Hypertonic Treatment Research Articles

Distinct fluorescent pattern of KAT1::GFP in the plasma membrane of Vicia faba guard cells

The organisation of membrane proteins into certain domains of the plasma membrane (PM) has been proposed to be important for signalling in yeast and animal cells. Here we describe the formation of a very distinct pattern of the K + channel KAT1 fused to the green fluorescent protein (KAT1::GFP) when transiently expressed in guard cells of Vicia faba. Using confocal laser scanning microscopy we observed a radially striped pattern of KAT1::GFP fluorescence in the PM in about 70% of all transfected guard cells. This characteristic pattern was found to be cell type and protein specific and independent of the stomatal aperture and the cytoskeleton. Staining of the cell wall of guard cells with Calcofluor White revealed a great similarity between the arrangement of cellulose microfibrils and the KAT1::GFP pattern. Furthermore, the radial pattern of KAT1::GFP immediately disappeared when turgor pressure was strongly decreased by changing from hypotonic to hypertonic conditions. The pattern reappeared within 15 min upon reestablishment of high turgor pressure in hypotonic solution. Evaluation of the staining pattern by a mathematical algorithm further confirmed this reversible abolishment of the radial pattern during hypertonic treatment. We therefore conclude that the radial organisation of KAT1::GFP depends on the close contact between the PM and cell wall in turgid guard cells. These results offer the first indication for a role of the cell wall in the localisation of ion channels. We propose a model in which KAT1 is located in the cellulose fibrils intermediate areas of the PM and discuss the physiological role of this phenomenon.

Effect of osmotic shrinkage and hormones on the expression of Na+/H+ exchanger-1,Na+/K+/2Cl– cotransporter and Na+/K+-ATPase in gill pavement cells of freshwater adapted Japanese eel,Anguilla japonica

It is well-known that gill epithelial cells are important in fish osmoregulation. However, studies on the effect of osmotic stress on the direct cellular responses of the gill epithelial cells are limited. In this paper, we aimed to determine the effects of osmotic hypertonicity, hormones and cellular signaling molecules on the expression of ion transporters in the cultured primary freshwater pavement cells (PVCs), prepared from freshwater-adapted eels (Anguilla japonica). Our data demonstrated that the hypertonic (500 mOsmol l(-1)) treatment of the isolated PVCs induced cell shrinkage, followed by regulatory volume increase (RVI). Application of blockers (i.e. ouabain, bumetanide and EIPA) demonstrated that Na+/K+ -ATPase, Na+/K+/2Cl- cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) were involved in RVI. Western blot analysis of the hypertonic-treated cells revealed a significant induction of NHE-1, NKCC and, alpha and beta subunits of Na+/K+ -ATPase. In nonshrunken cultured PVCs, we found that dexamethasone and dibutyryl cAMP treatments significantly stimulated the expression levels of the three ion transporters. Both prolactin and insulin-like growth factor-1, can only induce the expression of NKCC. The effect of thyroid hormone (T3) and dibutyryl cGMP was negligible. In this study, the induction of ion transporter expression was found to be post-transcriptionally regulated as no significant change in mRNA levels was detected. This observation implies that the regulation is rapid and is probably induced via nongenomic actions.

Immunolocalization of PIP aquaporins in protoplasts from the suspension culture of sugar beet mesophyll under isoosmotic conditions and osmotic stress

Protoplasts from the suspension culture of sugar beet (Beta vulgaris L.) mesophyll were found to change their volume in response to short-term osmotic stress. When the sorbitol concentration in the external medium was increased 1.5-fold (from 0.4 to 0.6 M) or decreased from 0.4 to 0.25 M, the volume of protoplasts decreased and increased, respectively, by 55–60%. These changes started immediately after the shift in osmoticum concentration and completed within 1–3 min. In the presence of an endocytosis marker FM1-43, its fluorescence increased conspicuously after replacement of isotonic medium with the hypotonic solution but did not change after the substitution with hypertonic medium. At the same time, the hypertonic shrinkage of protoplasts was accompanied by accumulation of fluorescent material in the periplasmic space. The western blot analysis with the use of immune serum for conservative sequence of PIP-type aquaporins revealed their presence in the plasmalemma and intracellular membranes. This conclusion was confirmed by indirect immunofluorescence microscopy: the membrane-bound secondary antibodies labeled with a fluorescent probe Alexa-Fluor 488 were distributed comparatively uniformly on the boundary between the plasmalemma and the protoplast internal compartment. As evident from micrographs of protoplasts exposed to the hypotonic treatment, the fluorescence was smoothly distributed over the plasmalemma after protoplast swelling but its intensity was not so bright. The protoplast shrinkage during the hypertonic treatment resulted in heterogeneous alternate distribution of fluorescent and transparent plasmalemma regions, the fluorescence of stained regions being very intense. The results are interpreted as the evidence that the short-term osmotic stress activates exo-and endocytosis. The migrating regions of the plasmalemma were depleted of PIP-type aquaporins; hence, the induction of osmotic stress has no effect on the amount of this type aquaporins in the plasma membrane.

Hypertonic Saline Treatment of Cystic Fibrosis

To review the literature concerning the use of hypertonic saline (HS) in patients with cystic fibrosis and explain the rationale for its use. A MEDLINE search was conducted through February 2007. Search terms included hypertonic saline, mucociliary clearance, cystic fibrosis, and human DNASE 1 protein. Additional data were identified through subsequent bibliographic reviews. All articles in English identified from the data sources were evaluated. Pertinent studies using HS in patients with cystic fibrosis were included in the analysis. Cystic fibrosis is caused by a deficiency in the cystic fibrosis transmembrane regulator gene, resulting in reduced chloride secretion and excessive sodium absorption. The most significant changes are seen in the airway lumen of the lungs. HS has been shown to improve mucociliary clearance versus placebo. A short-term efficacy trial showed a modest and variable increase in forced expiratory volume in 1 second (FEV(1)) over a 2 week period (15.0 +/- 16.0% from baseline vs 2.8 +/- 13.1% with HS 6% and NaCl 0.9%, respectively; p = 0.004). A long-term efficacy trial of either HS 7% or NaCl 0.9% twice daily for 48 weeks has shown a modest sustained improvement in FEV(1) and a significantly increased exacerbation-free survival rate (76% vs 62% for HS 7% and NaCl 0.9%, respectively; p = 0.03). HS preceded by a bronchodilator is an inexpensive, safe, effective additional therapy in cystic fibrosis patients with stable lung function. Its use has been associated with a modest improvement in lung function and reduced frequency of pulmonary exacerbations.

Osmotherapy with hypertonic saline attenuates water content in brain and extracerebral organs*

Because of their beneficial effects in patients with hemorrhagic shock and multiple-system trauma, hypertonic saline solutions are increasingly being used perioperatively for volume resuscitation. Although the anti-edema effects of hypertonic saline on brain are well documented in a variety of brain injury paradigms, its effects on the water content on other organs has not been studied rigorously. In this study, we tested the hypothesis that a) hypertonic saline when given as an intravenous bolus and continuous infusion attenuates water content of small bowel, lung, and brain in rats without neuro-injury; and b) attenuation of stroke-associated increases in lung water is dependent on achieving a target serum osmolality. Prospective laboratory animal study. Research laboratory in a teaching hospital. Adult male Wistar rats. In the first series of experiments, under controlled conditions of normoxia, normocarbia, and normothermia, spontaneously breathing, halothane-anesthetized (1.0-1.5%) adult male Wistar rats (280-320 g) were treated in a blinded randomized fashion with 7.5% hypertonic saline or 0.9% normal saline in a 8-mL/kg intravenous infusion for 3 hrs followed by a continuous intravenous infusion (1 mL/kg/hr) of 5% hypertonic saline or normal saline, respectively (n=10 each), for 48 hrs. A second group of rats were treated with continuous infusion only for 48 hrs of either 7.5% hypertonic saline or normal saline (1 mL/kg/hr) (n=10 each) without an intravenous bolus. Naïve rats served as controls (n=10). Tissue water content of small bowel, lung, and brain was determined by comparing the wet-to-dry ratios at the end of the experiment. In a second series of experiments, rats (n=94) were subjected to 2 hrs of transient middle cerebral artery occlusion by the intraluminal occlusion technique. At 6 hrs following middle cerebral artery occlusion, rats were treated in a blinded randomized fashion with a continuous intravenous infusion of normal saline, 3% hypertonic saline, or 7.5% hypertonic saline for 24, 48, 72, and 96 hrs. Surgical shams served as controls (n=7). Hypertonic saline was instituted as chloride/acetate mixture (50:50) in all experiments. Serum osmolality was determined at the end of the experiment in all animals. In rats without neuro-injury that received intravenous bolus followed by a continuous infusion, lung water content was significantly reduced with hypertonic saline (73.9+/-1.1%; 359+/-10 mOsm/L) (mean+/-sd) compared with normal saline treatment (76.1+/-0.53%; 298+/-4 mOsm/L) as was water content of small bowel (hypertonic saline, 69.1+/-5.8%; normal saline, 74.7+/-0.71%) and brain (hypertonic saline, 78.1+/-0.87%; normal saline, 79.2+/-0.38%) at 48 hrs. Stroke-associated increases in lung water content were attenuated with 7.5% hypertonic saline at all time points. There was a strong correlation between serum osmolality and attenuation of stroke-associated increases in lung water content (r=-.647) Bowel, lung, and brain water content is attenuated with hypertonic saline when serum osmolality is >350 mOsm/L without adverse effect on mortality in animals with and without neuro-injury. Attenuation of water content of extracerebral organs with hypertonic saline treatment may have therapeutic implications in perioperative fluid management in patients with and without brain injury.

Hypertonic Stress Increases Phosphatidylinositol 4,5-Bisphosphate Levels by Activating PIP5KIβ

Hyperosmotic stress increases phosphoinositide levels, reorganizes the actin cytoskeleton, and induces multiple acute and adaptive physiological responses. Here we showed that phosphatidylinositol 4,5-bisphosphate (PIP(2)) level increased rapidly in HeLa cells during hypertonic treatment. Depletion of the human type I phosphatidylinositol 4-phosphate 5-kinase beta isoform (PIP5KIbeta) by RNA interference impaired both the PIP(2) and actin cytoskeletal responses. PIP5KIbeta was recruited to membranes and was activated by hypertonic stress through Ser/Thr dephosphorylation. Calyculin A, a protein phosphatase 1 inhibitor, blocked the hypertonicity-induced PIP5KIbeta dephosphorylation/activation as well as PIP(2) increase in cells. Urea, which raises osmolarity without inducing cell shrinkage, did not promote dephosphorylation nor increase PIP(2) levels. Disruption or stabilization of the actin cytoskeleton, or inhibition of the Rho kinase, did not block the PIP(2) increase nor PIP5KIbeta dephosphorylation. Therefore, PIP5KIbeta is dephosphorylated in a volume-dependent manner by a calyculin A-sensitive protein phosphatase, which is activated upstream of actin remodeling and independently of Rho kinase activation. Our results establish a cause-and-effect relation between PIP5KIbeta dephosphorylation, lipid kinase activation, and PIP(2) increase in cells. This PIP(2) increase can orchestrate multiple downstream responses, including the reorganization of the actin cytoskeleton.

Clathrin Required for Phosphorylation and Internalization of β2-Adrenergic Receptor by G Protein-coupled Receptor Kinase 2 (GRK2)

Clathrin Required for Phosphorylation and Internalization of β2-Adrenergic Receptor by G Protein-coupled Receptor Kinase 2 (GRK2)

An early bolus of hypertonic saline hydroxyethyl starch improves long-term outcome after global cerebral ischemia

The beneficial effect of hypertonic saline solutions in the emergency treatment of shock and traumatic brain injury is well described. The present study determines effects of a single bolus of hypertonic saline on long-term survival, neurologic function, and neuronal survival 10 days after global cerebral ischemia. In addition, we evaluated the therapeutic window for hypertonic saline treatment (early vs. delayed application). Laboratory experiment. University laboratory. Male Wistar rats weighing 240-330 g. Rats were submitted to temporal global cerebral ischemia using temporary bilateral carotid occlusion combined with hypobaric hypotension. Animals received 7.5% saline/6% hydroxyethyl starch (HHS) or vehicle (NaCl 0.9%) at either 1.5 mins (early treatment) or 31.5 mins (delayed treatment) of reperfusion. Regional cerebral blood flow (rCBF) and physiologic variables were measured during insult and early reperfusion. Animal survival and neurologic function were evaluated throughout the 10-day observation period. Quantification of brain injury was performed on day 10. Early treatment with HHS resulted in a robust restoration of rCBF after ischemia, reduced postischemic mortality by 77% (9% vs. 39% in vehicle-treated controls), ameliorated neurologic performance (Neuro-Deficit-Score 10 days after insult, 96 +/- 0.7 vs. 85 +/- 1.4, mean +/- se), and almost blunted neuronal cell death (hippocampal CA1, 2150 +/- 191 vs. 884 +/- 141 neurons/mm; cortex, 1746 +/- 91 vs. 1060 +/- 112). In contrast, delayed treatment resulted in no sustained effects. Timing of HHS treatment is critical after experimental global cerebral ischemia to reduce mortality, improve neurologic function, and neuronal survival. Our results suggest that early application of HHS may be a potential neuroprotective strategy after global cerebral ischemia.

Hypertonic saline enhances host defense to bacterial challenge by augmenting Toll-like receptors*

To determine whether hypertonic saline infusion modulates thermal injury-induced bacterial translocation and host response to bacterial challenge through the augmentation of Toll-like receptors (TLRs). Prospective, experimental study. Research laboratory at a university hospital. Thermal injury models in the mice. In experiment 1, mice underwent burn were given with 10 mL/kg hypertonic saline (7.5% NaCl), 10 mg/kg saline (N/S1), or 80 mL/kg saline (N/S2) at 4 or 8 hrs after burn. At 24 hrs after burn, mesenteric lymph nodes were harvested for bacterial translocation assay. In experiment 2, mice receiving hypertonic saline or saline after thermal injury received peritoneal challenge with Escherichia coli, and bacterial clearance was measured. In experiment 3, peritoneal cells from mice receiving hypertonic saline or saline after thermal injury were incubated with E. coli, and bacterial count, TLR2, TLR4, MIP2, CXCR2, pp38, and ERK expression were evaluated. In experiment 4, reactive oxygen species production, CXCR2, MIP2, TLR2, and TLR4 expression of bone marrow neutrophil from mice receiving hypertonic saline or saline treatment after thermal injury were evaluated. In experiment 5, neutrophil were cultured with hypertonic saline or N/S and incubated with E. coli. TLR2 and TLR4 expression and bacterial count were evaluated. In experiment 6, mice were fed with oral antibiotics with or without lipopolysaccharide, a TLR ligand, supplements. At 24 hrs after burn, mesenteric lymph nodes were harvested for bacterial translocation assay, and neutrophils were harvested for TLR2 and TLR4 protein assay. Hypertonic saline decreased thermal injury-induced bacterial translocation. Hypertonic saline increased bacterial clearance, phagocytic activity, and TLR2, TLR4, CXCR2, pp38, and p44/42 expression of peritoneal cells. Hypertonic saline treatment at 4 or 8 hrs after thermal injury decreased reactive oxygen species production of neutrophil. Hypertonic saline injection increased TLR2, TLR4, and pp38 expression of neutrophil. In vitro treatment of neutrophil with hypertonic saline increased phagocytic activity and TLR2 and TLR4 expression. Commensal depletion with oral antibiotics decreased TLR2 and TLR4 expression of neutrophil; lipopolysaccharide increased TLR4 expression of neutrophil and decreased thermal injury-induced bacterial translocation. Restoration of extracellular fluid in burn shock with hypertonic saline decreased thermal injury-induced bacterial translocation. Hypertonic saline increased the phagocytic activity and TLR2, TLR4, CXCR2, pp38, and P44/42 expression of peritoneal cells. Hypertonic saline decreased reactive oxygen species but increased TLR2, TLR4, and pp38 expression and phagocytic activity of bone marrow neutrophil. Stimulation of the TLRs with lipopolysaccharide in commensal depleted mice increased TLRs expression of neutrophil and decreased thermal injury-induced bacterial translocation. Taken together with the fact that stimulation of TLRs with hypertonic saline increases phagocytic activity of systemic inflammatory cells, we conclude that TLRs play a critical role in the innate immunity by recognizing bacteria and that hypertonic saline enhances host response to bacterial challenge by increasing TLRs of inflammatory cells.

Influences of isotonic, hypertonic and hypovolemic treatments on vasopressin response and fluid-electrolyte balance in l-thyroxine-induced hyperthyroid rat

The present study was performed to determine how l-thyroxine-induced hyperthyroidism affects the vasopressin response to different stimulations (isotonic, hypertonic and hypovolemic) in rats. Spraque-Dawley rats were initially separated into 3 groups; control ( n = 24), sham hyperthyroidism ( n = 24, hyperthyroidism ( n = 24). At the end of the experiment additional sub-groups were formed before decapitation. These sub-groups were formed as; without stimulation ( n = 6), isotonic stimulation ( n = 6), hypertonic stimulation ( n = 6) and hypovolemic stimulation ( n = 6). Total T3, total T4 and AVP levels were evaluated in the plasma. Haematocrit and osmolality levels were also determined. When the parameters related to thyroid hormones were evaluated, it was determined that total T3 and T4 levels were higher in hyperthyroid group than the other groups. Plasma AVP levels showed more increase in hyperthyroid group both in basal grade and against to hypertonic and hypovolemic stimulations than the other groups ( P < 0.001). The results of the present study indicate that l-thyroxine-induced experimental hyperthyroidism increased basal and stimulated AVP release in rats.

Comparison of hypertonic saline treatment in meningitis with cerebral edema

Purpose:Cerebral edema in meningitis is a potentially complication. Hypertonic saline of various concentrations are frequently used to treat cerebral edemas in meningitis. We analyzed the safety and efficacy of osmotic therapy in cerebral edema by comparison of various hypertonic saline concentrations and mannitol. Methods:The medical records of 42 patients, who were followed up in the Department of Pediatrics, Busan Medical Center, from Jan. 2002 to Oct. 2005, were analyzed retrospectively. We measured intracranial pressure, mean flow velocity, and various laboratory parameters. Results:In cerebral edema developed in meningitis, intracranial pressure and symptoms were improved in treatment of hypertonic saline and mannitol. Serial bolus infusion of 3 percent hypertonic saline resulted in the best outcome. There was not a statistically considerable difference on the mean values of the intracranial pressure gap. On transcranial doppler, mean flow velocity was increased and pulsatilty index was decreased. Laboratory findings (osmolarity, Na, Cl, pH, lactic acid, Ca) were diffenent during the treatment period as opposed to K, Hb, bicarbonate, base excess. There was not a specific form of hypertonic saline used in meningitis treatment with cerebral edema. Conclusion:The therapy for cerebral edema in meningitis remains largely empirical. Serial bolus infusion of 3 percent hypertonic saline is better than other hypertonic salines. Various concentrations and different infusion methods of hypertonic saline statistically does not influence the result of treatment. More research aimed at improving cerebral edema treatment is needed to identify new, effective forms of treatment

Ca2+ transient induced by extracellular changes in osmotic pressure in Arabidopsis leaves: differential involvement of cell wall–plasma membrane adhesion

We investigated the mechanism underlying the perception of extracellular changes in osmotic pressure in Vallisneria gigantea Graebner and transgenic Arabidopsis thaliana (L.) Heynh. expressing cytoplasmic aequorin. Hypertonic and hypotonic treatments of A. thaliana leaves each rapidly induced a Ca2+ transient. Both responses were essentially dependent on the presence of extracellular Ca2+ and were sensitive to Gd3+ a potential blocker of stretch-activated Ca2+ channels. Immediately after plasmolysis caused by hypertonic treatment and subsequent deplasmolysis caused by hypotonic treatment, the cells did not respond to a second hypertonic treatment and exhibited an impaired adhesion of the plasma membrane (PM) to the cell wall (CW). Recovery of the responsiveness required about 6 h. By contrast, no refractory phenomenon was observed in response to hypotonic treatment. Pretreatment with cellulase completely inhibited the Ca2+ transient induced by hypertonic treatment, but it did not affect the response to hypotonic treatment. V. gigantea mesophyll cells pretreated with cellulase exhibited an impaired adhesion of the PM to the CW. The leaf cells of multicellular plants can respond to both hypertonic and hypotonic treatments through the stretch-activated Ca2+ channels, whereas cellulase-sensitive adhesion of the PM to the CW is involved only in the response to hypertonic treatment.

Hypertonic Induction of COX-2 in Collecting Duct Cells by Reactive Oxygen Species of Mitochondrial Origin

Our previous studies have documented MAPK mediation of the hypertonicity-induced stimulation of COX-2 expression in cultured renal medullary epithelial cells. The present study extends this observation by examining the role of reactive oxygen species (ROSs). ROS levels, determined using dichlorodihydrofluorescence diacetate and cytochrome c, were rapidly and significantly increased following exposure of mIMCD-K2 cells to media made hypertonic by adding NaCl. Hypertonic treatment (550 mosmol/kg) for 16 h induced a 5.6-fold increase in COX-2 protein levels and comparable increases in prostaglandin E(2) release, both of which were completely abolished by the NADPH oxidase inhibitor diphenyleneiodonium (25-50 microM). The general antioxidant N-acetyl-l-cysteine (6 mM), and the superoxide dismutase mimetic TEMPO (2.0 mm) reduced COX-2 levels by 75.6 and 79.8%, respectively. Exposure of mIMCD-K2 cells to exogenous O(2)(-.) generated by the xanthine/xanthine oxidase system mimicked the effect of hypertonicity on COX-2 expression and prostaglandin E(2) release. The increases in phosphorylation of ERK1/2 and p38 were detected 20 min following the hypertonic treatment and were both prevented by N-acetyl-l-cysteine. The increases in ROSs in response to hypertonic treatment were completely blocked by any one of the mitochondrial inhibitors tested, such as rotenone, thenoyltrifluoroacetone, or carbonyl cyanide m-chlorophenylhydrazone, associated with remarkable inhibition of COX-2 expression. In contrast, the increases in ROSs were not significantly altered in IMCD cells deficient in either gp91(phox) or p47(phox), nor were the increases in COX-2 expression. We conclude that ROSs derived from mitochondria, but not NADPH oxidase, mediate the hypertonicity-induced phosphorylation of MAPK and the stimulation of COX-2 expression.

DNA-PK is responsible for enhanced phosphorylation of histone H2AX under hypertonic conditions

Exposure of cells to hypertonic medium after X-irradiation results in a 3–4-fold increase in the phosphorylation of histone H2AX (γH2AX) at sites of radiation-induced DNA double-strand breaks. This increase was previously associated with salt-induced radiosensitization and inhibition of repair of DNA double-strand breaks. To examine possible mechanisms for the increase in foci size, chemical inhibitors of kinase and phosphatase activity and cell lines deficient in ATM and DNA-PK, two kinases known to phosphorylate H2AX, were examined. H2AX kinase and phosphatase activity were maintained in the presence of high salt. ATM mutant HT144 melanoma cells showed the expected 3–4-fold increase in H2AX phosphorylation in the presence of 0.5 M Na +. However, DNA-PKcs deficient M059J cells failed to respond to hypertonic treatment and M059J Fus1 cells corrected for this deficiency showed the expected increase in foci size. Although the active phosphoform of ATM, phosphoserine-1981, increased after irradiation, the level was unaffected by the addition of 0.5 M Na +. Instead, 0.5 M Na + caused a partial redistribution of serine-1981-ATM to perinuclear regions. Hypertonic medium added after irradiation was effective in inhibiting rejoining of the radiation-induced double-strand breaks even in DNA-PK deficient M059J cells. We suggest that hypertonic treatment following irradiation inhibits double-strand break rejoining that in turn maintains DNA-PK activity at the site of the break, enhancing the size of the γH2AX foci.

Distinct Conformations of the Corticotropin Releasing Factor Type 1 Receptor Adopted following Agonist and Antagonist Binding Are Differentially Regulated

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.

Effect of hypo-, iso- and hypertonic saline irrigation on secretory mucins and morphology of cultured human nasal epithelial cells

Conclusion. We speculate that isotonic saline is the most physiological irrigation solution in terms of mucus secretion and the cellular morphology of nasal epithelial cells. Objective. To determine the most physiologic and effective saline concentration for nasal irrigation in terms of mucus secretion and cellular morphology by using normal human nasal epithelial cells treated with hypo-, iso- and hypertonic saline. Material and methods. Fully differentiated passage-2 normal human nasal epithelial cells were treated with pure water and with 0.3% (hypotonic), 0.9% (isotonic) and 3% (hypertonic) saline solutions. mRNA expression levels of MUC5AC and MUC5B, which are known to be major airway mucins, were analyzed after 30 min using reverse transcriptase polymerase chain reaction. Total mucin and MUC5AC and MUC5B mucin secretions were analyzed using dot-blotting. Cellular morphology was observed with light microscopy after hematoxylin–eosin staining and with scanning electron microscopy. Results. MUC5AC and MUC5B mRNA levels did not change after treatment with pure water and various concentrations of saline. Total mucin and MUC5AC mucin secretions only increased following pure water treatment, while MUC5B mucin secretion increased with pure water, hypo- and hypertonic saline treatment. Morphologic analysis revealed that pure water severely damaged normal human nasal epithelial cells and that only isotonic saline did not affect their morphology.

Insulin exerts neuroprotection by counteracting the decrease in cell‐surface GABAA receptors following oxygen–glucose deprivation in cultured cortical neurons

A loss of balance between excitatory and inhibitory signaling leads to excitoxicity, and contributes to ischemic cell death. Reduced synaptic inhibition as a result of dysfunction of the ionotropic GABAA receptor has been suggested as one of the major causes for this imbalance, although the underlying mechanisms remain poorly understood. In the present study, we investigated whether oxygen-glucose deprivation (OGD), an ischemia-like challenge, alters cell-surface expression of GABAA receptors in cultured hippocampal neurons, and thereby leads to excitotoxic cell death. Using cell culture ELISA as a cell surface receptor assay, we found that OGD produced a marked decrease in cell surface GABAA receptors, without altering the total amount of receptors. Furthermore, the reduction could be prevented by inhibition of receptor endocytosis with hypertonic sucrose treatment. Notably, insulin significantly limited OGD-induced changes in cell-surface GABAA receptors. In parallel, insulin protected cultured neurons against both glutamate toxicity and OGD, as assayed by mitochondrial reduction of Alamar Blue. Importantly, insulin-mediated neuroprotection was eliminated when bicuculline, a GABAA receptor antagonist, was co-applied with insulin during OGD. Together, our results strongly suggest that ischemia-like insults decrease cell surface GABAA receptors in neurons via accelerated internalization, and that insulin provides neuroprotection by counteracting this reduction.

The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

Deactivation of photosynthetic activities is triggered by loss of a small amount of water in a desiccation-tolerant cyanobacterium, Nostoc commune.

Changes in photosynthetic activities under hypertonic conditions were studied in a terrestrial, highly desiccation-tolerant cyanobacterium, Nostoc commune, and in some desiccation-sensitive cyanobacteria. The amounts of water sustained in the colony matrix outside the N. commune cells and the cellular solute concentration were estimated by measuring the water potential, and the solute concentration was supposed to correspond to around 0.22 M sorbitol. Incubation of the colonies in 0.8 M sorbitol solution inhibited the energy transfer from the phycobilisome (PBS) anchor to PSII core complexes. At higher sorbitol concentrations, light energy absorbed by PSI, PSII, and PBS was dissipated to heat. PSI and cyclic electron flow around PSI was also deactivated by hypertonic treatment. Fv/Fm and (Fm'-F)/Fm' values started to decrease at 0.6 and 0.3 M sorbitol and reached zero at 1.0 and 0.8 M, respectively. Decreases in these two fluorescence parameters corresponded to the decreases in PSII fluorescence (F695) and photosynthetic CO2 fixation, respectively. The intensity of delayed light emission started to decrease at 1.0 M sorbitol and became negligible at 4.0 M. Comparing these changes in N. commune with those in desiccation-sensitive species, we found that N. commune cells actively deactivates photosynthetic systems on sensing water loss.

Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity.

Glutamate is the predominant excitatory neurotransmitter in the CNS, and it is removed from the synaptic cleft by sodium-dependent glutamate transport activity. Glutamate transporter-1 (GLT-1) is expressed predominantly in astroglial cells and is responsible for the largest proportion of glutamate transport in the adult forebrain. In the present study, we demonstrate the ability of endogenous and recombinant GLT-1 to form clusters in astrocytic processes and characterize the mobility and physiological importance of these clusters in the regulation of GLT-1 activity in the presence or absence of neurons. At the distal end of C6 glioma cell processes, GLT-1 clusters undergo rapid morphological changes in both shape and size, and these changes are inhibited by cytochalasin D treatment, suggesting that the morphogenesis of GLT-1 clusters is highly dependent on the actin network. Treatment of astrocytes with phorbol 12-myristate 13-acetate (PMA) quickly and preferentially decreases GLT-1 localization on the process membrane, leading to de novo generation of GLT-1 clusters along the process shaft. Pretreatment with the PKC inhibitor bisindolylmaleimide II (Bis II), with sucrose (0.4 m), or through the expression of a dominant-negative form of dynamin prevents PMA-induced GLT-1 internalization and cluster formation. In terms of glutamate transporter function, PMA treatment elicits a significant decrease in GLT-1 activity that is prevented by preexposure to either Bis II or hypertonic treatment. Together, these data indicate that GLT-1 trafficking and cluster formation in glial cell processes are dynamic events that play important roles in regulating glutamate uptake in astrocytes and glioma cells.