Hypoxic and osmotic expression of Kir2.1 potassium channels in retinal pigment epithelial cells: Contribution to vascular endothelial growth factor expression.

Retinal pigment epithelial (RPE) cells express different subtypes of inwardly rectifying potassium (Kir) channels. We investigated whether human and rat RPE cells express genes of strongly rectifying Kir2 channels. We also determined the hypoxic and hyperosmotic regulation of Kir2.1 gene expression in cultured human RPE cells and the effects of siRNA-mediated knockdown of Kir2.1 on VEGFA expression, VEGF secretion, proliferation, and viability of the cells. Extracellular hyperosmolarity was induced by addition of NaCl or sucrose. Hypoxia and chemical hypoxia were produced by cell culture in 0.25% O2 and addition of CoCl2, respectively. Gene expression levels were evaluated by real-time RT-PCR. Rat RPE cells contained Kir2.1, Kir2.2, Kir2.3, and Kir2.4 gene transcripts while human RPE cells contained Kir2.1, Kir2.2, and Kir2.4 transcripts. Immunocytochemical data may suggest that Kir2.1 protein in cultured human cells is expressed in both perinuclear and plasma membranes. Kir2.1 gene expression and Kir2.1 protein level in human cells increased under hypoxic and hyperosmotic conditions. The expression of the Kir2.1 gene was mediated in part by diverse intracellular signal transduction pathways and transcription factor activities under both conditions; the hyperosmotic, but not the CoCl2-induced Kir2.1 gene expression was dependent on intracellular calcium signaling. Autocrine/paracrine activation of purinergic receptors contributed to Kir2.1 gene expression under hyperosmotic (P2Y1, P2Y2, P2X7) and CoCl2-induced conditions (P2Y2, P2X7). Exogenous VEGF, TGF-ß1, and blood serum decreased Kir2.1 gene expression. Inhibition of VEGF receptor-2 increased the Kir2.1 gene expression under control conditions and in CoCl2-simulated hypoxia, and decreased it under high NaCl conditions. Knockdown of Kir2.1 by siRNA inhibited the CoCl2-induced and hyperosmotic transcription of the VEGFA gene and caused a delayed decrease of the constitutive VEGFA gene expression while VEGF protein secretion was not altered. Kir2.1 knockdown stimulated RPE cell proliferation under control and hyperosmotic conditions without affecting cell viability. The data indicate that Kir2.1 channel activity is required for the expression of the VEGFA gene and inhibits the proliferation of RPE cells. Under control and hypoxic conditions, the extracellular VEGF level may regulate the production of VEGF via its inhibitory effect on the Kir2.1 gene transcription; this feedback loop may prevent overproduction of VEGF.

Combined hyperosmolarity and inflammatory conditions in stressed human corneal epithelial cells and macrophages to evaluate osmoprotective agents as potential DED treatments.

PURPOSE: To develop an easy-to-perform combined model in human corneal epithelial cells (HCECs) and Balb/c mice macrophages J774.A1 (MP) for preliminary screening of potential ophthalmic therapeutic substances. METHODS: HCECs were exposed to different osmolarities (350-500 mOsm/L) and MTT assay was employed for cell survival and flow cytometry to assess apoptosis-necrosis and relative cell size (RCS) distribution. Effectiveness of Betaine, L-Carnitine, Taurine at different concentrations (ranging from 20 mM to 200 mM) was studied. Also, mucoadhesive polymers such as Hyaluronic acid (HA) and Hydroxypropylmethylcellulose (HPMC) (0.4 and 0.8%) were evaluated. Cells were pre-incubated with the compounds (8h) and then exposed to hyperosmotic stress (470 mOsm/L) for 16h. Moreover, anti-inflammatory activity was performed in LPS-stimulated MP. RESULTS: Exposure to hyperosmotic solutions between 450 and 500 mOsm/L promoted the highest cell death after 16h exposures (p < 0.0001) with a drop in viability to 34.96% ± 11.77 for 470 mOsm/L. Pre-incubation with Betaine at 150 mM and 200 mM provided the highest cell survival against hyperosmolarity (66.01% ± 3.65 and 65.90% ± 0.78 respectively) while HA 0.4% was the most effective polymer in preventing cell death (42.2% ± 3.60). Flow cytometry showed that Betaine and Taurine at concentrations between 150-200 mM and 20-80 mM respectively presented the highest anti-apoptotic activity. Also, HA and HPMC polymers reduced apoptotic-induced cell death. All osmoprotectants modified RCS, and polymers increased their value over 100%. L-Carnitine 50 mM, Taurine 40 mM and HA 0.4% presented the highest TNF-α inhibition activity (60%) albeit all of them showed anti-inflammatory inhibition percentages higher than 20% CONCLUSIONS: HCECs hyperosmolar model combined with inflammatory conditions in macrophages allows the screening of osmoprotectants by simulating chronic hyperosmolarity (16h) and inflammation (24h).

Cell volume regulation modulates NLRP3 inflammasome activation.

Cell volume regulation is a primitive response to alterations in environmental osmolarity. The NLRP3 inflammasome is a multiprotein complex that senses pathogen- and danger-associated signals. Here, we report that, from fish to mammals, the basic mechanisms of cell swelling and regulatory volume decrease (RVD) are sensed via the NLRP3 inflammasome. We found that a decrease in extracellular osmolarity induced a K(+)-dependent conformational change of the preassembled NLRP3-inactive inflammasome during cell swelling, followed by activation of the NLRP3 inflammasome and caspase-1, which was controlled by transient receptor potential channels during RVD. Both mechanisms were necessary for interleukin-1ß processing. Increased extracellular osmolarity prevented caspase-1 activation by different known NLRP3 activators. Collectively, our data identify cell volume regulation as a basic conserved homeostatic mechanism associated with the formation of the NLRP3 inflammasome and reveal a mechanism for NLRP3 inflammasome activation.

Hypertonic versus isotonic crystalloid infusion for cerebral perfusion pressure in a porcine experimental cardiac arrest model.

BACKGROUND: The effect of intravenous (IV) fluid administration type on cerebral perfusion pressure (CePP) during cardiopulmonary resuscitation (CPR) is controversial. The purpose of this study was to evaluate the association between IV fluid type and CePP in a porcine cardiac arrest model. METHODS: We randomly assigned 12 pigs to the hypertonic crystalloid, isotonic crystalloid and no-fluid groups. After 4 min of untreated ventricular fibrillation (VF), chest compression was conducted for 2 cycles (CC only). Chest compression with IV fluid infusion (CC + IV) was followed for 2 cycles. Advanced life support, including defibrillation and epinephrine, was added for 8 cycles (ALS phase). Mean arterial pressure (MAP), intracranial pressure (ICP) and CePP were measured. A paired t-test was used to measure the mean difference in CePP. RESULTS: Twelve pigs underwent the experiment. The hypertonic crystalloid group showed higher CePP values than those demonstrated by the isotonic crystalloid group from ALS cycles 2 to 8. The MAP values in the hypertonic group were higher than those in the isotonic group starting at ALS cycle 2. The ICP values in the hypertonic group were lower than those in the isotonic group starting at ALS cycle 4. From ALS cycles 2 to 8, the reduction in the mean difference in the isotonic group was larger than that in the other groups. CONCLUSION: In a VF cardiac arrest porcine study, the hypertonic crystalloid group showed higher CePP values by maintaining higher MAP values and lower ICP values than those of the isotonic crystalloid group.

Activating PAX gene family paralogs to complement PAX5 leukemia driver mutations.

PAX5, one of nine members of the mammalian paired box (PAX) family of transcription factors, plays an important role in B cell development. Approximately one-third of individuals with pre-B acute lymphoblastic leukemia (ALL) acquire heterozygous inactivating mutations of PAX5 in malignant cells, and heterozygous germline loss-of-function PAX5 mutations cause autosomal dominant predisposition to ALL. At least in mice, Pax5 is required for pre-B cell maturation, and leukemic remission occurs when Pax5 expression is restored in a Pax5-deficient mouse model of ALL. Together, these observations indicate that PAX5 deficiency reversibly drives leukemogenesis. PAX5 and its two most closely related paralogs, PAX2 and PAX8, which are not mutated in ALL, exhibit overlapping expression and function redundantly during embryonic development. However, PAX5 alone is expressed in lymphocytes, while PAX2 and PAX8 are predominantly specific to kidney and thyroid, respectively. We show that forced expression of PAX2 or PAX8 complements PAX5 loss-of-function mutation in ALL cells as determined by modulation of PAX5 target genes, restoration of immunophenotypic and morphological differentiation, and, ultimately, reduction of replicative potential. Activation of PAX5 paralogs, PAX2 or PAX8, ordinarily silenced in lymphocytes, may therefore represent a novel approach for treating PAX5-deficient ALL. In pursuit of this strategy, we took advantage of the fact that, in kidney, PAX2 is upregulated by extracellular hyperosmolarity. We found that hyperosmolarity, at potentially clinically achievable levels, transcriptionally activates endogenous PAX2 in ALL cells via a mechanism dependent on NFAT5, a transcription factor coordinating response to hyperosmolarity. We also found that hyperosmolarity upregulates residual wild type PAX5 expression in ALL cells and modulates gene expression, including in PAX5-mutant primary ALL cells. These findings specifically demonstrate that osmosensing pathways may represent a new therapeutic target for ALL and more broadly point toward the possibility of using gene paralogs to rescue mutations driving cancer and other diseases.

Hypertonicity-responsive ubiquitin ligase RNF183 promotes Na, K-ATPase lysosomal degradation through ubiquitination of its ß1 subunit.

We previously reported that RNF183, a member of the RING finger (RNF) protein family, is specifically expressed in the renal collecting duct and that RNF183 mRNA is induced by the activity of nuclear factor of activated T cells 5 (NFAT5), which regulates the transcription of essential proteins for adaptation to hypertonic conditions. The renal medulla is the only tissue that is continuously hypertonic; therefore, RNF183 possibly plays an important role in adaptation to continuous hypertonic conditions. However, the mechanism of how cells adapt to long-term hypertonicity via RNF183 remains unclear. In this study, the Na, K-ATPase α1 subunit was identified as a candidate substrate of RNF183 by the BirA proximity-biotinylation technique. The Na, K-ATPase α1 subunit acts as an ion transporter along with the Na, K-ATPase ß1 subunit at the plasma membrane. We confirmed that RNF183 interacted with both α1 and ß1 subunits; however, we found that RNF183 ubiquitinated only the ß1 subunit, not the α1 subunit. Furthermore, RNF183 translocated both α1 and ß1 subunits from the plasma membrane to lysosomes. In addition, the expression levels of α1 and ß1 subunits in HEK293 cells stably expressing RNF183 were significantly decreased compared with mock control cells, and were restored by siRNA-mediated knockdown of RNF183. Moreover, in RNF183-expressing cells, chloroquine treatment increased the protein levels of the α1 and ß1 subunits. Therefore, our results suggest that Na, K-ATPase α1 and ß1 subunits are degraded in lysosomes by RNF183-mediated ubiquitination of ß1 subunit.

Physical-mathematical Model of Substance Redistribution between the Cell and its Hypertonic Solution Environment of Penetrating Cryoprotectants with Relevance to Membrane Potential.

BACKGROUND: The redistribution of basic ions between the cell cytoplasm and its surrounding medium due to osmotic action affects transmembrane potential and plasma membrane integrity at all stages of low temperature preservation. OBJECTIVE: To develop a physical-mathematical model describing the redistribution of osmotically active solutes between the cell and its hypertonic solutions of penetrating cryoprotectants that enables the calculation of kinetic changes in cell volume, cryoprotectant and ion concentrations, as well as the cell transmembrane potential during cell equilibration with cryoprotectant solutions. MATERIALS AND METHODS: The study has modeled the mass transfer process of mouse oocytes upon exposure to 1.5 M DMSO and 1,2-Propanediol (1,2-PD) solutions. RESULTS: Equations for changes of the normalized volume as well as intracellular concentrations of DMSO, 1,2-PD, potassium, sodium, chlorine and transmembrane potential have been obtained in a dimensionless form. The membrane permeability coefficients for DMSO and 1,2-propanediol have been determined and compared with the data of Paynter et al (5). CONCLUSION: The study shows that the incorporation of transmembrane ion movement and electrical potential change in the mathematical model leads to lower values of mouse oocyte membrane permeability coefficients for water and cryoprotectants in comparison with data determined by the traditional model.

Application of continuous-wave photoacoustic sensing to red blood cell morphology.

The feasibility of continuous wave laser-based photoacoustic (CWPA) response technique in detecting the morphological changes in cells during the biological studies, through the features extracted from CWPA signal (i.e., amplitude) is demonstrated here. Various hematological disorders (e.g., sickle cell anemia, thalesemia) produce distinct changes at the cellular level morphologically. In order to explore the photoacoustic response technique to detect these morphological changes, we have applied CWPA technique onto the blood samples. Results of our preliminary study show a distinct change in the signal amplitude of photoacoustic (PA) signal due to a change in the concentration of blood, which signifies the sensitivity of the technique towards red blood cell (RBC) count (related to hematological disease like anemia). Further hypotonic and hypertonic solutions were induced in blood to produce morphological changes in RBCs (i.e., swollen and shrink, respectively) as compared to the normal RBCs. Experiments were performed using continuous wave laser-based photoacoustic response technique to verify the morphological changes in these RBCs. A distinct change in the PA signal amplitude was found for the distinct nature of RBCs (swollen, shrink, and normal). Thus, this can serve as a diagnostic signature for different biological studies based on morphological changes at cellular level. The experiments were also performed using conventional pulsed laser photoacoustic response technique which uses nano-second pulsed laser and the results obtained from both PA techniques were validated to produce identical changes. This demonstrates the utility of continuous wave laser-based photoacoustic technique for different biological studies related to morphological cellular disorders.

Osmolyte taurine induction in UVA exposed human retinal pigment epithelial cells.

AIM: To explore the osmolytes expression in ultraviolet (UVA) stressed human retinal pigment epithelial cells. METHODS: Osmolyte transporters and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) were determined by real time polymerase chain reaction (PCR). Osmolyte uptake was measured by radioimmunoassay. VEGF concentrations were determined by immunoassay and enzyme-linked immunosorbent assay (ELISA). Osmolyte taurine transporter (TAUT) were silenced by siRNA technology. RESULTS: Hypertonicity accelerated osmolyte betaine uptake, myoinositol uptake, and taurine uptake, compared to normotonic stress. UVA irradiation also accelerated osmolyte transporters expression and osmolytes uptake. Especially, osmolyte taurine remarkably inhibited VEGF release induced by UVA irradiation. VEGF in the UVA stressed retinal pigment epithelial cell supernatant was accumulated slow after taurine preincubation. VEGF expression increased significantly in UVA-stressed cells after TAUT silencing. Moreover, taurine reduced the VEGF level in human ocular aqueous humor. CONCLUSION: The inhibition of VEGF by osmolyte taurine plays the crucial role in retina adaption to UVA irradiation.

Role of nuclear factor of activated T-cells 5 in regulating hypertonic-mediated secretin receptor expression in kidney collecting duct cells.

A growing body of evidence suggests that secretin (SCT) is an important element in the osmoregulatory pathway. It is interesting to note that both SCT and its receptor (SCTR) gene are activated upon hyperosmolality in the kidney. However, the precise molecular mechanisms underlying the induction of the SCTR gene expression in response to changes in osmolality have yet to be clarified. Detailed DNA sequence analysis of the promoter regions of the SCTR gene reveals the presence of multiple osmotic response elements (ORE). The ORE is the binding site of a key osmosensitive transactivator, namely, the nuclear factor of activated T-cells 5 (NFAT5). SCTR and NFAT5 are co-expressed in the kidney cortex and medulla collecting duct cells. We therefore hypothesize that NFAT5 is responsible for modulating SCTR expression in hypertonic environments. In this study, we found hypertonicity stimulates the promoter activities and endogenous gene expression of SCTR in mouse kidney cortex collecting duct cells (M1) and inner medulla collecting duct cells (mIMCD3). The overexpression and silencing of NFAT5 further confirmed it to be responsible for the up-regulation of the SCTR gene under hypertonic conditions. A significant increase in the interaction between NFAT5 and the SCTR promoter was also observed following chromatin immunoprecipitation assay. In vivo, osmotic stress up-regulates the SCTR gene in the kidney cortex and medulla of wild-type mice, but does not do so in NFAT5(+/-) animals. Hence, this study provides comprehensive information on how NFAT5 regulates SCTR expression in different osmotic environments.

Protective effects of hydrogen-rich saline against N-methyl-N-nitrosourea-induced photoreceptor degeneration.

The N-methyl-N-nitrosourea (MNU)-treated rat is typically used as an animal model of chemically-induced retinitis pigmentosa (RP). Reactive oxygen species (ROS) have been recognized as the crucial contributor to the retinal photoreceptor apoptosis seen in MNU-treated rats. In the present study, we explored the therapeutic effects of hydrogen-rich saline (HRS), a selective ROS scavenger, on MNU-induced photoreceptor degeneration. Intraperitoneal (IP) administration of HRS ameliorated MNU-induced photoreceptor degeneration in terms of morphology and function: Sharply decreased thickness of the retinal outer nuclear layer (ONL) and flattened photopic and scotopic electroretinogram (ERG) waveforms, typically seen in response to MNU treatment, were substantially rescued in rats cotreated with MNU and HRS (MNU + HRS). Moreover, the terminal deoxyuridine triphosphate nick-end labeling (TUNEL) assay revealed a smaller number of apoptotic photoreceptors in the MNU + HRS group compared that in the MNU group. Compared to MNU-treated rats, retinal malondialdehyde (MDA) content in MNU + HRS rats significantly decreased while superoxide dismutase (SOD) activity significantly increased. Morphological and multi-electrode array (MEA) analyses revealed more efficient preservation of the architecture and field potential waveforms in particularly the peripheral regions of the retinas within the MNU + HRS group, compared to that in the MNU group. However, this enhanced protection of structure and function in the peripheral retina is unlikely the result of site-dependent variation in the efficacy of HRS; rather, it is most likely due to reduced susceptibility of peripheral photoreceptors to MNU-induced degeneration. Inner retinal neuron function in the MNU + HRS rats was better preserved, with fewer apoptotic photoreceptors in the ONL. Collectively, these results support the rationale for future clinical evaluation of HRS as a therapeutic agent for human RP.

Two isoforms of aquaporin 2 responsive to hypertonic stress in the bottlenose dolphin.

This study investigated the expression of aquaporin 2 (AQP2) and its newly found alternatively spliced isoform (alternative AQP2) and the functions of these AQP2 isoforms in the cellular hyperosmotic tolerance in the bottlenose dolphin, ITALIC! Tursiops truncatus mRNA sequencing revealed that alternative AQP2 lacks the fourth exon and instead has a longer third exon that includes a part of the original third intron. The portion of the third intron, now part of the coding region of alternative AQP2, is highly conserved among many species of the order Cetacea but not among terrestrial mammals. Semi-quantitative PCR revealed that AQP2 was expressed only in the kidney, similar to terrestrial mammals. In contrast, alternative AQP2 was expressed in all organs examined, with strong expression in the kidney. In cultured renal cells, expression of both AQP2 isoforms was upregulated by the addition to the medium of NaCl but not by the addition of mannitol, indicating that the expression of both isoforms is induced by hypersalinity. Treatment with small interfering RNA for both isoforms resulted in a decrease in cell viability in hypertonic medium (500 mOsm kg(-1)) when compared with controls. These findings indicate that the expression of alternatively spliced AQP2 is ubiquitous in cetacean species, and it may be one of the molecules important for cellular osmotic tolerance throughout the body.

Co-expression of mouse TMEM63A, TMEM63B and TMEM63C confers hyperosmolarity activated ion currents in HEK293 cells.

Osmoreception is essential for systemic osmoregulation, a process to stabilize the tonicity and volume of the extracellular fluid through regulating the ingestive behaviour, sympathetic outflow and renal function. The sensation of osmotic changes by osmoreceptor neurons is mediated by ion channels that detect the change of osmolarity in extracellular fluid. However, the molecular identity of these channels remains mysterious. AtCSC1and OSCA1,two closely related paralogues from Arabidopsis, have been demonstrated to form hyperosmolarity activated ion channels, which makes their mammalian orthologues-the members of TMEM63 proteins, possible candidates for osmoreceptor transduction channel. To test this possibility, we cloned the cDNAs of all the three members of the mouse TMEM63 family, TMEM63A, TMEM63B and TMEM63C from the mRNA from mouse brain. When all of the three subtypes of TMEM63 proteins were co-expressed in HEK293 cells, we recorded membrane currents evoked by hypertonic stimulation in these cells. However, the cells expressing the combinations of any two subtypes of TMEM63 proteins could not exhibit any hyperosmolarity evoked currents. Thus, all the three members of TMEM63 proteins are required to constitute a hyperosmolarity activated ion channel. We propose that the TMEM63 proteins may serve as an osmolarity sensitive ion channel for the osmoreception. Copyright © 2016 John Wiley & Sons, Ltd.

Activation of protein kinase Cα increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct.

Hypertonicity increases urea transport, as well as the phosphorylation and membrane accumulation of UT-A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Hypertonicity stimulates urea transport through PKC-mediated phosphorylation. To determine whether PKC phosphorylates UT-A1, eight potential PKC phosphorylation sites were individually replaced with alanine and subsequently transfected into LLC-PK1 cells. Of the single mutants, only ablation of the S494 site dampened induction of total UT-A1 phosphorylation by the PKC activator phorbol dibutyrate (PDBu). This result was confirmed using a newly generated antibody that specifically detected phosphorylation of UT-A1 at S494. Hypertonicity increased UT-A1 phosphorylation at S494. In contrast, activators of cAMP pathways (PKA and Epac) did not increase UT-A1 phosphorylation at S494. Activation of both PKC and PKA pathways increased plasma membrane accumulation of UT-A1, although activation of PKC alone did not do so. However, ablating the PKC site S494 decreased UT-A1 abundance in the plasma membrane. This suggests that the cAMP pathway promotes UT-A1 trafficking to the apical membrane where the PKC pathway can phosphorylate the transporter, resulting in increased UT-A1 retention at the apical membrane. In summary, activation of PKC increases the phosphorylation of UT-A1 at a specific residue, S494. Although there is no cross talk with the cAMP-signaling pathway, phosphorylation of S494 through PKC may enhance vasopressin-stimulated urea permeability by retaining UT-A1 in the plasma membrane.

Dynamic regulation of the root hydraulic conductivity of barley plants in response to salinity/osmotic stress.

Salinity stress significantly reduces the root hydraulic conductivity (Lpr) of several plant species including barley (Hordeum vulgare). Here we characterized changes in the Lpr of barley plants in response to salinity/osmotic stress in detail using a pressure chamber. Salt-tolerant and intermediate barley cultivars, K305 and Haruna-nijyo, but not a salt-sensitive cultivar, I743, exhibited characteristic time-dependent Lpr changes induced by 100 mM NaCl. An identical response was evoked by isotonic sorbitol, indicating that this phenomenon was triggered by osmotic imbalances. Further examination of this mechanism using barley cv. Haruna-nijyo plants in combination with the use of various inhibitors suggested that various cellular processes such as protein phosphorylation/dephosphorylation and membrane internalization appear to be involved. Interestingly, the three above-mentioned barley cultivars did not exhibit a remarkable difference in root cell sap osmolality under hypertonic conditions, in contrast to the case of Lpr. The possible biological significance of the regulation of Lpr in barley plants upon salinity/osmotic stress is discussed.

Regulatory volume increase in astrocytes exposed to hypertonic medium requires ß1 -adrenergic Na(+) /K(+) -ATPase stimulation and glycogenolysis.

The cotransporter of Na(+) , K(+) , 2Cl(-) , and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K(+) concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1-mediated transport occurs as secondary active transport driven by Na(+) /K(+) -ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na(+) , K(+) , and Cl(-) × Cl(-) . In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na(+) /K(+) -ATPase activity is stimulated by elevated K(+) or the ß-adrenergic agonist isoproterenol. Extracellular K(+) concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well-differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the ß1 -adrenergic antagonist betaxolol, but not by ICI118551, a ß2 -adrenergic antagonist. A large part of the ß1 -adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na(+) /K(+) -ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post-excitatory undershoot.

Involvement of TRPV1 and AQP2 in hypertonic stress by xylitol in odontoblast cells.

AIM: To examine the responses of mouse odontoblast-lineage cell line (OLC) cultures to xylitol-induced hypertonic stress. METHODOLOGY: OLCs were treated with xylitol, sucrose, sorbitol, mannitol, arabinose and lyxose. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The expression of transient receptor potential vanilloids (TRPV) 1, 3 and 4 was detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The expression of aquaporin (AQP) 2 was detected using immunofluorescence and Western blotting analysis. The expression of interleukin-6 (IL-6) under xylitol-induced hypertonic stress was assessed using an enzyme-linked immunosorbent assay (ELISA). Small interfering ribonucleic acid (siRNA) for AQP-2 was used to inhibition assay. RESULTS: Xylitol-induced hypertonic stress did not decrease OLC viability, unlike the other sugars tested. OLCs expressed TRPV1, 3 and 4 as well as AQP2. Xylitol inhibited lipopolysaccharide (LPS)-induced IL-6 expression after 3 h of hypertonic stress. TRPV1 mRNA expression was upregulated by xylitol. Costimulation with HgCl2 (AQP inhibitor) and Ruthenium red (TRPV1 inhibitor) decreased cell viability with xylitol stimulation. OLCs treated with siRNA against TRPV1 exhibited decreased cell viability with xylitol stimulation. CONCLUSION: OLCs have high-cell viability under xylitol-induced hypertonic stress, which may be associated with TRPV1 and AQP2 expressions.

Angiotensin (1-7) re-establishes heart cell communication previously impaired by cell swelling: implications for myocardial ischemia.

UNLABELLED: The influence of hypertonic solution on dye coupling was investigated in cell pairs isolated from the left ventricle of adult Sprague Dawley rats.The hypertonic solution together with Lucifer Yellow CH, were dialyzed into one cell of the pair using the whole cell clamp tecnique, and the diffusion of dye in the dialyzed as well as in non-dialyzed cell, was followed by measuring the intensity of fluorescence in both cells as a function of time.The results indicated that: (1) Lucifer Yellow CH dialyzed into one cell of the pair diffuses easily into the nondialyzed cell through gap junctions; (2) the intracellular dialysis of an hypertonic solution into one cell of the pair, increases the area of the dialyzed cell and reduced the area of the non-dialyzed cell suggesting intercellular movement of water; (3) the hypertonic solution dialyzed into one cell of the pair abolished the dye coupling; (4) the gap junction permeability (Pj) estimated before and after administration of hypertonic solution showed an appreciably decrease of Pj; (5) angiotensin (1-7) (Ang (1-7) (10-9M) administered to the bath re-established the dye coupling abolished by hypertonic solution and reduced the cell area; (6) the effect of Ang (1-7) was related to the activation of Mas receptor and was dependent on the activation of PKA. CONCLUSIONS: the reestablishment of dye coupling elicited by Ang (1-7) seen in cell pairs dialyzed with hypertonic solution, might indicate that under similar conditions like that seen during myocardial ischemia, the peptide might be of benefit preventing the impairment of cell communication and impulse propagation associated with cardiac reentrant arrhytmias.

Effects of hypertonic buffer composition on lymph node uptake and bioavailability of rituximab, after subcutaneous administration.

The subcutaneous administration of biologics is highly desirable; however, incomplete bioavailability after s.c. administration remains a major challenge. In this work we investigated the effects of excipient dependent hyperosmolarity on lymphatic uptake and plasma exposure of rituximab as a model protein. Using Swiss Webster (SW) mice as the animal model, we compared the effects of NaCl, mannitol and O-phospho-L-serine (OPLS) on the plasma concentration of rituximab over 5 days after s.c. administration. An increase was observed in plasma concentrations in animals administered rituximab in hypertonic buffer solutions, compared with isotonic buffer. Bioavailability, as estimated by our pharmacokinetic model, increased from 29% in isotonic buffer to 54% in hypertonic buffer containing NaCl, to almost complete bioavailability in hypertonic buffers containing high dose OPLS or mannitol. This improvement in plasma exposure is due to the improved lymphatic trafficking as evident from the increase in the fraction of dose trafficked through the lymph nodes in the presence of hypertonic buffers. The fraction of the dose trafficked through the lymphatics, as estimated by the model, increased from 0.05% in isotonic buffer to 13% in hypertonic buffer containing NaCl to about 30% for hypertonic buffers containing high dose OPLS and mannitol. The data suggest that hypertonic solutions may be a viable option for improving s.c. bioavailability.

Hyperosmolarity-induced up-regulation of claudin-4 mediated by NADPH oxidase-dependent H2O2 production and Sp1/c-Jun cooperation.

Claudin-4 is exclusively localized in the tight collecting ducts in the renal tubule. We examined what molecular mechanism is involved in the regulation of claudin-4 expression. In Madin-Darby canine kidney cells, hyperosmolarity increased the expression level of claudin-4 and the production of reactive oxygen species, which were inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, and manganese (III) tetrakis (4-benzoic acid)porphyrin (MnTBAP), a scavenger of H2O2. Both hyperosmolarity and H2O2 increased p-ERK1/2 and p-JNK, which were inhibited by U0126, a MEK inhibitor, and SP600125, a JNK inhibitor, respectively. Immunoprecipitation assay showed that hyperosmolarity increased the association of nuclear Sp1 with c-Jun, which was inhibited by U0126 and SP600125. In mouse inner medullary collecting duct cells and rat kidney slices, hyperosmolarity increased the expression level of claudin-4, which was inhibited by DPI, MnTBAP, U0126, and SP600125. Hyperosmolarity increased luciferase reporter activity of claudin-4, which was inhibited by U0126, SP600125, Sp1 siRNA, and c-Jun siRNA. The activity was inhibited by the mutation in the Sp1 binding site. Chromatin immunoprecipitation assay and avidin-biotin conjugated DNA assay showed that Sp1 and c-Jun are associated with the Sp1 binding site. These results suggest that hyperosmolarity increases nuclear Sp1/c-Jun complex and the association of the complex with the Sp1 binding site, resulting in the segment-specific expression of claudin-4 in the kidney.