Isotonic Medium Research Articles

Influence of the retinoid acitretin on erythrocyte microrheology in vitro

To determine in vitro changes in the microrheology of red blood cells (RBCs) treated with acitretin (Ro 10-1670). Venous blood samples were obtained from 27 healthy donors. Washed erythrocytes (5 x 10(6) cells/ml) were incubated for 30 min at 37 degrees C in solutions of low (25 ng/ml) and high (400 ng/ml) acitretin concentration. The whole red cell deformability was measured using osmotic gradient ektacytometry. To assess the contribution of factors influencing changes in red cell deformability, the biophysical state of the phospholipid bilayer was determined using the cation osmotic hemolysis technique (COH) and the internal viscosity using electron paramagnetic resonance (EPR) spectroscopy. Osmotic deformability studies revealed that cells treated with acitretin at low concentration deformed almost normally in isotonic medium, but RBCs incubated with high acitretin concentration showed distinct loss of deformation. However, in hypotonic medium, cell deformability was reduced to abnormally low values and this change was most marked with high acitretin concentration. CHO measurements showed that incubation of red cells at low acitretin concentration caused a significant increase in hemolysis at ionic strengths from 123.2 - 154.0 mmol/1 NaCl in relation to the control (p < 0.001). A high concentration of acitretin caused a significant increase in COH with high ionic strengths in the range 107.8 - 154.0 mmol/l NaCl (p < 0.001) and at low ionic strengths in the range 15.4 mmol/l NaCl (p < 0.05). The measurement of RBC intracellular microviscosity showed no statistically significant differences between control (6.22+/-0.22 cP) and acitretin-treated (6.29+/-0.25 cP) cells. The data show that acitretin, even at low concentration, is capable of inducing the reduction of red cell deformability. A reduced tendency of acitretin-treated RBCs to deform might result from the ability of acitretin to perturb the cell shape and membrane viscoelastic properties.

Ion Specificity and Ionic Strength Dependence of the Osmoregulatory ABC Transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.

Regulation of Nucleocytoplasmic Trafficking of Transcription Factor OREBP/TonEBP/NFAT5

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels, including nucleocytoplasmic trafficking. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity.

Detection and release of allergenic proteins in Parietaria judaica pollen grains

Rapid diffusion of allergenic proteins in isotonic media has been demonstrated for different pollen grains. Upon contact with stigmatic secretion or with the mucosa of sensitive individuals, pollen grains absorb water and release soluble low-molecular-weight proteins, these proteins enter in the secretory pathway in order to arrive at the cell surface. In this study we located allergenic proteins in mature and hydrated-activated pollen grains of Parietaria judaica L. (Urticaceae) and studied the diffusion of these proteins during the first 20 min of the hydration and activation processes. A combination of transmission electron microscopy and immunocytochemical methods was used to locate these proteins in mature pollen and in pollen grains after different periods of hydration and activation processes. Activated proteins reacting with antibodies in human serum from allergic patients were found in the cytoplasm, wall, and exudates from the pollen grains. The allergenic component of these pollen grains changes according to the pollen state; the presence of these proteins in the exine, the cytoplasm, and especially in the intine and in the material exuded from the pollen grains, is significant in the hydrated-activated studied times, whereas this presence is not significant in mature pollen grains. The rapid activation and release of allergenic proteins of P. judaica pollen appears to be the main cause of the allergenic activity of these pollen grains.

Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system

Giant HEK293 cells of 30–65 μm in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 ± 0.1 μF/cm 2 for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 μm) was found to be between 12.8 and 16.1 μS/cm 2, which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system.

A biophysical approach to the optimisation of dendritic-tumour cell electrofusion

Electrofusion of tumour and dendritic cells (DCs) is a promising approach for production of DC-based anti-tumour vaccines. Although human DCs are well characterised immunologically, little is known about their biophysical properties, including dielectric and osmotic parameters, both of which are essential for the development of efficient electrofusion protocols. In the present study, human DCs from the peripheral blood along with a tumour cell line used as a model fusion partner were examined by means of time-resolved cell volumetry and electrorotation. Based on the biophysical cell data, the electrofusion protocol could be rapidly optimised with respect to the sugar composition of the fusion medium, duration of hypotonic treatment, frequency range for stable cell alignment, and field strengths of breakdown pulses triggering membrane fusion. The hypotonic electrofusion consistently gave a tumour-DC hybrid rate of up to 19%, as determined by counting dually labelled fluorescent hybrids in a microscope. This fusion rate is nearly twice as high as that usually reported in the literature for isotonic media. The experimental findings and biophysical approach presented here are generally useful for the development of efficient electrofusion protocols, especially for rare and valuable human cells.

Participation of protein phosphatases in regulation of sodium transport across the erythrocyte membrane of the lamprey Lampetra fluviatilis

Erythrocytes of lamprey Lampetra fluviatilis were incubated in standard isotonic medium at 20°C with 22Na to determine the unidirectional Na+ influx. Cell incubation in the presence of various protein phosphatase inhibitors (NaF, cantharidin, calyculin A) led to a considerable increase of Na+ transport into erythrocytes. The stimulation of Na+ influx into erythrocytes rose with increase of concentration of calyculin A within the range of 10–100 nM. The calyculin A concentration producing a 50% activation of Na+ transport amounted to 41.5 nM. Under optimal experimental conditions, the Na+ influx increased from control level of 5–8 to 20–40 mmol/l cells/h under effect of protein phosphatase blockers. The Na+ transport induced by these inhibitors was completely suppressed on addition of amiloride to the incubation medium. The treatment of lamprey erythrocytes with protein phosphatase inhibitors was accompanied by a small (∼12%), but statistically significant decrease of intracellular Na+ content. A small decrease of intracellular K+ content in erythrocyte was observed only under the effect of NaF. The obtained data allow making the conclusion that protein phosphatases of the PP1 and PP2A types play a significant role in regulation of Na+ transport across the lamprey erythrocyte membrane in both directions.

Chloride/Bicarbonate Exchanger SLC26A7 Is Localized in Endosomes in Medullary Collecting Duct Cells and Is Targeted to the Basolateral Membrane in Hypertonicity and Potassium Depletion

SLC26A7 is a Cl(-)/HCO(3)(-) exchanger that is expressed on the basolateral membrane and in the cytoplasm of two distinct acid-secreting epithelial cells: The A-intercalated cells in the kidney outer medullary collecting duct and the gastric parietal cells. The intracellular localization of SLC26A7 suggests the possibility of trafficking between cell membrane and intracellular compartments. For testing this hypothesis, full-length human SLC26A7 cDNA was fused with green fluorescence protein and transiently expressed in MDCK epithelial cells. In monolayer cells in isotonic medium, SLC26A7 showed punctate distribution throughout the cytoplasm. However, in medium that was made hypertonic for 16 h, SLC26A7 was detected predominantly in the plasma membrane. The presence of mitogen-activated protein kinase inhibitors blocked the trafficking of SLC26A7 to the plasma membrane. Double-labeling studies demonstrated the localization of SLC26A7 to the transferrin receptor-positive endosomes. A chimera that was composed of the amino terminal fragment of SLC26A7 and the carboxyl terminal fragment of SLC26A1, and a C-terminal-truncated SLC26A7 were retained in the cytoplasm in hypertonicity. In separate studies, SLC26A7 showed predominant localization in plasma membrane in potassium-depleted isotonic medium (0.5 or 2 mEq/L KCl) versus cytoplasmic distribution in normal potassium isotonic medium (4 mEq/L). It is concluded that SLC26A7 is present in endosomes, and its targeting to the basolateral membrane is increased in hypertonicity and potassium depletion. The trafficking to the cell surface suggests novel functional upregulation of SLC26A7 in states that are associated with hypokalemia or increased medullary tonicity. Additional studies are needed to ascertain the role of SLC26A7 in enhanced bicarbonate absorption in outer medullary collecting duct in hypokalemia and in acid-base regulation in conditions that are associated with increased medullary tonicity.

Regulation of aquaporin‐2 by hyperosmolality in renal epithelial cells

To investigate the stability, degradation, expression, and targeting of AQP2 by hyperosmolality, stably transfected mIMCD-3 cells expressing AQP2 (AQP2/IMCD3) were generated. In AQP2/IMCD3 cells, nonglycosylated (ng-AQP2) and glycosylated (g-AQP2) forms were detected by immunoblot. The stability of ng-AQP2 decreased with the lapse of time, whereas that of g-AQP2 was stable. NaCl, but not urea, destabilized ng-AQP2. The half-life of ng-AQP2 in isotonic conditions was ~5 hours, whereas that in medium supplemented with NaCl was ~1.5 hours. Urea enhanced it compared to isotonic conditions. These findings indicate that the stability of ng-AQP2 is enhanced by urea, but not NaCl. The stability of ng-AQP2 was significantly enhanced by the addition of proteasome or lysosome inhibitor. There was no significant effect on the stability of g-AQP2 by these inhibitors. This result suggests that the degradation of ng-AQP2 is dependent on proteasome and lysosome degradation pathways. The addition of NaCl or urea to the isotonic medium remarkably increased ng-AQP2 protein. The maximal accumulation of ng-AQP2 protein occurred with addition of 175 mM NaCl or 350 mM urea to the isotonic medium. Cell surface biotinylation experiments revealed that hyperosmolality enhanced the apical membrane insertion of ng-AQP2. The percentage of ng-AQP2 on the apical membrane by hyperosmotic NaCl and urea were 84% and 86%, respectively. These results indicate that hyperosmolality plays an important role in the stability, degradation, expression, and targeting of ng-AQP2.

Feed Forward Cycle of Hypotonic Stress-induced ATP Release, Purinergic Receptor Activation, and Growth Stimulation of Prostate Cancer Cells

ATP is released in many cell types upon mechanical strain, the physiological function of extracellular ATP is largely unknown, however. Here we report that ATP released upon hypotonic stress stimulated prostate cancer cell proliferation, activated purinergic receptors, increased intracellular [Ca(2+)](i), and initiated downstream signaling cascades that involved MAPKs ERK1/2 and p38 as well as phosphatidylinositol 3-kinase (PI3K). MAPK activation, the calcium response as well as induction of cell proliferation upon hypotonic stress were inhibited by preincubation with the ATP scavenger apyrase, indicating that hypotonic stress-induced signaling pathways are elicited by released ATP. Hypotonic stress increased prostaglandin E(2) (PGE(2)) synthesis. Consequently, ATP release was inhibited by antagonists of PI3K (LY294002 and wortmannin), phospholipase A(2) (methyl arachidonyl fluorophosphonate (MAFP)), cyclooxygenase-2 (COX-2) (indomethacin, etodolac, NS398) and 5,8,11,14-eicosatetraynoic acid (ETYA), which are involved in arachidonic acid metabolism. Furthermore, ATP release was abolished in the presence of the adenylate cyclase (AC) inhibitor MDL-12,330A, indicating regulation of ATP-release by cAMP. The hypotonic stress-induced ATP release was significantly blunted when the ATP-mediated signal transduction cascade was inhibited on different levels, i.e. purinergic receptors were blocked by suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), the Ca(2+) response was inhibited upon chelation of intracellular Ca(2+) by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ERK1,2 as well as p38 were inhibited by UO126 and SB203580, respectively. In summary our data demonstrate that hypotonic stress initiates a feed forward cycle of ATP release and purinergic receptor signaling resulting in proliferation of prostate cancer cells.

Suprazero cooling conditions significantly influence subzero permeability parameters of mammalian ovarian tissue

To model the cryobiological responses of cells and tissues, permeability characteristics are often measured at suprazero temperatures and the measured values are used to predict the responses at subzero temperatures. The purpose of the present study was to determine whether the rate of cooling from +25 to +4 degrees C influenced the measured water transport response of ovarian tissue at subzero temperatures in the presence or absence of cryoprotective agents (CPAs). Sections of freshly collected equine ovarian tissue were first cooled either at 40 degrees C/min or at 0.5 degrees C/min from 25 to 4 degrees C, and then cooled to subzero temperatures. A shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine ovarian tissue sections. After ice was induced to form in the extracellular fluid within the specimen, the sample was frozen from the phase change temperature to -50 degrees C at 5 degrees C/min. Replicate samples were frozen in isotonic medium alone or in medium containing 0.85 M glycerol or 0.85 M dimethylsulfoxide. The water transport response of ovarian tissue samples cooled at 40 degrees C/min from 25 to 4 degrees C was significantly different (confidence level >95%) from that of tissue samples cooled at 0.5 degrees C/min, whether in the presence or absence of CPAs. We fitted a model of water transport to the experimentally-derived volumetric shrinkage data and determined the best-fit membrane permeability parameters (L(pg) and E(Lp)) of equine ovarian tissue during freezing. Subzero water transport parameters of ovarian tissue samples cooled at 0.5 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.06 to 0.73 microm/min.atm and E(Lp) = 6.1 to 20.5 kcal/mol. The corresponding parameters of samples cooled at 40 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.04 to 0.61 microm/min.atm and E(Lp) = 8.2 to 54.2 kcal/mol. Calculations made of the theoretical response of tissue at subzero temperatures suggest that the optimal cooling rates to cryopreserve ovarian tissue are significantly dependent upon suprazero cooling conditions.

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.

A possible restriction of ferro- and ferricyanide oxidoreductase activities of rat liver mitochondria by the outer membrane

In this work, various ferro-ferricyanide oxidoreductase activities of rat liver mitochondria were studied to find conditions under which the outer membrane might restrict the flux of these highly charged non-biological anions. When the isotonic low ionic strength medium was supplemented with 25 mM KCl, a several-fold increase in the succinate-ferricyanide reductase activity of mitochondria and in the rate of external NADH oxidation in the presence of ferrocyanide was observed. Mitochondrial respiration with 5 mM ferrocyanide was almost completely inhibited after consumption of 3.8–18.5% of the dissolved oxygen, depending on the medium and the presence of 2,4-dinitrophenol. These and other experimental data together with mathematical modeling of the redox-state equilibrium suggest that the measured activities might be restricted by two factors: first, the permeability of the outer mitochondrial membrane and second, a strong influence of the ionic strength of incubation media on the intermembrane space redox reactions.

Polymeric micelles for the solubilization and delivery of cyclosporine A: pharmacokinetics and biodistribution

The aim of this study was to assess the potential of polymeric micelles to modify the pharmacokinetics and tissue distribution of cyclosporine A (CsA). Drug-loaded methoxy poly(ethylene oxide)- b-poly( ε -caprolactone) (PEO- b-PCL) micellar solutions in isotonic medium were prepared and administered intravenously to healthy Sprague-Dawley rats. Blood and tissues were harvested and assayed for CsA, and resultant pharmacokinetic parameters and tissue distribution of CsA in its polymeric micellar formulation were compared to its commercially available intravenous formulation (Sandimmune ®). In the pharmacokinetic assessment, a 6.1 fold increase in the area under the blood concentration versus time curve (AUC) was observed for CsA when given as polymeric micellar formulation as compared to Sandimmune ®. The volume of distribution and clearance of CsA as PEO- b-PCL formulation were observed to be 10.0 and 7.6 fold lower, respectively, compared to the commercial formulation. No significant differences in t 1/2 or MRT could be detected. In the biodistribution study, analysis of tissue samples indicated that the mean AUC of CsA in polymeric micelles was lower in liver, spleen and kidney (1.5, 2.1 and 1.4-fold, respectively). Similar to the pharmacokinetic study in these rats, the polymeric micellar formulation gave rise to 5.7 and 4.9-fold increase in the AUC of CsA in blood and plasma, respectively. Our results show that PEO- b-PCL micelles can effectively solubilize CsA, at the same time confining CsA to the blood circulation and restricting its access to tissues such as kidney, perhaps limiting the onset of toxicity.

Comparison of nasal tissue concentrations in rabbits following administration of hypotonic and isotonic ciclesonide suspensions

RATIONALE: Typically, intranasal sprays used to treat allergic rhinitis are formulated in an isotonic medium. Metabolism and activation of a hypotonic ciclesonide (CIC) suspension, an investigational new corticosteroid being studied for treatment of allergic rhinitis, was compared with an isotonic CIC suspension in rabbit nasal mucosa to determine differential effects.

Modulation of Hypertonicity-Induced Aquaporin-1 by Sodium Chloride, Urea, Betaine, and Heat Shock in Murine Renal Medullary Cells

Aquaporin-1 (AQP1) expression is induced by hypertonicity in renal medullary cells. The purpose of the present study was to elucidate the role of sodium chloride (NaCl), urea, betaine, and heat shock on hypertonicity-induced AQP1 expression in cultured murine renal medullary-K2 (mIMCD-K2) cells. AQP1 expression was maximally induced under mild hypertonic medium supplemented with 100 mM NaCl (N100), whereas severe hypertonic medium supplemented with 150 mM NaCl (N150) caused little AQP1 induction. The reduction of AQP1 expression in N150 was associated with reduced cell viability. When cells were exposed continuously to N100, hypertonicity-induced AQP1 expression was elevated, whereas the return to isotonic medium reduced AQP1 expression in a time-dependent manner. The half-life of AQP1 protein in isotonic conditions was approximately 4 h, whereas hypertonicity markedly increased its half-life. These results indicate that hypertonicity plays an important role in AQP1 induction, stability, and degradation. On the contrary, urea inhibited hypertonicity-induced AQP1 expression in a dose-dependent manner. The addition of organic osmolyte betaine in N150 enhanced hypertonicity-induced AQP1 expression, whereas it decreased AQP1 expression in N100. This suggests that the excessive accumulation of betaine may counteract hypertonic stress and thus attenuate hypertonicity-induced AQP1 expression. Heat shock treatment promoted hypertonicity-induced AQP1 and heat shock protein 70 (HSP70) expression in both N100 and N150, suggesting an effect on the stability of hypertonicity-induced AQP1 expression. Taken together, NaCl, urea, betaine, and heat shock that regulate hypertonicity-induced AQP1 expression are potentially important factors in urinary concentration and contribute to the steady-state level of AQP1 expression.

Differential Effects of Hyperosmolality on Na-K-ATPase and Vasopressin-Dependent cAMP Generation in the Medullary Thick Ascending Limb and Outer Medullary Collecting Duct

Hyperosmolality in the renal medullary interstitium is generated by the renal countercurrent multiplication system, in which the medullary thick ascending limb (MAL) and the outer medullary collecting duct (OMCD) primarily participate. Since arginine vasopressin (AVP) regulates Na-K-ATPase activity directly via protein kinase A and indirectly via hyperosmolality, we investigated the acute and chronic effects of hyperosmolality on Na-K-ATPase and AVP-dependent cAMP generation in the MAL and OMCD. Microdissected MAL and OMCD from control and dehydrated rats were used for the measurement of Na-K-ATPase activity, mRNA expression of alpha-1, beta-1, and beta-2 subunits of Na-K-ATPase, and AVP-dependent cAMP generation. Na-K-ATPase activity in the MAL from dehydrated rats, as measured in isotonic medium, was higher than that of control rats. Moreover, incubation of samples in hypertonic medium (490 mOsm/kg H2O) further increased Na-K-ATPase activity. Dehydration increased alpha-1, beta-1, and beta-2 mRNA expression in the MAL without changing that in the OMCD. Western blot analysis revealed that in the outer medulla, the expression of beta-1, but not that of alpha-1 or beta-2, was stimulated by dehydration. Incubation of MAL or OMCD in hypertonic medium increased AVP-dependent cAMP generation. Higher levels of AVP-dependent cAMP were generated in the MAL from dehydrated rats than that of controls, although incubation in hypertonic medium did not lead to additional increases in AVP-dependent cAMP accumulation. In contrast, AVP-dependent cAMP generation in the OMCD was stimulated by dehydration, and was further stimulated by incubation in hypertonic medium. These findings demonstrate that Na-K-ATPase is upregulated short- and long-term hyperosmolality in the MAL, but not in OMCD.

Calcium regulates the mode of exocytosis induced by hypotonic shock in isolated neuronal presynaptic endings

A decrease in the osmolarity of incubation medium is accompanied by calcium influx in neuronal presynaptic endings. We studied the influence of Ca 2+ on exocytosis induced by hypotonic shock using the hydrophilic fluorescent dye acridine orange and the hydrophobic fluorescent dye FM2–10. It was shown using acridine orange that lowering of osmolarity to 230 mOsm/l induces exocytosis both in calcium-containing and calcium-free medium. By contrast, we were able to demonstrate calcium-dependence of exocytosis using styryl dye FM2–10. Lowering of osmolarity leads to increase of [ 3H] d-aspartate and [ 3H]GABA release in calcium-free medium. Addition of calcium inhibits hypotonic-induced neurotransmitter release. Decreasing of NaCl concentration to 92 mM in isotonic medium is able to induce d-aspartate and GABA release. Thus, our data suggest that hypotonic swelling induces calcium-independent exocytosis possibly by a “kiss and run” mechanism. Calcium influx mediated by stretch channels is able to provoke full fusion between plasma membrane and synaptic vesicles. [ 3H] d-aspartate and [ 3H]GABA released by hypotonic shock is determined by sodium lowering rather than by osmolarity decreasing itself.

Proteolytic Activation of Sterol Regulatory Element-binding Protein Induced by Cellular Stress through Depletion of Insig-1

Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum (ER) that block proteolytic activation of sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that activate synthesis of cholesterol and fatty acids in animal cells. When cellular cholesterol levels are high, Insig proteins bind to SREBP cleavage-activating protein, retaining it in the ER and preventing it from escorting SREBPs to the site of proteolytic activation in the Golgi complex. Here we report that hypotonic stress reverses the sterol-mediated inhibition of SREBP proteolytic activation by reducing the level of Insig-1 but not Insig-2. The reduction of Insig-1, a protein with a rapid turnover rate, results from a general inhibition of protein synthesis mediated by hypotonic stress. Insig-2 is not affected by hypotonic stress because of its slower turnover rate. Inhibition of protein synthesis by hypotonic shock has not been reported previously. Thapsigargin, an activator of the ER stress response, also inhibits protein synthesis and activates proteolysis of SREBP. Such activation also correlates with the disappearance of Insig-1. The current study demonstrates that animal cells, in response to either hypotonic shock or ER stress, can bypass the cholesterol inhibition of SREBP processing, an effect that is attributable to the rapid turnover of Insig-1.

Extracellular ATP Increases Cation Fluxes in Human Erythrocytes by Activation of the P2X7 Receptor

Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor.