Osmotic Shrinkage Research Articles

Cl- channels in apoptosis.

A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K+, Cl-, and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl- channels LRRC8, TMEM16/anoctamin, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also determines sensitivity towards cytostatic drugs such as cisplatin. Recent data point to a molecular and functional relationship of LRRC8A and anoctamins (ANOs). ANO6, 9, and 10 (TMEM16F, J, and K) augment apoptotic Cl- currents and AVD, but it remains unclear whether these anoctamins operate as Cl- channels or as regulators of other apoptotic Cl- channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated by cell swelling, it is possible that CFTR serves RVD/AVD through accumulation of ROS and activation of independent membrane channels such as ANO6. Thus activation of ANO6 will support cell shrinkage and induce additional apoptotic events, such as membrane phospholipid scrambling.

VRACs and other ion channels and transporters in the regulation of cell volume and beyond.

Cells need to regulate their volume to counteract osmotic swelling or shrinkage, as well as during cell division, growth, migration and cell death. Mammalian cells adjust their volume by transporting potassium, sodium, chloride and small organic osmolytes using plasma membrane channels and transporters. This generates osmotic gradients, which drive water in and out of cells. Key players in this process are volume-regulated anion channels (VRACs), the composition of which has recently been identified and shown to encompass LRRC8 heteromers. VRACs also transport metabolites and drugs and function in extracellular signal transduction, apoptosis and anticancer drug resistance.

Influence of internal water phase gelation on the shear- and osmotic sensitivity of W/O/W-type double emulsions

It has already been hypothesized and in some cases proven that gelation of the internal water droplets of W/O/W-type double emulsions might allow the application of a higher shear in the second emulsification step while maintaining a high internal water content. In this contribution, thermal denaturation of a concentrated whey protein isolate solution, which is used as the internal water phase, was investigated. Low-resolution NMR T2-relaxometry measurements (using a CPMG-sequence) clearly showed that this thermal treatment effectively gelled the internal water phase. Using this approach, double emulsion droplets with a small average diameter (D43 of about 5 μm) and narrow particle size distribution were produced while a high internal water content was maintained. Moreover, our results indicated that gelation could not prevent osmotic shrinking of the double emulsion droplets upon dilution in hypertonic solution. However, gelation of the internal water phase clearly reduced the degree of osmotic swelling of the double emulsion droplets upon hypotonic dilution. However, this effect is probably due to droplet-globule coalescence occurring at lower internal water volume increases in case the internal water phase is gelled.

Some Cell Behavior is Encoded in Proteome Physics

More than half the biomass of a bacterial cell is protein. So, some cell behaviors may arise from protein physics -- folding, aggregation, oxidation, and diffusional transport, for example. We develop simple physical models for how protein physics affects cell growth. Cells are sensitive to temperature. We believe this is because cell's live near protein denaturation temperatures. Cells grow slowly in high salts. We believe this is because osmotic shrinkage of the cell crowds proteins, slowing their diffusional transport. A cell's proteome oxidizes with age. We believe this results in the electrostatic unfolding of high-charge proteins. Reasoning with simple models also gives insights into a cell's energy balance, replication speed, and others.

Size Determination of a Liposomal Drug by Small-Angle X-ray Scattering Using Continuous Contrast Variation.

The continuously growing complexity of nanodrugs urges for complementary characterization techniques which can elude the current limitations. In this paper, the applicability of continuous contrast variation in small-angle X-ray scattering (SAXS) for the accurate size determination of a complex nanocarrier is demonstrated on the example of PEGylated liposomal doxorubicin (Caelyx). The mean size and average electron density of Caelyx was determined by SAXS using a gradient of aqueous iodixanol (Optiprep), an iso-osmolar suspending medium. The study is focused on the isoscattering point position and the analysis of the Guinier region of the scattering curves recorded at different solvent densities. An average diameter of (69 ± 5) nm and electron density of (346.2 ± 1.2) nm(-3) were determined for the liposomal formulation of doxorubicin. The response of the liposomal nanocarrier to increasing solvent osmolality and the structure of the liposome-encapsulated doxorubicin after the osmotic shrinkage of the liposome are evaluated with sucrose contrast variation in SAXS and wide-angle X-ray scattering (WAXS). In the case of using sucrose as contrast agent, a clear osmolality threshold at 670 mOsm kg(-1) was observed, above which the liposomal drug carriers start to shrink, though preserving the intraliposomal doxorubicin structure. The average size obtained by this technique is smaller than the value measured by dynamic light scattering (DLS), though this difference is expected due to the hydrodynamic size of the PEG moieties attached to the liposomal surface, which are not probed with solvent contrast variation in SAXS. The advantages and drawbacks of the proposed technique are discussed in comparison to DLS, the most frequently used sizing method in nanomedicine.

Driving the Early Auditory Network the Old-Fashioned Way

Spontaneous neuronal activity during the development of the auditory sensory system is important in establishing mature connectivity. Wang et al. show that glia-like cells drive spontaneous spiking in neighboring cochlear inner hair cells via a process that involves osmotic cell shrinkage and the secretion of potassium ions.

Spontaneous Activity of Cochlear Hair Cells Triggered by Fluid Secretion Mechanism in Adjacent Support Cells

Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl(-) efflux and osmotic cell shrinkage by opening TMEM16A Ca(2+)-activated Cl(-) channels. Release of Cl(-) from ISCs also forces K(+) efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea and prevents ATP-dependent shrinkage of supporting cells. These results indicate that supporting cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells.

57. A transmembrane mega highway for cryoprotective agent delivery via self-assembling organic nanopores with tunable function

Cryoprotective agents (CPAs) are critical additives that improve the post-thaw viability of cryopreserved biological systems by preventing ice crystal nucleation and growth. Membrane permeable CPAs also prevent osmotic shrinkage of the cells and reduce the volume of available water by penetrating and equilibrating across the cell membrane. All known CPAs exhibit various levels of cytotoxicity at their effective concentration which may be decreased by reducing the CPA loading temperature and exposure time. However, most CPAs become effectively impermeable at sub-zero temperatures. This is largely associated with the dysfunction of transmembrane protein channels at low temperature that serve as mass transport gates. An unbalanced gradient of CPA and salt across cell membrane leads to detrimental cell shrinkage as water efflux out of the cell. This could be detrimental to the fate of large, complex solid tissues/organs by damaging cell–cell and cell–matrix junctions. To facilitate the intracellular delivery and transmembrane equilibration of the CPA at sub-zero temperatures, we propose size and function tunable organic nanopores as a “mega highway” to deliver CPAs across cell membrane which allows a significant decrease in both the CPA exposure time and temperature during freezing. This method may be particularly effective when CPA loading in tissues using the “liquidus tracking” or step-wise methods where increasingly concentrated solutions of CPA are loaded in the tissue/organ at progressively decreasing temperatures. Recent work in Gong’s group has demonstrated the reliability of constructing well-defined nanotubular assemblies via the enforced stacking of shape-persistent macrocycles based on the interplay of multiple hydrogen-bonding, dipole-dipole, and aromatic π - π stacking interactions. The resultant nanotubes have modifiable surfaces and non-collapsible inner pores of a uniform diameter defined by the constituent macrocycles. The robust self-assembling nanopores have been found to act as transmembrane channels which can mediate highly selective transmembrane ion transport with high stability, unprecedented for synthetic ion channels, as well as exhibiting highly efficient transmembrane water permeability, which is comparable to aquaporin. Aquaporins have been found to efficiently assist transport of water and glycerol during freezing to prevent cell rupture under osmotic pressure. In this proposal, selected organic nanopores are delivered into a target system at physiological temperature (37 °C), followed by CPA loading at hypothermic temperature (e.g. 1 °C). A high influx rate of CPAs through shape-persistent nanopores can be maintained during cooling as a function of the concentration gradient across cell membrane, thereby reducing the required time to reach vitrification concentrations. Upon rewarming, these organic nanopores will facilitate rapid removal of CPAs to reduce exposure time and the consequent toxic side effects. The proposed incorporation of synthetic nanopores can significantly reduce toxicity and cell injury due to osmotic shrinkage caused by CPAs and salt during both the cooling and rewarming processes via (1) reducing CPA exposure time and (2) enabling rapid CPA loading and unloading at lower temperatures. Moreover, versatile functional organic nanotubes of diverse sizes and properties by modifying inner macrocyclic cavities allow selective CPA transport by while preventing ion exchange. Incorporation of functional supramolecular assemblies to enhance membrane permeability of CPAs could lead to a revolutionary solution to cryopreserve complex biological systems.

Osmotic shrinkage elicits FAK- and Src phosphorylation and Src-dependent NKCC1 activation in NIH3T3 cells.

The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10-30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2-10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 μM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by ∼50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by ∼15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkage-activation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process.

Osmotic shrinkage of sterically stabilized liposomes as revealed by time-resolved small-angle X-ray scattering

Time-resolved synchrotron small-angle X-ray scattering (SAXS) was used to study the structural changes during the osmotic shrinkage of a pharmacologically relevant liposomal drug delivery system. Sterically stabilized liposomes (SSLs) with a diameter of 100 nm and composed of hydrogenated soy phosphocholine, cholesterol and distearoyl-phosphoethanolamine-PEG 2000 prepared in a salt-free buffer were mixed with a buffered 0.3 MNaCl solution using a stopped flow apparatus. The changes in the liposome size and the bilayer structure were followed by using SAXS with a time resolution of 20 ms. A linear decrease in liposome size is observed during the first ∼4 s of the osmotic shrinkage, which reveals a water permeability value of 0.215 (15) µm s−1. The change in the size of the liposomes upon the osmotic shrinkage is also confirmed by dynamic light scattering. After this initial step, broad correlation peaks appear on the SAXS curves in theqrange of the bilayer form factor, which indicates the formation of bi- or oligolamellar structures. Freeze-fracture combined with transmission electron microscopy revealed that lens-shaped liposomes are formed during the shrinkage, which account for the appearance of the quasi-Bragg peaks superimposed on the bilayer form factor. On the basis of these observations, it is proposed that the osmotic shrinkage of SSLs is a two-step process: in the initial step, the liposome shrinks in size, while the area/lipid adapts to the decreased surface area, which is then followed by the deformation of the spherical liposomes into lens-shaped vesicles.

The Putative Role of the Non-Gastric H+/K+-ATPase ATP12A (ATP1AL1) as Anti-Apoptotic Ion Transporter: Effect of the H+/K+ ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

Background/Aims: The ATP12A gene codes for a non-gastric H⁺/K⁺ ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H⁺/K⁺ ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM) human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H⁺/K⁺ ATPase inhibitor SCH28080 in apoptosis. Methods: Real-time reverse-transcription PCR (qRT-PCR) was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pH_i) after acute intracellular acid load (NH₄Cl prepulsing). Mean cell volumes (MCV) and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI) were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle), to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining) and differentiation (CD86 expression). Results: We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM) induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H⁺/K⁺ ATPase inhibitor SCH28080 (100 µM) diminishes K⁺-dependent pH_i recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently decrease it over the course of the next 48 h. This effect can be observed in the overall- and non-apoptotic fraction of both untreated and 1 mM butyrate-treated HL-60 cells, but not in 1 mM butyrate-stimulated phosphatidylserine-positive cells. These cells do not shrink from 24 h to 72 h and have finally a higher MCV than untreated cells unless they are exposed to SCH28080. 10 mM butyrate induces apoptosis within 24 h. Conclusion: In summary we show that in HL-60 cells ATP12A is a functionally active H⁺/K⁺ ATPase that may counteract events during early apoptosis like intracellular acidosis, loss of intracellular K⁺ ions and apoptotic volume decrease. Its expression and/or susceptibility to the H⁺/K⁺ ATPase inhibitor SCH28080 becomes most evident in cells exposing phosphatidylserine on the outer leaflet of the cell membrane and therefore during early apoptosis.

Downregulation of KCNQ4 by Janus Kinase 2

Janus kinase-2 (JAK2) participates in the signaling of several hormones, growth factors and cytokines. Further stimulators of JAK2 include osmotic cell shrinkage, and the kinase activates the cell volume regulatory Na(+)/H(+) exchanger. The kinase may thus participate in cell volume regulation. Cell shrinkage is known to inhibit K(+) channels. Volume-regulatory K(+) channels include the voltage-gated K(+) channel KCNQ4. The present study explored the effect of JAK2 on KCNQ4 channel activity. KCNQ4 was expressed in Xenopus oocytes with or without wild-type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2; and cell membrane conductance was determined by dual-electrode voltage clamp. Expression of KCNQ4 was followed by the appearance of voltage-gated K(+) conductance. Coexpression of JAK2 or of (V617F)JAK2, but not of (K882E)JAK2, resulted in a significant decrease in conductance. Treatment of KCNQ4 and JAK2 coexpressing oocytes with the JAK2 inhibitor AG490 (40 μM) was followed by an increase in conductance. Treatment of KCNQ4 expressing oocytes with brefeldin A (5 μM) was followed by a decrease in conductance, which was similar in oocytes expressing KCNQ4 together with JAK2 as in oocytes expressing KCNQ4 alone. Thus, JAK2 apparently does not accelerate channel protein retrieval from the cell membrane. In conclusion, JAK2 downregulates KCNQ4 activity and thus counteracts K(+) exit, an effect which may contribute to cell volume regulation.

Impacts of the Osmolality and the Lumenal Ionic Strength on Osmosensory Transporter ProP in Proteoliposomes

H(+) symporter ProP serves as a paradigm for the study of osmosensing. ProP attains the same activity at the same osmolality when the medium outside cells or proteoliposomes is supplemented with diverse, membrane-impermeant solutes. The osmosensory mechanism of ProP has been probed by varying the solvent within membrane vesicles and proteoliposomes. ProP activation was not ion specific, did not require K(+), and could be elicited by large, uncharged solutes polyethylene glycols (PEGS). We hypothesized that ProP is an ionic strength sensor and lumenal macromolecules activate ProP by altering ion activities. The attainable range of lumenal ionic strength was expanded by lowering the phosphate concentration within proteoliposomes. ProP activity at high osmolality, but not the osmolality, yielding half-maximal activity (Π(1/2)/RT), decreased with the lumenal phosphate concentration. This was attributed to acidification of the proteoliposome lumen due to H(+)-proline symport. The ionic strength yielding half-maximal ProP activity was more anion-dependent than Π(1/2)/RT for proteoliposomes loaded with citrate, sulfate, phosphate, chloride, or iodide. The anion effects followed the Hofmeister series. Lumenal bovine serum albumin (BSA) lowered the lumenal ionic strength at which ProP became active. Osmolality measurements documented the non-idealities of solutions including potassium phosphate and other solutes. The impacts of PEGS and BSA on ion activities did not account for their impacts on ProP activity. The effects of the tested solutes on ProP appear to be non-coulombic in nature. They may arise from effects of preferential interactions and macromolecular crowding on the membrane or on ProP.

Highlights from the Literature

Highlights from the Literature

The Apoptotic Volume Decrease Is an Upstream Event of MAP Kinase Activation during Staurosporine-Induced Apoptosis in HeLa Cells

Persistent cell shrinkage, called apoptotic volume decrease (AVD), is a pivotal event of apoptosis. Activation of the volume-sensitive outwardly rectifying Cl− channel (VSOR) is involved in the AVD induction. On the other hand, activation of the MAP kinase (MAPK) cascade is also known to play a critical role in apoptosis. In the present study, we investigated the relationship between the AVD induction and the stress-responsive MAPK cascade activation during the apoptosis process induced by staurosporine (STS) in HeLa cells. STS was found to induce AVD within 2–5 min and phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK after over 20–30 min. VSOR blockers suppressed not only STS-induced AVD but also phosphorylation of JNK and p38 as well as activation of caspase-3/7. Moreover, a p38 inhibitor, SB203580, and a JNK inhibitor, SP600125, failed to affect STS-induced AVD, whereas these compounds reduced STS-induced activation of caspase-3/7. Also, treatment with ASK1-specific siRNA suppressed STS-induced caspase-3/7 activation without affecting the AVD induction. Furthermore, sustained osmotic cell shrinkage per se was found to trigger phosphorylation of JNK and p38, caspase activation, and cell death. Thus, it is suggested that activation of p38 and JNK is a downstream event of AVD for the STS-induced apoptosis of HeLa cells.

Video analysis of osmotic cell response during cryopreservation

Cellular response during the freeze–thaw process strongly affects the cryopreservation outcome including cell morphology and cell viability. Cryomicroscopy was used to individually analyze the osmotic response of human pulmonary microvascular endothelial cells (HPMECs) during slow cooling (1°C/min) to −60°C and fast rewarming to 4°C (100°C/min). The ice nucleation temperature was controlled (Tn=−8°C). Different concentrations of different cryoprotectant agents, dimethyl sulfoxide, ethylene glycol, proline, ectoin, and trehalose resulted in various cell volume changes. The described methods for image processing and computer vision allows for a fully automatic and individual analysis of the osmotically driven cell response under a temporal resolution of 2frames per second. As a result, we show that in the presence of dimethyl sulfoxide or ethylene glycol cells shrink during cooling to a high degree, especially at intermediate molar concentrations in the range between 0 and 2M, while during rewarming cells swell to isotonic volumes gradually. Comparative cell vitality tests, membrane integrity, and viability tests after 24h recultivation, under these conditions show a high cell survival. In the absence of cryoprotective agents or with proline, ectoin or trehalose, osmotic shrinkage did not meet our expectations: a freeze-induced swelling was detected during cooling and an extreme swelling was observed after rewarming, which was accompanied by lower comparative cell viability. A linear correlation between the cellular membrane integrity after cryopreservation and the maximal relative cell volume was derived (R2=96). The results clearly show that it is crucial to analyze cells within a sample individually due to their individual different osmotic response.

Dual Response of Human Leukemia U937 Cells to Hypertonic Shrinkage: Initial Regulatory Volume Increase (RVI) and Delayed Apoptotic Volume Decrease (AVD)

Background/Aims: Osmotic cell shrinkage is a powerful trigger of suicidal cell death or apoptosis, which is paralleled and enforced by apoptotic volume decrease (AVD). Cells counteract cell shrinkage by volume regulatory increase (RVI). The present study explored the response of human U937 cells to hypertonic solution thus elucidating the relationship between RVI and AVD. Methods: Cell water, concentration of monovalent ions and the appearance of apoptotic markers were followed for 0.5-4 h after the cells were transferred to a hypertonic medium. Intracellular water, K+, Na+, and Cl– content, ouabain-sensitive and -resistant Rb+ influxes were determined by measurement of the cell buoyant density in Percoll density gradient, flame emission analysis and 36Cl– assay, respectively. Fluorescent microscopy of live cells stained by acridine orange and ethidium bromide was used to verify apoptosis. Results: After 2-4 h incubation in hypertonic media the cell population was split into light (L) and heavy (H) fractions. According to microscopy and analysis of monovalent ions the majority of cells in the L population were healthy, while the H fractions were enriched with apoptotic cells. The density of L cells was decreasing with time, while the density of H cells was increasing, thus reflecting the opposite effects of RVI and AVD. At the same time, some of the cells were shifting from L to H fractions, indicating that apoptosis was gradually extending to cells that were previously displaying normal RVI. Conclusion: The findings suggest that apoptosis can develop in cells capable of RVI.

Contribution of the Epithelial Sodium Channel to Chondrocyte Regulatory Volume Increase

Chondrocytes are the cells of articular cartilage, responsible for the production and maintenance of the extracellular milieu. They exist in a constantly changing osmotic environment and to survive such osmotic changes they must be able to quickly and effectively regulate their volume. Cells use regulatory volume increase (RVI) to oppose osmotic shrinkage. It has previously been shown that the epithelial sodium channel (ENaC) is important to this process in rat hepatocytes1. Here we investigate the possible contribution of ENaC to RVI mechanisms in canine chondrocytes.Chondrocytes were isolated from cartilage according to standard methods2. Amiloride sensitive single-channel activity reversed at a membrane potential of −1±5mV (n = 5), mean conductance was 9±0.4pS (n = 5) and kinetics were slow. The calculated ENa under these conditions was −6mV, which coupled to the very small conductance would be consistent with this channel being an ENaC. Channel open probability in control conditions was 0.3±0.06 and decreased by 97±2% after application of the ENaC inhibitor, amiloride (10μM; n = 3).Upon exposure to hypertonic solution, cell volume decreased significantly by 35±3% (n = 5; p<0.001). Within 20 minutes of reaching their smallest size, cells under control conditions had returned to 92±4% of their original volume, not significantly different to starting volume (p=0.07). When 100nM benzamil, a specific ENaC inhibitor, was added to the hypertonic solution, cells shrank by 41±3% (n = 5) and were unable to return to their original volume.These data suggest that ENaC contributes to RVI in canine chondrocytes.1. F. Wehner, C. Bohmer, H. Heinzinger et al., Cellular Physiology and Biochemistry 10 (5-6), 335 (2000).2. R. Lewis, K. Asplin, G. Bruce et al., J Cell Physiol 226 (8) (2011).

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

The Na+/H + Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na+/H+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na+/H+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na+ transport capacity without affecting transport affinity (Km = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.

Hyperosmotic stress strongly potentiates serum response factor (SRF)‐dependent transcriptional activity in ehrlich lettré ascites cells through a mechanism involving p38 mitogen‐activated protein kinase

Long-term osmotic stress results in altered gene transcription, however, with the exception of the TonE/TonEBP system, the underlying mechanisms are poorly understood. We previously showed that upon osmotic shrinkage of Ehrlich Lettré Ascites (ELA) fibroblasts, the MEK1-ERK1/2 pathway is transiently inhibited while p38 MAPK is activated, in turn impacting on cell survival (Pedersen et al., 2007, Cell Physiol Biochem 20: 735-750). Here, we show that downstream of these kinases, two transcription factors with major roles in control of cell proliferation and death, serum response factor (SRF) and cAMP response element-binding protein (CREB) are differentially regulated in ELA cells. SRF Ser(103) phosphorylation and SRF-dependent transcriptional activity were strongly augmented 5-30 min and 24 h, respectively, after hyperosmotic stress (50% increase in extracellular ionic strength), in a p38 MAPK-dependent manner. In contrast, CREB Ser(133) was transiently dephosphorylated upon osmotic shrinkage. The ERK1/2 effector ribosomal S kinase (RSK) and the ERK1/2- and p38 MAPK effector mitogen- stress-activated protein kinase 1 (MSK1) both phosphorylate CREB at Ser(133) . RSK and MSK1 were dephosphorylated within 5 min of shrinkage. MSK1 phosphorylation recovered within 30 min in a p38-MAPK-dependent manner. CREB was transiently dephosphorylated after shrinkage in a manner exacerbated by p38 MAPK inhibition or MSK1 knockdown, but unaffected by inhibition of RSK. In conclusion, in ELA cells, hyperosmotic stress activates SRF in a p38 MAPK-dependent manner and transiently inactivates CREB, likely due to MSK1 inactivation. We suggest that these events contribute to shrinkage-induced changes in gene transcription and death/survival balance.