Hypotonic Challenge Research Articles

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.

Cell-Based Fluorescence Screen for K+ Channels and Transporters Using an Extracellular Triazacryptand-Based K+ Sensor

K+ channels and K+-coupled membrane transporters are important targets for drug discovery. We previously developed a triazacryptand (TAC)-based K+ sensor, TAC-Red, and demonstrated its utility to image K+ waves in mouse brain in vivo (Padmawar et al. Nat. Methods. 2005, 2, 825-827). Here, we synthesized a green-fluorescing dextran conjugate of TAC-bodipy ("TAC-Limedex") for use as an extracellular K+ sensor and demonstrated its utility in measuring K+ transport across cell membranes. TAC-Limedex fluorescence increased by 50% with increasing [K+] from 0 to 2 mM and was insensitive to [Na+], [Cl-], or pH. K+ efflux from cells was quantified from increasing extracellular TAC-Limedex fluorescence following cell immersion in K+-free buffer. In HT-29 cells, K+ efflux was 2.0 +/- 0.1 micromol/cm2/s, increasing 8-fold following K+ channel activation by ATP; the increase in K+ efflux was inhibited by a K+ channel blocker or by preventing cytoplasmic calcium elevation. Electroneutral K+/Cl- cotransport was demonstrated in SiHa cells, in which K+ efflux was increased 3-fold by hypotonic challenge; the increase in K+ efflux was fully inhibited by a K+/Cl- transport blocker. K+ efflux measurements were adapted to a commercial fluorescence platereader for automated screening. The fluorescence-based K+ transport assay largely replaces assays requiring radioactive rubidium and is suitable for high-throughput identification of K+ transport modulators.

Dynamic Effects of Hg2+-induced Changes in Cell Volume

Using a microfluidic volume sensor, we studied the dynamic effects of Hg2+ on hypotonic stress-induced volume changes in CHO cells. A hypotonic challenge to control cells caused them to swell but did not evoke a significant regulatory volume decrease (RVD). Treatment with 100 muM HgCl2 caused a substantial increase in the steady-state volume following osmotic stress. Continuous hypotonic challenge following a single 10-min exposure to HgCl2 produced a biphasic volume increase with a steady-state volume 100% larger than control cells. Repeated hypotonic challenges to cells exposed once to Hg2+ resulted in a sequential approach to the same steady-state volume. Stimulation after reaching steady state caused a reduction in peak cell volume. Repeated stimulation was different than continuous stimulation resulting in a more rapid approach to steady state. Substituting extracellular Na+ with impermeant NMDG+ in the hypotonic solution produced a rapid RVD-like volume decrease and eliminated the Hg2+-induced excess swelling. The volume decrease in the presence of Hg2+ was inhibited by tetraethylammonium and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium, blockers of K+ and Cl(-) channels, respectively, suggesting that part of the Hg2+ effect was increasing NaCl influx over KCl efflux. The presence of multiple phases of steady-state volume and their sensitivity to the stimulation history suggests that factors beyond solute fluxes, such as modification of mechanical stress within the cytoskeleton also plays a role in the response to hypotonic stress.

ATP Release via Connexin Hemichannels from Rat Ventricular Fibroblasts

ATP can be released by and regulate the function of many cell types via the activation of membrane receptors. However, the release of ATP from cardiac fibroblasts (CF) has not been studied. We hypothesized that CF release ATP in response to physical perturbation, and that this release may be mediated by connexin (Cx) hemichannels. We isolated ventricular CF from adult Sprague Dawley rats, exposed them to hypotonic challenge or cyclic mechanical stress, and assayed extracellular media for ATP by a luciferin‐luciferase method. We found that hypotonic challenge and cyclic mechanical stretch increased ATP release from CF. Decreasing extracellular calcium from 1 mM to 0 mM, a maneuver that opens Cx hemichannel, increased ATP release, while carbenoxolone, a Cx blocker, markedly decreased ATP release. Real‐time PCR revealed that CF express multiple Cx proteins: Cx 43 > Cx 45 > Cx 40 and that TGF‐β treatment decreased expression of Cx 43 by 40%. We conclude that ATP release occurs from CF in response to hypotonic conditions and mechanical stretch and that this release likely occurs through Cx hemichannels, whose expression can be regulated by TGF‐β, a profibrogenic stimulus. Since CF and myocytes express receptors for ATP and its metabolites, release of ATP by CF likely contributes to autocrine/paracrine regulation of both fibroblasts and myocytes in the heart. (Supported by grants from NIH, AHA, and Ellison Medical Foundation)

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.

Journeying down the long and winding road: the whole picture of volume-activated Cl- channel activation in cardiac myocytes

Patch-clamp studies of anion channels show a bewildering variety in their properties and regulation mechanisms.2 Amongst them, the anion channels activated by hypo-osmotic cell swelling ( I Cl,swell) have drawn attention because of their wide expression in virtually every vertebrate cell(s), including cardiac myocytes, and their critical roles in cell volume regulation.2–4 Cells need to regulate their volume in the face of several external and internal challenges. For this purpose, cells are endowed with various ion channels and transporters that become activated upon cell swelling or shrinkage. I Cl,swell are also called by different names like volume-regulated anion channels and volume-stimulated osmolyte and anion channels, implicating their permeability to a wide variety of anionic molecules including organic substances and ATP4−. In spite of their importance and ubiquitous expression, their molecular nature is still the subject of controversy and remains unclear.2,3 One of the key issues in I Cl,swell is the activation mechanism, i.e. how is the hypo-osmotic stress transduced into anion channel activation? A direct mechanical gating is unlikely with regard to the considerable time lag between hypotonic challenge and steady-state activation of I Cl,swell. The list of suggested mechanisms or critical factors supported by experimental evidence include … *Corresponding author. Tel: +82 2 740 8224; fax: +82 2 763 9667. E-mail address : earmye{at}snu.ac.kr

Control of chondrocyte regulatory volume decrease (RVD) by [Ca 2+] i and cell shape

Optimal matrix metabolism by articular chondrocytes is controlled by the 'set-point' volume which is determined mainly by membrane transporters. The signal transduction pathway(s) for the key membrane transporter which responds to cell swelling ('osmolyte channel') and mediates regulatory volume decrease (RVD) is poorly understood, so here the role of Ca2+ and the effects of 2D culture have been clarified. Changes to the volume and intracellular calcium levels ([Ca2+]i) of freshly isolated and 2D cultured bovine articular chondrocytes subjected to hypotonic challenge using a 43% reduction in medium osmolarity were studied by single-cell fluorescence microscopy. The effects of ethylene glycol tetraacetic acid (EGTA), REV5901 and Gd(3+) were studied and the role of Ca2+ influx determined by Mn2+ quench. In freshly isolated cells, approximately 50% of chondrocytes exhibited 'robust RVD' (6[120]). RVD was inhibited by REV 5901 (4+/-2% responding) (3[23]) and 2 mM EGTA (18+/-5% responding) (4[166]) whereas Gd3+ had no effect (3[89]). The hypotonic challenge resulted in a Gd3+-insensitive rise in [Ca2+]i that did not correlate with RVD in all cells. Following 2D culture, chondrocytes also demonstrated Gd3+-insensitive RVD, but in contrast, the [Ca2+]i rise was blocked by this agent. The data suggested that in freshly isolated and 2D cultured chondrocytes, the rise in [Ca2+]i occurring during hypotonic challenge could be related to RVD, but only in some cells. However, with 2D culture, the Ca2+ response switched to being Gd3+-sensitive, suggesting that as a result of changes to chondrocyte shape, stretch-activated cation channels although present, do not appear to play a role in volume regulation.

4α‐PDD induces Ca2+ influx in human corneal epithelial cells by activating TRPV4 channels

Abstract Purpose: Transient receptor potential (TRP) isoform expression is evident in human corneal epithelial cells (HCEC‐SV40). However, their role in maintaining corneal epithelial homeostasis is not fully understood. We probed for gene and protein expression as well as functional activity of the vanilloid subtype, TRPV4, in immortalized HCEC‐SV40 since they elicit Ca2+ dependent regulatory volume decrease (RVD) responses during exposure to a hypotonic challenge. Methods: RT‐PCR and Western blotting analyses identified TRPV4 gene and protein expression. Functional activity was assessed based on determining whether the TRPV4 selective agonist, 4α‐PDD, induced transients increases in intracellular Ca2+ concentration. Results: Single cell fluorescence imaging results showed that 4α‐PDD (3 μM) increased intracellular Ca2+ concentration. The fura2 fluorescence ratio (f340/f380) was 0.39 ± 0.03578 in the resting state (n = 5). After application of 4α‐PDD it increased to 0.904 ± 0.14363 (n = 5; p = 5.72077×10‐5). This increase was abolished by the TRP channel blocker ruthenium red or by Ca2+‐free Ringer's medium. Conclusions: In conclusion, there is functional TRPV4 expression in HCEC‐SV40. TRPV4 expression may provide an osmosensor role to induce RVD behavior during exposure to a hypotonic challenge since this response is mediated through intracellular Ca2+ transients.Supported in part DFG Pl 150/14‐1 and NIH, EY04795. CR: none

Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes

Cell–cell communication in astroglial syncytia is mediated by intracellular Ca 2+ ([Ca 2+] i) responses elicited by extracellular signaling molecules as well as by diverse physical and chemical stimuli. Despite the evidence that astrocytic swelling promotes [Ca 2+] i elevation through Ca 2+ influx, the molecular identity of the channel protein underlying this response is still elusive. Here we report that primary cultured cortical astrocytes express the transient receptor potential vanilloid-related channel 4 (TRPV 4), a Ca 2+-permeable cation channel gated by a variety of stimuli, including cell swelling. Immunoblot and confocal microscopy analyses confirmed the presence of the channel protein and its localization in the plasma membrane. TRPV4 was functional because the selective TRPV4 agonist 4-alpha-phorbol 12,13-didecanoate (4αPDD) activated an outwardly rectifying cation current with biophysical and pharmacological properties that overlapped those of recombinant human TRPV4 expressed in COS cells. Moreover, 4αPDD and hypotonic challenge promoted [Ca 2+] i elevation mediated by influx of extracellular Ca 2+. This effect was abolished by low micromolar concentration of the TRPV4 inhibitor Ruthenium Red. Immunofluorescence and immunogold electron microscopy of rat brain revealed that TRPV4 was enriched in astrocytic processes of the superficial layers of the neocortex and in astrocyte end feet facing pia and blood vessels. Collectively, these data indicate that cultured cortical astroglia express functional TRPV4 channels. They also demonstrate that TRPV4 is particularly abundant in astrocytic membranes at the interface between brain and extracerebral liquid spaces. Consistent with its roles in other tissues, these results support the view that TRPV4 might participate in astroglial osmosensation and thus play a key role in brain volume homeostasis.

Aquaporin-1 Channel Function Is Positively Regulated by Protein Kinase C

Aquaporin-1 (AQP1) channels contribute to osmotically induced water transport in several organs including the kidney and serosal membranes such as the peritoneum and the pleura. In addition, AQP1 channels have been shown to conduct cationic currents upon stimulation by cyclic nucleotides. To date, the short term regulation of AQP1 function by other major intracellular signaling pathways has not been studied. In the present study, we therefore investigated the regulation of AQP1 by protein kinase C. AQP1 wild type channels were expressed in Xenopus oocytes. Water permeability was assessed by hypotonic challenges. Activation of protein kinase C (PKC) by 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced a marked increase of AQP1-dependent water permeability. This regulation was abolished in mutated AQP1 channels lacking both consensus PKC phosphorylation sites Thr(157) and Thr(239) (termed AQP1 DeltaPKC). AQP1 cationic currents measured with double-electrode voltage clamp were markedly increased after pharmacological activation of PKC by either OAG or phorbol 12-myristate 13-acetate. Deletion of either Thr(157) or Thr(239) caused a marked attenuation of PKC-dependent current increases, and deletion of both phosphorylation sites in AQP1 DeltaPKC channels abolished the effect. In vitro phosphorylation studies with synthesized peptides corresponding to amino acids 154-168 and 236-250 revealed that both Thr(157) and Thr(239) are phosphorylated by PKC. Upon stimulation by cyclic nucleotides, AQP1 wild type currents exhibited a strong activation. This regulation was not affected after deletion of PKC phosphorylation sites in AQP1 DeltaPKC channels. In conclusion, this is the first study to show that PKC positively regulates both water permeability and ionic conductance of AQP1 channels. This new pathway of AQP1 regulation is independent of the previously described cyclic nucleotide pathway and may contribute to the PKC stimulation of AQP1-modulated processes such as endothelial permeability, angiogenesis, and urine concentration.

Mechanisms of hypotonic inhibition of the sodium, proton exchanger type 1 (NHE1) in a biliary epithelial cell line (Mz‐Cha‐1)

To elucidate the cellular events that results in inhibition of Na(+), H(+) exchanger type 1 (NHE1) by hypotonicity. Intracellular pH (pH(i)) was measured in biliary epithelial cells, with the pH-sensitive fluorochrome 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) using a spectrophotometer. Regulatory volume decrease (RVD) was analysed from confocal images. Changes in NHE1 membrane content were visualized by confocal laser scanning microscopy after transfection of Mz-Cha-1 cells with a NHE1-cMyc fusion protein. In Mz-Cha-1 cells hypotonicity (-80 mmol L(-1) NaCl) inhibited endogenous Na(+), H(+) exchange. Tyrosine and serine kinase inhibitors were incapable to prevent inhibition. As several signalling pathways influence Na(+), H(+) exchange, we tested the effect of the Ca(++), Calmodulin, protein kinase C or the cAMP, protein kinase A system on inhibition of Na(+), H(+) exchange by hypotonic challenge, but neither system was involved. In contrast, cytoskeleton did influence the effect of hypotonicity. Inhibition of microtubule polymerization by colchicine prevented inhibition of NHE1, and also restored Na(+), H(+) exchange kinetics. Specific inhibition of Src kinases with PP2, attenuated pH(i) recovery rate from 1.93 +/- 0.16 pH units min(-1) (normotonic environment) to 1.02 +/- 0.50 pH units min(-1) (hypotonic environment). Membrane staining of NHE1-cMyc fusion protein was maintained after hypotonic exposure in colchicine pre-treated cells as was RVD. Microfilament inhibition by cytochalasin preserved NHE1 activity. Inhibition of phosphatidylinositol-3'-kinase was unable to restore Na(+), H(+) exchange activity. We conclude that regulation of Na(+), H(+) exchange during RVD is mediated by cytoskeletal elements. This receptor independent pathway is regulated by Src.

Role of Calcium in Volume-Activated Chloride Currents in a Mouse Cholangiocyte Cell Line

Volume-activated Cl(-) channels (VACCs) play vital roles in many cells including cholangiocytes. Previously, we characterized the VACCs in mouse cholangiocytes. Since calcium plays an important role in VACC regulation in many cells, we have studied the effect of calcium modulation on the regulatory volume decrease (RVD) and VACC currents in mouse bile duct cells (MBDCs). Cell volume measurements were assessed by a Coulter counter with cell sizer, and conventional whole-cell patch-clamp techniques were used to study the role of calcium on RVD and VACC currents. Cell volume study indicated that MBDCs exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) but not by removal of extracellular calcium. During hypotonic challenge, MBDCs exhibited an outwardly rectified current, which was significantly inhibited by administration of classical chloride channel inhibitors such as NPPB and tamoxifen. Chelation of the intracellular calcium with BAPTA-AM or removal of extracellular calcium and calcium channel blocker had no significant effect on VACC currents during hypotonic challenge. In addition to VACC, MBDC had a calcium-activated chloride channel, which was inhibited by NPPB. The present study is the first to systemically study the role of calcium on the VACC and RVD in mouse cholangiocytes and demonstrates that a certain level of intracellular calcium is necessary for RVD but the activation of VACC during RVD does not require calcium. These findings suggest that calcium does not have a direct regulatory role on VACC but has a permissive role on RVD in cholangiocytes.

Functional and molecular characterization of multiple K–Cl cotransporter isoforms in corneal epithelial cells

The dependence of regulatory volume decrease (RVD) activity on potassium-chloride cotransporter (KCC) isoform expression was characterized in corneal epithelial cells (CEC). During exposure to a 50% hypotonic challenge, the RVD response was larger in SV40-immortalized human CEC (HCEC) than in SV40-immortalized rabbit CEC (RCEC). A KCC inhibitor—[(dihydroindenyl)oxy] alkanoic acid (DIOA)—blocked RVD more in HCEC than RCEC. Under isotonic conditions, N-ethylmaleimide (NEM) produced KCC activation and transient cell shrinkage. Both of these changes were greater in HCEC than in RCEC. Immunoblot analysis of HCEC, RCEC, primary human CEC (pHCEC), and primary bovine CEC (BCEC) plasma membrane enriched fractions revealed KCC1, KCC3, and KCC4 isoform expression, whereas KCC2 was undetectable. During a hypotonic challenge, KCC1 membrane content increased more rapidly in HCEC than in RCEC. Such a challenge induced a larger increase and more transient p44/42MAPK activation in HCEC than RCEC. On the other hand, HCEC and RCEC p38MAPK phosphorylation reached peak activations at 2.5 and 15 min, respectively. Only in HCEC, pharmacological manipulation of KCC activity modified the hypotonicity-induced activation of p44/42MAPK, whereas p38MAPK phosphorylation was insensitive to such procedures in both cell lines. Larger increases in HCEC KCC1 membrane protein content correlated with their ability to undergo faster and more complete RVD. Furthermore, pharmacological activation of KCC increased p44/42MAPK phosphorylation in HCEC but not in RCEC, presumably a reflection of low KCC1 membrane expression in RCEC. These findings suggest that KCC1 plays a role in (i) maintaining isotonic steady-state cell volume homeostasis, (ii) recovery of isotonic cell volume after a hypotonic challenge through RVD, and (iii) regulating hypotonicity-induced activation of the p44/42MAPK signaling pathway required for cell proliferation.

Changes in Membrane Conductance Play a Pathogenic Role in Osmotic Glial Cell Swelling in Detached Retinas

Detachment of the neural retina from the pigment epithelium may be associated with tissue edema; however, the mechanisms of fluid accumulation are not understood. Because retinal detachment is usually not accompanied by vascular leakage, we investigated whether the osmotic swelling characteristics of retinal glial (Müller) cells are changed after experimental detachment of the porcine retina. Osmotic stress, induced by application of a hypotonic bath solution to retinal slices, caused swelling of Müller cell bodies in 7-day-detached retinas, but no swelling was inducible in slices of control retinas. Müller cell somata in slices of retinal areas that surround local detachment in situ also showed osmotic swelling, albeit at a smaller amplitude. The amplitude of osmotic Müller cell swelling correlated with the decrease in the K+ conductance, suggesting a causal relationship between both gliotic alterations. Further factors implicated in Müller cell swelling were inflammatory mediators and oxidative stress. We propose that a dysregulation of the ion and water transport through Müller cells may impair the fluid absorption from the retinal tissue, resulting in chronic fluid accumulation after detachment. This knowledge may lead to a better understanding of the mechanisms involved in retinal degeneration after detachment.

Enhanced Binding of Sperm With Superior Volume Regulation to Oviductal Epithelium

The plasma membrane is a key organelle with respect to sperm fertilizing ability. A sensitive way of testing plasma membrane functionality is to examine the sperm ability to moderate its swelling in response to hypo-osmotic stress (volume regulatory ability) using an electronic cell counter to assess cell volume changes. In this study of frozen-thawed bull sperm, we examined the relationship among sperm-oviductal epithelium binding capacity, osmotically induced swelling response, volume regulatory ability, and standard spermatologic parameters. Sperm cell volume distributions were measured under iso-osmotic conditions and after hypo-osmotic stress. The relative volume shift was calculated by comparing modal values of the cell volume distributions during transition from iso-osmotic to hypo-osmotic conditions. Significant correlations were found between volumetric parameters and sperm-oviduct binding capacity. Both the relative volume shift and regulative volume decrease correlated positively and significantly with the sperm-oviduct binding capacity. No significant correlations were found between sperm volumetric parameters and any of the standard sperm parameters with the exception of forward motility of Percoll-washed sperm. However, the use of multiple regression models improved the prediction level for binding capacity when motility parameters were combined with membrane integrity and volumetric parameters (R2 = .84). Spermatozoa of bulls with high nonreturn rates responded to hypotonicity as "perfect osmometers." Subfertile bulls had lower binding indices and deficiencies in volume recovery after hypotonic challenge, indicating that intact volume regulatory ability is a necessary prerequisite for binding to oviductal epithelium and is related to fertility. Volumetric parameters therefore could be used as tools in semen evaluation programs.

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.

Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin ( approximately 7 microm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of approximately 250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached approximately 1 microm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.

Hypotonic stress enhances slope conductivity of ATP-sensitive K + channels activated pharmacologically

The aim of this paper was to find out the effects of hypotonic stress on the slope conductivity of ATP-sensitive K + channels. Using patch clamp technique, rilmakalim and pinacidil as the activators and glibenclamide as an inhibitor of mentioned channels, we have demonstrated that short hypotonic challenge doubled slope conductivity of exploring channels by augmentation of their open probability.

Prestin is expressed on the whole outer hair cell basolateral surface

Prestin has been identified as a motor protein responsible for outer hair cell (OHC) electromotility. Previous experiments revealed that OHC electromotility and its associated nonlinear capacitance resided in the OHC lateral wall and was not detected at the apical cuticular plate and basal region. In this experiment, the distribution of prestin in adult mouse, rat, and guinea pig OHCs was re-examined by use of immunofluorescent staining and confocal microscopy. We found that prestin labeling was located at the whole OHC basolateral wall, including the basal plasma membrane. However, staining at the basal membrane was weak. As compared with the intensity at the lateral wall, the intensities of prestin labeling at the membrane at the nuclear level and basal pole were 80.5% and 61.1%, respectively. Prestin labeling was not found at the cuticular plate and stereocilia. The prestin labeling was also absent in the cytoplasm and nuclei. The OHC lateral wall above the nuclear level is composed of the plasma membrane, cortical lattice, and subsurface cisternae. By co-staining with di-8-ANEPPS, prestin labeling was found at the outer layer of the OHC lateral wall, which was further evidenced by use of a hypotonic challenge to separate the plasma membrane from the underlying subsurface cisternae. The data revealed that prestin is expressed at the whole OHC basolateral membrane. Prestin in the basal plasma membrane may provide a reservoir on the OHC surface for prestin-recycling and may also facilitate performing its hypothesized transporter function.

Swelling‐activated chloride channels in aqueous humour formation: on the one side and the other

Aqueous humour is secreted by the ciliary epithelium comprising pigmented and non-pigmented cell layers facing the stroma and aqueous humour respectively. Net chloride secretion likely limits the rate of aqueous humour formation and proceeds in three steps: stromal chloride entry into pigmented cells, diffusion through gap junctions and final non-pigmented cell secretion. Swelling-activated chloride channels function on both epithelial surfaces. At the stromal surface, swelling- and cyclic adenosine monophosphate-activated maxi-chloride channels can recycle chloride, reducing net chloride secretion. At the aqueous-humour surface, swelling- and A3 adenosine receptor-activated chloride channels subserve chloride release into the aqueous humour. The similar macroscopic properties of the two non-pigmented cell chloride currents suggest that both flow through a common conduit. In addition, measurements of intraocular pressure (IOP) in living wild-type and mutant mice have confirmed that A3 adenosine receptor-activated agonists and antagonists increase and lower IOP respectively. Isolated ciliary epithelial cells are commonly perfused with hypotonic solution to probe and characterize chloride channels, but the physiological role of swelling-activated channels has been unclear without knowing their epithelial distribution. Recently, hypotonic challenge has been found to stimulate the chloride-sensitive short-circuit current across the intact bovine ciliary epithelium, suggesting that the net effect of the swelling-activated chloride currents is oriented to enhance aqueous humour formation. Taken together, the results suggest that swelling-activated chloride channels are predominantly oriented to enhance aqueous humour secretion, and these chloride channels at the aqueous surface may be identical with adenosine receptor-activated chloride channels which likely modulate aqueous inflow and IOP in the living mouse.