Extracellular Tonicity Research Articles

Osmoprotective Transcription Factor NFAT5/TonEBP Modulates Nuclear Factor-κB Activity

Tonicity-responsive binding-protein (TonEBP or NFAT5) is a widely expressed transcription factor whose activity is regulated by extracellular tonicity. TonEBP plays a key role in osmoprotection by binding to osmotic response element/TonE elements of genes that counteract the deleterious effects of cell shrinkage. Here, we show that in addition to this "classical" stimulation, TonEBP protects cells against hypertonicity by enhancing nuclear factor-κB (NF-κB) activity. We show that hypertonicity enhances NF-κB stimulation by lipopolysaccharide but not tumor necrosis factor-α, and we demonstrate overlapping protein kinase B (Akt)-dependent signal transduction pathways elicited by hypertonicity and transforming growth factor-α. Activation of p38 kinase by hypertonicity and downstream activation of Akt play key roles in TonEBP activity, IκBα degradation, and p65 nuclear translocation. TonEBP affects neither of these latter events and is itself insensitive to NF-κB signaling. Rather, we reveal a tonicity-dependent interaction between TonEBP and p65 and show that NF-κB activity is considerably enhanced after binding of NF-κB-TonEBP complexes to κB elements of NF-κB-responsive genes. We demonstrate the key roles of TonEBP and Akt in renal collecting duct epithelial cells and in macrophages. These findings reveal a novel role for TonEBP and Akt in NF-κB activation on the onset of hypertonic challenge.

Gene expression profiling and immune fluorescence implicate cellular redifferentiation induced by hypertonicity in primary cultured IMCD cells

The cells of the renal inner medulla are challenged with an environment consisting of high osmolality mediated mainly by Na+ and urea. High tonicity leads to the activation of the tonicity enhancer binding protein, which induces the expression of several transporters and enzymes. This in turn leads to the cellular accumulation of organic osmolytes thereby protecting the cells from high extracellular tonicity. Although several genes that are induced by tonicity are known, a global gene expression profile and analysis of pathways is missing. We used primary cultured rat renal inner medullary collecting duct cells which were cultivated for 6 days at 300, 600 or 900 mosmol/kg. Total RNA was isolated and prepared for hybridization on Rat Gene 1.0 ST GeneChip. Differentially expressed genes were identified using the BRB Array Tools and functional molecular network were identified with Ingenuity Pathway Analysis.Comparing cells grown in 300 vs. 600 mosmol/kg, more than 1600 and vs. 900 mosmol/kg more than 3300 genes were differentially expressed. In both cases the ratio between the up and down regulated genes was approximately one. While 1300 were common in both comparisons 290 at 600 and more than 2000 genes at 900 mosmol/kg were unique. Besides known genes the expression of several other genes were identified for which regulation by tonicity had not been described before. Cellular morphology was markedly modified with increases in culture medium tonicity. Tonicity induced actin reorganization and morphological changes at the cell to cell contacts. It also induced a hypertrophic phenotype. The adaptation to a higher tonicity is not only associated with massive changes in gene expression but also with changes in cell morphology. This implicates that tonicity induces cellular redifferentiation.

Hypotonicity-Induced Renin Exocytosis from Juxtaglomerular Cells Requires Aquaporin-1 and Cyclooxygenase-2

The mechanism by which extracellular hypotonicity stimulates release of renin from juxtaglomerular (JG) cells is unknown. We hypothesized that osmotically induced renin release depends on water movement through aquaporin-1 (AQP1) water channels and subsequent prostanoid formation. We recorded membrane capacitance (C(m)) by whole-cell patch clamp in single JG cells as an index of exocytosis. Hypotonicity increased C(m) significantly and enhanced outward current. Indomethacin, PLA(2) inhibition, and an antagonist of prostaglandin transport impaired the C(m) and current responses to hypotonicity. Hypotonicity also increased exocytosis as determined by a decrease in single JG cell quinacrine fluorescence in an indomethacin-sensitive manner. In single JG cells from COX-2(-/ -) and AQP1(-/ -) mice, hypotonicity increased neither C(m) nor outward current, but 0.1-muM PGE(2) increased both in these cells. A reduction in osmolality enhanced cAMP accumulation in JG cells but not in renin-producing As4.1 cells; only the former had detectable AQP1 expression. Inhibition of protein kinase A blocked the hypotonicity-induced C(m) and current response in JG cells. Taken together, our results show that a 5 to 7% decrease in extracellular tonicity leads to AQP1-mediated water influx in JG cells, PLA(2)/COX-2-mediated prostaglandin-dependent formation of cAMP, and activation of PKA, which promotes exocytosis of renin.

Tonicity-dependent induction of Sgk1 expression has a potential role in dehydration-induced natriuresis in rodents

In various mammalian species, including humans, water restriction leads to an acute increase in urinary sodium excretion. This process, known as dehydration natriuresis, helps prevent further accentuation of hypernatremia and the accompanying rise in extracellular tonicity. Serum- and glucocorticoid-inducible kinase (Sgk1), which is expressed in the renal medulla, is regulated by extracellular tonicity. However, the mechanism of its regulation and the physiological role of hypertonicity-induced SGK1 gene expression remain unclear. Here, we identified a tonicity-responsive enhancer (TonE) upstream of the rat Sgk1 transcriptional start site. The transcription factor NFAT5 associated with TonE in a tonicity-dependent fashion in cultured rat renal medullary cells, and selective blockade of NFAT5 activity resulted in suppression of the osmotic induction of the Sgk1 promoter. In vivo, water restriction of rats or mice led to increased urine osmolality, increased Sgk1 expression, increased expression of the type A natriuretic peptide receptor (NPR-A), and dehydration natriuresis. In cultured rat renal medullary cells, siRNA-mediated Sgk1 knockdown blocked the osmotic induction of natriuretic peptide receptor 1 (Npr1) gene expression. Furthermore, Npr1-/- mice were resistant to dehydration natriuresis, which suggests that Sgk1-dependent activation of the NPR-A pathway may contribute to this response. Collectively, these findings define a specific mechanistic pathway for the osmotic regulation of Sgk1 gene expression and suggest that Sgk1 may play an important role in promoting the physiological response of the kidney to elevations in extracellular tonicity.

Aquaporin-2 abundance in the renal collecting duct: new insights from cultured cell models

The renal cortico-papillary osmotic gradient is generated by sodium reabsorption in the thick ascending limb. The antidiuretic hormone arginine vasopressin (AVP) increases collecting duct water permeability by enhancing aquaporin-2 (AQP2) water channel insertion in the apical membrane of principal cells, allowing water to passively flow along the osmotic gradient from the tubule lumen to the interstitium. In addition to short-term AQP2 redistribution between intracellular compartments and the cell surface, AQP2 whole cell abundance is tightly regulated. AVP is a major transcriptional activator of the AQP2 gene, and stimulation of insulin- and calcium-sensing receptors respectively potentiate and reduce its action. Extracellular tonicity is another key factor that determines the levels of AQP2 abundance. Its effect is dependent on activation of the tonicity-responsive enhancer binding protein that reinforces AVP-induced AQP2 transcriptional activation. Conversely, activation of the NF-kappaB transcriptional factor by proinflammatory factors reduces AQP2 gene transcription. Aldosterone additionally regulates AQP2 whole cell abundance by simultaneously reducing AQP2 gene transcription and stimulating AQP2 mRNA translation. These examples illustrate how cross talk between various stimuli regulates AQP2 abundance in collecting duct principal cells and consequently contributes to maintenance of body water homeostasis.

Control of Cell Volume in Skeletal Muscle

Regulation of cell volume is a fundamental property of all animal cells and is of particular importance in skeletal muscle where exercise is associated with a wide range of cellular changes that would be expected to influence cell volume. These complex electrical, metabolic and osmotic changes, however, make rigorous study of the consequences of individual factors on muscle volume difficult despite their likely importance during exercise. Recent charge-difference modelling of cell volume distinguishes three major aspects to processes underlying cell volume control: (i) determination by intracellular impermeant solute; (ii) maintenance by metabolically dependent processes directly balancing passive solute and water fluxes that would otherwise cause cell swelling under the influence of intracellular membrane-impermeant solutes; and (iii) volume regulation often involving reversible short-term transmembrane solute transport processes correcting cell volumes towards their normal baselines in response to imposed discrete perturbations. This review covers, in turn, the main predictions from such quantitative analysis and the experimental consequences of comparable alterations in extracellular pH, lactate concentration, membrane potential and extracellular tonicity. The effects of such alterations in the extracellular environment in resting amphibian muscles are then used to reproduce the intracellular changes that occur in each case in exercising muscle. The relative contributions of these various factors to the control of cell volume in resting and exercising skeletal muscle are thus described.

Phosphorylation by Casein Kinase 1 Regulates Tonicity-induced Osmotic Response Element-binding Protein/Tonicity Enhancer-binding Protein Nucleocytoplasmic Trafficking

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, is the only known osmo-sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. Although it is well documented that the subcellular localization and transactivation activity of OREBP/TonEBP are tightly regulated by extracellular tonicity, the molecular mechanisms involved remain elusive. Here we show that nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by the dual phosphorylation of Ser-155 and Ser-158. Alanine scanning mutagenesis revealed that Ser-155 is an essential residue that regulates OREBP/TonEBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser-155 and Ser-158 of OREBP/TonEBP are both phosphorylated in living cells under hypotonic conditions. In vitro phosphorylation assays further suggest that phosphorylation of the two serine residues proceeds in a hierarchical manner with phosphorylation of Ser-155 priming the phosphorylation of Ser-158 and that these phosphorylations are essential for nucleocytoplasmic trafficking of the transcription factor. Finally, we have shown that the pharmacological inhibition of casein kinase 1 (CK1) abolishes the phosphorylation of Ser-158 and impedes OREBP/TonEBP nuclear export and that recombinant CK1 phosphorylates Ser-158. Knockdown of CK1alpha1L, a novel isoform of CK1, inhibits hypotonicity-induced OREBP/TonEBP nuclear export. Together these data highlight the importance of Ser-155 and Ser-158 in the nucleocytoplasmic trafficking of OREBP/TonEBP and indicate that CK1 plays a major role in regulating this process.

Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice

BackgroundThe transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells.ResultsThe 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360–380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages.ConclusionOur results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells.

Membrane potentials in Rana temporaria muscle fibres in strongly hypertonic solutions.

Conventional microelectrode methods were used to measure variations in resting membrane potentials, E(m), of intact amphibian skeletal muscle fibres over a wide range of increased extracellular tonicities produced by inclusion of varying extracellular concentrations of sucrose. Moderate increases in extracellular tonicity to up to 2.6x normal (2.6tau) under Cl(-) free conditions produced negative shifts in E(m) that followed expectations for the K(+) Nernst equation (E(K)) applied to a perfect osmometer containing a conserved intracellular K(+) content despite any accompanying cell volume change. In contrast, E(m) remained stable in fibres studied in otherwise similar Cl(-) containing solutions, consistent with E(m) stabilization despite negative shifts in E(K) through inward cation-Cl(-) co-transport activity. Short exposures to higher tonicities (>3tau) similarly produced negative shifts in E(m) in Cl(-) free but not Cl(-) containing solutions. However, prolonged exposures to solutions of >3tau caused gradual net positive changes in E (m) in both Cl(-) containing and Cl(-) free solutions suggesting that these changes were independent of cation-Cl(-) transport. Indeed, there was no evidence of cation-Cl(-) co-transport activity in strongly hypertonic solutions despite its predicted energetic favourability, suggesting its possible regulation by E (m) in muscle. Additional findings implicated a failure to maintain greatly increased transmembrane [K(+)] gradients in these E(m) changes. Thus: (1) halving or doubling [K(+)](e) produced negative or positive shifts in E(m), respectively in isotonic or moderately hypertonic (<2.7tau), but not strongly hypertonic (>3tau) solutions; (2) subsequent restoration of isotonic extracellular conditions produced further positive changes in E(m) consistent with a dilution of the depleted [K(+)](i) by fibres regaining their original resting volumes; (3) quantitative modelling similarly predicted a gradual net efflux of K(+) as the balance between active and passive [K(+)] fluxes altered due to increased transmembrane [K(+)] gradients in hypertonic and low [K(+)](e) solutions. However, the observed positive changes in E(m) in the most strongly hypertonic solutions eventually exceeded these predictions suggesting additional limitations on Na(+)/K(+)-ATPase activity in strongly hypertonic solutions.

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.

Identification of novel nuclear export domains of transcription factor OREBP/TonEBP/NFAT5

The Osmotic Response Element-Binding Protein (OREBP), also known as the 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. Specifically, OREBP/TonEBP undergoes nuclear translocation and export under hypertonic and hypotonic condition respectively. Here we show that, by immunocytochemistry and GFP fusion, the transactivation domain of OREBP/TonEBP is dispensable for proper subcellular localization of OREBP/TonEBP in response to change in osmolality. Under isotonicity, OREBP/TonEBP undergoes nucleocytoplasmic shuttling which can be blocked by leptomycin B (LMB) treatment. Unexpectedly, two leucine-rich motifs that exhibit significant sequence similarity to canonical nuclear export signal (NES) located in the N-terminal of OREBP/TonEBP are not responsible for the export. Nevertheless, we identified a CRM1 responsive protein domain (designated NES-A) responsible for the nucleocytoplasmic shuttling of OREBP/TonEBP under isotonicity. Disruption of NES-A increases nuclear translocation of OREBP/TonEBP, yet hypotonicity is able to direct nuclear export of this mutant. Moreover, we identified a second protein motif (designated NES-B) that is critical for OREBP/TonEBP nuclear export under both isotonic and hypotonic conditions. Disruption of the NES-B alone is sufficient to cause aberrant nuclear translocation of OREBP/TonEBP irrespective of extracellular tonicity. Our study provides novel insights on the regulation of nucleo-cytoplasmic trafficking of OREBP/TonEBP.

Role of integrin α1β1in the regulation of renal medullary osmolyte concentration

The mechanism by which cells sense extracellular tonicity and trigger the accumulation of protective organic osmolytes is poorly understood. It has been proposed that changes in cell volume following alteration of extracellular toncity are important initiators of signaling events that lead to osmolyte accumulation. Because the extracellular matrix receptors integrins are linked to the cytoskeleton and can transduce signals that alter cell behavior, we investigated the role of these receptors in the modulation of osmolyte accumulation in the kidney medulla under different osmotic conditions. We show that integrin alpha1-null mice have impaired ability to accumulate organic osmolytes in the inner medulla due to altered signaling and decreased induction of osmolyte transporters or aldose reductase gene transcription. Utilizing inner medullary collecting duct cells, we demonstrate that the lack of integrin alpha1beta1 results in an impaired ability to induce the tonicity enhancer-binding protein TonEBP under hypertonic conditions. Furthermore, under the same conditions, integrin alpha1-null cells show prolonged ERK1/2 phosphorylation and decreased inositol uptake compared with control cells. The reduction of inositol uptake is significantly reversed by treatment with the MEK inhibitor PD-98059. Finally, integrin alpha1-null mice develop morphological changes of early tubular necrosis and increased apoptosis of renal medullary cells following dehydration. Together, these results show that integrin alpha1beta1 is an important mediator of the compatible osmolyte response in the medulla of the mammalian kidney.

Proteomic Analysis of Cellular Response to Osmotic Stress in Thick Ascending Limb of Henle’s Loop (TALH) Cells

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.

Dual Effects of Hypertonicity on Aquaporin-2 Expression in Cultured Renal Collecting Duct Principal Cells

The driving force for renal water reabsorption is provided by the osmolarity gradient between the interstitium and the tubular lumen, which is subject to rapid physiologic variations as a consequence of water intake fluctuations. The effect of increased extracellular tonicity/osmolarity on vasopressin-inducible aquaporin-2 (AQP2) expression in immortalized mouse collecting duct principal cells (mpkCCD(cl4)) is investigated in this report. Increasing the osmolarity of the medium either by the addition of NaCl, sucrose, or urea first decreased AQP2 expression after 3 h. AQP2 expression then increased in cells exposed to NaCl- or sucrose-supplemented hypertonic medium after longer periods of time (24 h), while urea-supplemented hyperosmotic medium had no effect. Altered AQP2 expression induced by both short-term (3 h) and long-term (24 h) exposure of cells to hypertonicity arose from changes in AQP2 gene transcription because hypertonicity did not modify AQP2 mRNA stability nor AQP2 protein turnover. On the long-term, vasopressin (AVP) and hypertonicity increased AQP2 expression in a synergistic manner. Hypertonicity altered neither the dose-responsiveness of AVP-induced AQP2 expression nor cAMP-protein kinase (PKA) activity, while PKA inhibition did not reduce the extent of the hypertonicity-induced increase of AQP2 expression. These results indicate that in collecting duct principal cells: (1) a short-term increase of extracellular osmolarity decreases AQP2 expression through inhibition of AQP2 gene transcription; (2) a long-term increase of extracellular tonicity, but not osmolarity, enhances AQP2 expression via stimulation of AQP2 gene transcription; and (3) long-term hypertonicity and PKA increases AQP2 expression through synergistic but independent mechanisms.

The effect of intracellular acidification on the relationship between cell volume and membrane potential in amphibian skeletal muscle.

The relationship between cell volume (V(c)) and membrane potential (E(m)) in Rana temporaria striated muscle fibres was investigated under different conditions of intracellular acidification. Confocal microscope xz-scanning monitored the changes in V(c), whilst conventional KCl and pH-sensitive microelectrodes measured E(m) and intracellular pH (pH(i)), respectively. Applications of Ringer solutions with added NH(4)Cl induced rapid reductions in V(c) that rapidly reversed upon their withdrawal. These could be directly attributed to the related alterations in extracellular tonicity. However: (1) a slower and persistent decrease in V(c) followed the NH(4)Cl withdrawal, leaving V(c) up to 10% below its resting value; (2) similar sustained decreases in resting V(c) were produced by the addition and subsequent withdrawal of extracellular solutions in which NaCl was isosmotically replaced with NH(4)Cl; (3) the same manoeuvres also produced a marked intracellular acidification, that depended upon the duration of the preceding exposure to NH(4)Cl, of up to 0.53 +/- 0.10 pH units; and (4) the corresponding reductions in V(c) similarly increased with this exposure time. These reductions in V(c) persisted and became more rapid with Cl(-) deprivation, thus excluding mechanisms involving either direct or indirect actions of pH(i) upon Cl(-)-dependent membrane transport. However they were abolished by the Na(+),K(+)-ATPase inhibitor ouabain. The E(m) changes that accompanied the addition and withdrawal of NH(4)(+) conformed to a Nernst equation modified to include realistic NH(4)(+) permeability terms, and thus the withdrawal of NH(4)(+) restored E(m) to close to control values despite a persistent change in V(c). Finally these E(m) changes persisted and assumed faster kinetics with Cl(-) deprivation. The relative changes in V(c), E(m) and pH(i) were compared to predictions from the recent model of Fraser and Huang published in 2004 that related steady-state values of V(c) and E(m) to the mean charge valency (z(x)) of intracellular membrane-impermeant anions, X(-)(i). By assuming accepted values of intracellular buffering capacity (beta(i)), intracellular acidification was shown to produce quantitatively predictable decreases in V(c). These findings thus provide experimental evidence that titration of the anionic z(x) by increased intracellular [H(+)] causes cellular volume decrease in the presence of normal Na(+),K(+)- ATPase activity, with Cl(-)-dependent membrane fluxes only influencing the kinetics of such changes.

Membrane potential stabilization in amphibian skeletal muscle fibres in hypertonic solutions.

This study investigated membrane transport mechanisms influencing relative changes in cell volume (V) and resting membrane potential (E(m)) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of E(m) despite cell shrinkage, which was attributable to elevation of intracellular [Cl(-)] above electrochemical equilibrium through Na(+)-Cl(-) and Na(+)-K(+)-2Cl(-) cotransporter action following exposures to extracellular hypertonicity. Fibre volumes (V) determined by confocal microscope x z - scanning of cutaneous pectoris muscle fibres varied linearly with [1/extracellular osmolarity], showing insignificant volume corrections, in fibres studied in Cl(-)-free, normal and Na(+)-free Ringer solutions and in the presence of bumetanide, chlorothiazide and ouabain. The observed volume changes following increases in extracellular tonicity were compared with microelectrode measurements of steady-state resting potentials (E(m)). Fibres in isotonic Cl(-)-free, normal and Na(+)-free Ringer solutions showed similar E(m) values consistent with previously reported permeability ratios P(Na)/P(K)(0.03-0.05) and P(Cl)/P(K) ( approximately 2.0) and intracellular [Na(+)], [K(+)] and [Cl(-)]. Increased extracellular osmolarities produced hyperpolarizing shifts in E(m) in fibres studied in Cl(-)-free Ringer solution consistent with the Goldman-Hodgkin-Katz (GHK) equation. In contrast, fibres exposed to hypertonic Ringer solutions of normal ionic composition showed no such E(m) shifts, suggesting a Cl(-)-dependent stabilization of membrane potential. This stabilization of E(m) was abolished by withdrawing extracellular Na(+) or by the combined presence of the Na(+)-Cl(-) cotransporter (NCC) inhibitor chlorothiazide (10 microM) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC) inhibitor bumetanide (10 microM), or the Na(+)-K(+)-ATPase inhibitor ouabain (1 or 10 microM) during alterations in extracellular osmolarity. Application of such agents after such increases in tonicity only produced a hyperpolarization after a time delay, as expected for passive Cl(-) equilibration. These findings suggest a model that implicates the NCC and/or NKCC in fluxes that maintain [Cl(-)](i) above its electrochemical equilibrium. Such splinting of [Cl(-)](i) in combination with the high P(Cl)/P(K) of skeletal muscle stabilizes E(m) despite volume changes produced by extracellular hypertonicity, but at the expense of a cellular capacity for regulatory volume increases (RVIs). In situations where P(Cl)/P(K) is low, the same co-transporters would instead permit RVIs but at the expense of a capacity to stabilize E(m).

Regulation of the cellular content of the organic osmolyte taurine in mammalian cells.

Change in the intracellular concentration of osmolytes or the extracellular tonicity results in a rapid transmembrane water flow in mammalian cells until intracellular and extracellular tonicities are equilibrated. Most cells respond to the osmotic cell swelling by activation of volume-sensitive flux pathways for ions and organic osmolytes to restore their original cell volume. Taurine is an important organic osmolyte in mammalian cells, and taurine release via a volume-sensitive taurine efflux pathway is increased and the active taurine uptake via the taurine specific taurine transporter TauT decreased following osmotic cell swelling. The cellular signaling cascades, the second messengers profile, the activation of specific transporters, and the subsequent time course for the readjustment of the cellular content of osmolytes and volume vary from cell type to cell type. Using Ehrlich ascites tumor cells, NIH3T3 mouse fibroblasts and HeLa cells as biological systems, it is revealed that phospholipase A2-mediated mobilization of arachidonic acid from phospholipids and subsequent oxidation of the fatty acid via lipoxygenase systems to potent eicosanoids are essential elements in the signaling cascade that is activated by cell swelling and leads to release of osmolytes. The cellular signaling cascade and the activity of the volume-sensitive taurine efflux pathway are modulated by elements of the cytoskeleton, protein tyrosine kinases/phosphatases, GTP-binding proteins, Ca2+/calmodulin, and reactive oxygen species and nucleotides. Serine/threonine phosphorylation of the active taurine uptake system TauT or a putative regulator, as well as change in the membrane potential, are important elements in the regulation of TauT activity. A model describing the cellular sequence, which is activated by cell swelling and leads to activation of the volume-sensitive efflux pathway, is presented at the end of the review.

The effect of extracellular tonicity on the anatomy of triad complexes in amphibian skeletal muscle.

Ultrastructural features of tubular-sarcoplasmic (T-SR) triad junctions and measures of cell volume following graded increases of extracellular tonicity were compared under physiological conditions recently shown to produce spontaneous release of intracellularly stored Ca2+ in fully polarized amphibian skeletal muscle fibres. The fibres were fixed using solutions of equivalent tonicities prior to processing for electron microscopy. The resulting anatomical sections demonstrated a partially reversible cell shrinkage corresponding to substantial increases in intracellular solute or ionic strength graded with extracellular tonicity. Serial thin sections through triad structures confirmed the presence of geometrically close but anatomically isolated transverse (T-) tubular and sarcoplasmic reticular (SR) membranes contrary to earlier suggestions for the development of luminal continuities between these structures in hypertonic solutions. They also quantitatively demonstrated accompanying decreases in T-SR distances, increased numbers of sections that showed closely apposed T and SR membranes, tubular luminal swelling and reductions in luminal volume of the junctional SR, all correlated with the imposed increases in extracellular osmolarity. Fully polarized fibres correspondingly showed elementary Ca(2+)-release events ('sparks', in 100 mM-sucrose-Ringer solution), sustained Ca2+ elevations and propagated Ca2+ waves (> or = 350-500 mM sucrose) following exposure to physiological Ringer solutions of successively greater tonicities. These were absent in hypotonic, isotonic or less strongly hypertonic (approximately 50 mM sucrose-Ringer) solutions. Yet exposure to hypotonic solutions also disrupted T-SR junctional anatomy. It increased the tubular diameters and T-SR distances and reduced their area of potential contact. The spontaneous release of intracellularly stored Ca2+ thus appears more closely to correlate with the expected changes in intracellular solute strength or a reduction in absolute T-SR distance rather than disruption of an optimal anatomical relationship between T and SR membranes taking place with either increases or decreases in extracellular tonicity.

Endothelin inhibits NPR-A and stimulates eNOS gene expression in rat IMCD cells.

We have shown in previous studies that high extracellular tonicity is associated with increased expression of the type A natriuretic peptide receptor (NPR-A) and reduced expression of the endothelial NO synthase (eNOS) gene in cultured rat inner-medullary collecting duct cells. The vasoactive peptide endothelin has been shown to be avidly expressed in this nephron segment, and to be subject to osmotic regulation. We asked whether endothelin might play a role in the control of basal or osmotically regulated NPR-A or eNOS gene expression in these cells. Although exogenous endothelin had little or no effect on basal expression of eNOS mRNA or protein or NPR-A gene expression, both the type A (BQ610) and type B (IRL1038) endothelin receptor antagonists proved capable of reducing eNOS mRNA and protein expression, and increasing levels of the NPR-A mRNA. Increased extracellular tonicity reduced endothelin mRNA accumulation in these cells (approximately 15% of control levels); however, exogenous endothelin failed to normalize osmotically increased NPR-A activity or expression, or osmotically suppressed eNOS expression. Collectively, these data demonstrate the presence of a number of independent but highly interactive local regulatory networks governing fluid and electrolyte handling in this distal nephron segment.

Osmoregulation of Endothelial Nitric-oxide Synthase Gene Expression in Inner Medullary Collecting Duct Cells: ROLE IN ACTIVATION OF THE TYPE A NATRIURETIC PEPTIDE RECEPTOR

Previously, we showed that increased extracellular tonicity promotes increased type A natriuretic peptide receptor (NPR-A) expression through a p38 MAPKbeta pathway in inner medullary collecting duct cells. The endothelial and inducible nitric-oxide synthase (eNOS and iNOS respectively) genes are also expressed in this nephron segment and are thought to play a role in regulating urinary sodium concentration. We sought to determine whether changes in tonicity might regulate NOS gene expression, and if so, whether these latter changes might be linked mechanistically to the increase in NPR-A gene expression. Increased extracellular tonicity effected a time-dependent reduction in eNOS and iNOS protein levels, eNOS mRNA levels, and eNOS gene promoter activity over the first 8 h of the incubation. Although levels of the eNOS mRNA and promoter activity had returned to normal after 24 h, eNOS protein levels remained low at 24-36 h, and recovery was not complete even at 48 h. The decrease in eNOS expression was signaled in large part through a p38 MAPK-dependent mechanism. Reduction in eNOS expression together with the concomitant decline in intracellular cyclic GMP levels appears to account for a significant portion of the p38 MAPK-dependent osmotic stimulation of NPR-A gene expression noted previously. Collectively, these findings support the existence of a complex regulatory circuitry in the cells of the inner medullary collecting duct linking two independent cyclic GMP-generating signal transduction systems involved in regulation of urinary sodium concentration.