger the selective uptake of specific solutes by exposing cells to hyperosmotic media. We then measure protein structure, stability, unfolding kinetics, and aggregation in these different intracellular environments by using fast relaxation imaging. We compare these results with controls where solutes cannot enter the cell and only hydration is altered. Protein structure, thermal stability, and aggregation onset all depend on the concentration and chemical nature of the solute titrated into the cell. Our work highlights the important contributions of small solutes in defining how proteins interact within the cell and suggests that intracellular variation of the solute composition could be an important regulator of protein function.

Hyperosmotic Stress-Induced TRPM2 Channel Activation Stimulates NLRP3 Inflammasome Activity in Primary Human Corneal Epithelial Cells.

The purpose of this study was to determine whether either a hyperosmotic or oxidative stress induces NLRP3 inflammasome activation and increases in bioactive IL-1β secretion through transient receptor potential melastatin 2 (TRPM2) activation in primary human corneal epithelial cells (PHCECs). Real-time PCR, Western blots, and immunofluorescent staining were used to evaluate TRPM2 and NLRP3, ASC, caspase-1, and IL-1β mRNA and protein expression levels, respectively. A CCK-8 assay evaluated cell viability. Hyperosmotic 500 mOsm and oxidative 0.5 mM H2O2 stresses were imposed. TRPM2 expression was inhibited with a TRPM2 inhibitor, 20 μM N-(p-amylcinnamoyl) anthranilic acid (ACA), or TRPM2 siRNA knockdown. In the hypertonic medium, TRPM2, NLRP3, ASC, caspase-1, and IL-1β gene and protein expression levels rose after 4 hours (P ≤ 0.043), whereas ACA preincubation suppressed these rises (P ≤ 0.044). Similarly, H2O2 upregulated TRPM2 protein expression by 80%, and induced both NLRP3 inflammasome activation and increased bioactive IL-1β secretion (P ≤ 0.036), whereas ACA pretreatment suppressed these effects (P ≤ 0.029). TRPM2 siRNA transfection reduced TRPM2 gene expression by 70% (P = 0.018) in this hyperosmotic medium and inhibited the increases in NLRP3, caspase-1, and IL-1β gene (P ≤ 0.028) and protein expression (P ≤ 0.037). TRPM2 activation by either a hyperosmotic or oxidative stress contributes to mediating increases in NLRP3 inflammasome activity and bioactive IL-1β expression because inhibiting TRPM2 activation or its expression blunted both of these responses in PHCECs. This association points to the possibility that TRPM2 is a viable target to suppress hyperosmotic-induced corneal epithelial inflammation.

Functional characterization of CgPBS2, a MAP kinase kinase in Colletotrichum gloeosporioides, using osmotic stress sensitivity as a selection marker

Colletotrichum leaf disease of Hever brasiliensis (rubber tree) caused by C. gloeosporioides is one of the major causes of declining rubber tree yields. Little is known about the fungal molecular characters that are important for pathogenicity on rubber tree and fungicide resistance. In this study, we cloned the CgPBS2 gene, the key component of the Hog1 pathway which controls various aspects of osmoregulation and fungicide resistance in various fungal pathogens, including the causal agent of Colletotrichum leaf disease of rubber tree. We characterized the function of the CgPBS2 gene by reverse genetics. Because the Hog1 pathway plays an important role in stress responses, we obtained a CgPBS2 gene deletion mutant by PEG-mediated transformation of protoplasts after reducing the concentration of sucrose in the screening medium from 1.0 M to 0.2 M. Then, the complemented transformants and GFP-labelled CgPBS2 gene transformants were selected directly under highly hyperosmotic medium (PDA + 1.5 M sorbitol) without using other selectable gene markers. Phenotypic observations showed that the CgPBS2 protein was mainly localized in the conidial cytoplasm of the CgPBS2-GFP transformants. In addition, disruption of CgPBS2 led to sensitivity to hyperosmosis and high salt concentration as well as resistance to the fungicide fludioxonil. No obvious difference in virulence was observed between the null mutant and the wild-type strain. These results provide insights into the role of the CgPBS2 gene in osmotic stress, salt stress and fludioxonil resistance and suggest that osmotic stress sensitivity can be used as a selection marker.

Effect of pH, osmolality and temperature on sperm motility of pink cusk-eel (Genypterus blacodes, (Forster, 1801))

In this research we evaluated simple aspects of the sperm biology of Genypterus blacodes, in particular assessing the effects of pH (6, 7, 8 and 9), temperature (4, 8 and 16 °C) and osmolalities 100% sea water (1010 mOsm/kg, Control), 75% sea water (774 mOsm/kg, T1), 50% sea water (488 mOsm/kg, T2) and distilled water (0 mOsm/kg, T3)) on the motility of Genypterus blacodes intratesticular spermatozoa.In addition, we determined the fertilization rate. Our results show that G. blacodes spermatozoa have a sperm density of 5.35 ± 0.16 × 109 spermatozoa/mL. Sperm motility is initiated on contact with a hyperosmotic swimming medium under normal conditions (1010 mOsm/kg, pH 8 and 8 °C). The longest motility duration (432.48 ± 8.89 s) was recorded at 4 °C. The maximum percentage of motile cells was recorded at 8 °C (65.66 ± 4.95) at osmolality 1010 mOsm/kg, whereas an optimum was observed at pH 8. The fertility rate was 73.9 ± 17%. This is the first report on sperm motility of G. blacodes spermatozoa. In conclusion, the results of this study permit a baseline to be established for further research and protocols for artificial reproduction of this species to be developed and optimized. In addition, the information gathered in this research will be useful for developing the biotechnology of Genypterus blacodes.

COX-2 expression mediated by calcium-TonEBP signaling axis under hyperosmotic conditions serves osmoprotective function in nucleus pulposus cells

The nucleus pulposus (NP) of intervertebral discs experiences dynamic changes in tissue osmolarity because of diurnal loading of the spine. TonEBP/NFAT5 is a transcription factor that is critical in osmoregulation as well as survival of NP cells in the hyperosmotic milieu. The goal of this study was to investigate whether cyclooxygenase-2 (COX-2) expression is osmoresponsive and dependent on TonEBP, and whether it serves an osmoprotective role. NP cells up-regulated COX-2 expression in hyperosmotic media. The induction of COX-2 depended on elevation of intracellular calcium levels and p38 MAPK pathway, but independent of calcineurin signaling as well as MEK/ERK and JNK pathways. Under hyperosmotic conditions, both COX-2 mRNA stability and its proximal promoter activity were increased. The proximal COX-2 promoter (-1840/+123 bp) contained predicted binding sites for TonEBP, AP-1, NF-κB, and C/EBP-β. While COX-2 promoter activity was positively regulated by both AP-1 and NF-κB, AP-1 had no effect and NF-κB negatively regulated COX-2 protein levels under hyperosmotic conditions. On the other hand, TonEBP was necessary for both COX-2 promoter activity and protein up-regulation in response to hyperosmotic stimuli. Ex vivo disc organ culture studies using hypomorphic TonEBP+/- mice confirmed that TonEBP is required for hyperosmotic induction of COX-2. Importantly, the inhibition of COX-2 activity under hyperosmotic conditions resulted in decreased cell viability, suggesting that COX-2 plays a cytoprotective and homeostatic role in NP cells for their adaptation to dynamically loaded hyperosmotic niches.

Sucrose assists selection of high-quality oocytes in pigs.

We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.

Zidovudine protects hyperosmolarity-stressed human corneal epithelial cells via antioxidant pathway

Dry Eye Disease (DED) is a very common disorder that can result in severe disability and vision loss. Although the pathogenesis of DED is not fully understood, hyperosmolarity, inflammation, and tear film instability are recognized as hallmarks of DED. Recently, Nucleoside Reverse Transcriptase Inhibitors (NRTIs), a class of medication used to treat HIV, have been shown to inhibit inflammation in a mouse model of retinal atrophy. In this study, we investigated whether Zidovudine (AZT) can inhibit human corneal epithelial cell (HCEC) inflammatory responses under hyperosmotic conditions. HCECs were cultured in hyperosmotic media containing AZT. Cell viability, cytokine production, and reactive oxygen species (ROS) production were measured. We found that AZT decreased nuclear factor kappa B (NF-κB) and Interleukin-6 (IL-6) levels, increased Superoxide Dismutase 1 (SOD1) production, decreased ROS production, and increased cell viability. These results support the novel use of AZT in the reduction of ocular surface inflammation and the promotion of corneal health in the context of DED.

Changes of Somatotropin Concentration in Blood Serum of Juvenile Russian Sturgeon Acipenser gueldenstaedtii (Acipenseriformes) During Adaptation to Hyperosmotic Medium

To study the role of somatotropin (ST) in osmotic regulation of anadromous acipenserids, the dynamics of hormone concentration in blood serum of juvenile Russian sturgeon Acipenser gueldenstaedtii Brandt (age 4 months, average length 16.9 cm, average weight 17.1 g) during fish adaptation to hyperosmotic medium of 12.5‰ salinity (403 mOsm/l) was evaluated for the first time. During the first 24 h after the transfer of fish from freshwater to hyperosmotic medium, an increase of ST serum concentration (up to 2.06 ± 0.09 vs. 0.98 ± 0.03 pg/ml in control fish from freshwater) was observed. This increase occurs concurrently with an increase in blood serum osmolarity (up to 386.2 ± 4.0 mOsm/l vs. 218.5 ± 4.9 mOsm/l in control) resulting from the diffusion of water from the organism under new osmotic gradient, as well as with the period of morphofunctional remodelling of the osmoregulatory system corresponding to the transition of fish to hypoosmotic regulation. 24 h after the start of the experiment, fish that have shifted to hypoosmotic regulation display the adaptive decrease in osmolarity (aimed at maintaining the relative constancy of blood serum) and the ST concentration (down to 1.18 ± 0.16 pg/ml after 120 h, comparable with that in the control fish). It is concluded that during transition of juvenile Russian sturgeon to hypoosmotic regulation, ST becomes involved in morphofunctional remodelling of the osmoregulatory system concurrently with other components of this system. ST-immunopositive cells were revealed in the dorsal part of mesoadenohypophysis of Russian sturgeon juveniles by immunochemical method.