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Underlying Event measurements in pp collisions at and 7 TeV with the ALICE experiment at the LHC
Volume ragulatory pathways of anorganic and organic osmolytes in mammalian cells
Die Aufrechterhaltung des Zellvolumens unter variablen osmotischen Bedingungen stellt fĂŒr nahezu alle tierischen Zellen eine essenzielle Aufgabe dar. Um regulatorische Volumenanpassungen vorzunehmen besitzen sie daher effektive Mechanismen, mit deren Hilfe der zellulĂ€re Gehalt an organischen und anorganischen Osmolyten erhöht (= regulatorische Volumenzunahme; RVI) oder gesenkt (= regulatorische Volumenabnahme; RVD) werden kann. Trotz langjĂ€hriger Forschung auf diesem Gebiet konnten die hieran beteiligten Transportwege fĂŒr Osmolyte bisher nur unvollstĂ€ndig aufgeklĂ€rt werden.
Insbesondere bei T-Lymphozyten sind wichtige Zellfunktionen wie die Proliferation, Migration und die T-Zell-Aktivierung eng mit volumenregulatorischen Mechanismen verbunden. Bei all diesen Prozessen sind u. a. unterschiedliche KaliumkanĂ€le beteiligt, die insbesondere fĂŒr die pharmakologische Manipulation von Immunsystemprozessen von wissenschaftlichem Interesse sind. Bisherige Modelle der hypotonen Volumenregulation von T-Lymphozyten berĂŒcksichtigen lediglich den spannungsabhĂ€ngigen KV1.3 sowie den Ca2+-aktivierten IKCa1-Kanal, die zur Klasse der 6TM/P-K+-KanĂ€le gehören.
Im ersten Teil der vorliegenden Arbeit wurde eine potentielle Rolle von kĂŒrzlich entdeckten Zwei-Poren DomĂ€nen KaliumkanĂ€len (K2P) am RVD von murinen und humanen primĂ€ren CD4+-T-Lymphozyten untersucht. In einem kombinierten genetischen und pharmakologischen Ansatz mittels knockout-Tiermodellen und dem Einsatz kanalspezifischer Inhibitoren konnte mithilfe zellvolumetrischer Analysen gezeigt werden, dass die K2P-Vertreter TASK1, TASK2, TASK3 und TRESK maĂgeblich am schwellungsaktivierten Efflux von K+ beteiligt sind. Beurteilt an den Ergebnissen dieser Untersuchung sind der spannungsabhĂ€ngige TASK2- und der Ca2+-aktivierte TRESK-Kanal fĂŒr die hypotone Volumenregulation in T-Zellen deutlich bedeutender als TASK1 und TASK3. Der Beitrag der KanĂ€le TASK2 und TRESK am RVD-Prozess war ĂŒber dies vergleichbar mit dessen des bisher bekannten KV1.3-Kanals. In dieser Arbeit wurde damit erstmals eine Beteiligung der K2P-KanĂ€le am RVD muriner und humaner CD4+-Lymphozyten identifiziert. Aufgrund der engen Verbindung zwischen T-Zell-Funktion und der Volumenregulation können Zwei-Poren DomĂ€nen K+-KanĂ€le damit in den engeren Kreis potentieller immunmodulierende Angriffspunkte aufgefasst werden.
Im zweiten und umfangreicheren Teil dieser Arbeit wurden darĂŒber hinaus die schwellungsaktivierten Transportwege fĂŒr organische Osmolyte (small organic osmolytes; SOOs) untersucht. SOOs stellen chemisch inerte Verbindungen dar, zu denen vor allem Polyole (Sorbitol, myo-Inositol), Methylamine (Betain, α-Glycerophosphocholin) sowie AminosĂ€uren (α- bzw. ÎČ-Alanin und Prolin) und deren Derivate (Taurin) zĂ€hlen. Da SOOs weder die zellulĂ€re Struktur noch die Funktion von MakromolekĂŒlen beeintrĂ€chtigen, sind sie wichtige Instrumente der Volumenregulation, die sich in hohen Konzentrationen im Zytosol nahezu aller Zellen wiederfinden. Werden tierische Zellen mit hypotonen Bedingungen konfrontiert, dann ist bei nahezu allen Zellen die Freisetzung organischer Osmolyte zu beobachten, wodurch die zellulĂ€re OsmolaritĂ€t unabhĂ€ngig von Elektrolyten angepasst werden kann. Trotz der wichtigen Funktion der SOOs in der Osmoregulation tierischer Zellen konnte die molekulare IdentitĂ€t beteiligter Effluxwege (KanĂ€le bzw. Transporter) bisher nicht aufgeklĂ€rt werden.
Ungeachtet der molekularen IdentitĂ€t der SOO-Effluxwege war es aus zahlreichen biotechnologischen Anwendungen zu Beginn dieser Arbeit bekannt, dass die schwellungsaktivierten Transportwege fĂŒr organische Osmolyte eine gröĂenselektive PermeabilitĂ€t fĂŒr eine Reihe monomerer Zucker und verwandter Verbindungen aufweisen. Um diese GröĂenselektivitĂ€t nĂ€her zu charakterisieren, wurde im ersten Schritt die schwellungsaktivierte MembranpermeabilitĂ€t fĂŒr eine Reihe strukturell homogener Polyethylenglykole unterschiedlicher PolymerlĂ€nge (PEG200â1500; hydrodynamische Radien zwischen ~0,5-1,5 nm) unter iso- und hypotonen Bedingungen in Jurkat-Lymphozyten untersucht. Unter milden hypotonen Bedingungen (200 mOsm) war die Plasmamembran der untersuchten Lymphozyten fĂŒr PEG300-1500 undurchlĂ€ssig, was aus der FĂ€higkeit der Zellen zur hypotonen Volumenregulation geschlossen werden konnte. DarĂŒber hinaus wurde RVD in stark hypotonen Lösungen (100 mOsm) mit PEG600-1500 beobachtet, wĂ€hrend PEG300-400 unter vergleichbaren osmotischen Bedingungen die Volumenregulation der Zellen inhibierten. Dieses Ergebnis deutet darauf hin, dass starkes hypotones Zellschwellen der Lymphozyten zur Permeabilisierung der Plasmamembran fĂŒr PEG300-400, nicht jedoch fĂŒr PEG600-1500, fĂŒhrt. Anhand der hydrodynamischen Radien Rh der verwendeten PEGs konnte ein cutoff-Radius von ~0,74 nm fĂŒr schwellungsaktivierte Transportwege organischer Osmolyte bestimmt werden. Da diese schwellungsaktivierten Transportwege vielfĂ€ltig fĂŒr Zellbeladungstechniken verwendet werden, könnte dieses Ergebnis fĂŒr zahlreiche biotechnologische und biomedizinische Anwendungen von Interesse sein.
Im zweiten Schritt wurde der Versuch unternommen, potentielle Transportwege fĂŒr organische Osmolyte im RVD-Prozess molekular zu identifizieren. Da es grundlegend ungeklĂ€rt war, wie viele unterschiedliche Transporter bzw. KanĂ€le am Efflux der zahlreichen organischen Osmolyte beteiligt sind, erfolgte zunĂ€chst die vergleichende Analyse des schwellungsaktivierten Membrantransports strukturell verschiedener SOOs einschlieĂlich der AminosulfonsĂ€ure Taurin und des Polyols myo-Inositol. Hierbei wurde erstmals gezeigt, dass die schwellungsaktivierten Transportwege fĂŒr Taurin und myo-Inositol deutlich unterschiedliche AktivitĂ€tsprofile aufweisen. WĂ€hrend der Taurintransport bereits unter milden hypotonen Bedingungen, d.h. nach einer geringen Absenkung der OsmolalitĂ€t von 300 auf ~230 mOsm, aktiviert wurde, erfolgte die Aktivierung der MembranpermeabilitĂ€t fĂŒr myo-Inositol bei einer viel niedrigeren OsmolalitĂ€t von ~150 mOsm. DarĂŒber hinaus wiesen die beiden Transportwege unter vergleichbarem hypotonen Stress von 100 mOsm deutlich unterschiedliche AktivitĂ€tsdauern auf (Transport von Taurin ~95 min und myo-Inositol ~40 min). Somit deuteten diese Ergebnisse erstmals auf substrat-spezifische Transportwege fĂŒr SOOs hin, die voneinander stark abweichende osmotische Aktivierungsprofile besitzen.
Als aussichtsreiche Kandidaten fĂŒr diese Transportwege wurden zwei Mitglieder der Gruppe der Solute Carrier (SLC) untersucht, die klare Ăbereinstimmungen mit den gesuchten Transportern fĂŒr SOOs aufweisen. Daher wurde im Weiteren eine RVD-Beteiligung dieser Transportergruppe mit einer Kombination aus molekularbiologischer und konventioneller bzw. hochaufgelöster mikroskopischen Techniken ĂŒberprĂŒft. Die semiqantitativen RT-PCR-Ergebnisse dieser Arbeit zeigen dabei, dass die Gentranskription der potentiellen SOO-Transporter SLC5A3 und SLC6A6 in den untersuchten Zelllinien Jurkat, HEK wie auch HepG2-Zellen durch hypotone Bedingungen deutlich verstĂ€rkt wird. Hierbei nimmt der zellulĂ€re mRNA-Gehalt der Gene SLC5A3 zwischen 20-60% und SLC6A6 um 30-100% innerhalb von 10-20 min zu, was auf eine potentielle RVD-Beteiligung von SLC-Transportern hindeutet. Ausgehend von diesem Ergebnis wurde daraufhin die zellulĂ€re Lokalisation des SLC5A3-Transporters unter isotonen und hypotonen Bedingungen mikroskopisch untersucht. Wie anhand der konfokalen lasermikroskopischen Untersuchung zu erkennen ist, findet unter hypotoner Stimulation eine zellulĂ€re Umverteilung des mit EGFP fluoreszenzmarkierten Proteins SLC5A3 statt. Innerhalb von 10 min wird der Transporter dabei von intrazellulĂ€ren Regionen in Richtung Plasmamembran verlagert. DarĂŒber hinaus konnte mit Hilfe der hochauflösenden Mikroskopie-Technik dSTORM gezeigt werden, dass der Transporter SLC5A3 unter hypotoner Stimulation verstĂ€rkt mit der Plasmamembran assoziiert vorliegt. Diese verstĂ€rkte Membranassoziation des SLC5A3-Proteins deutet damit auf einen schwellungsinduzierten exozytotischen Einbau des Transporters hin.
Die Ergebnisse dieser Arbeit zeigen damit erstmals, dass SLC-Transporter wie SLC5A3, SLC6A6 und vermutlich andere Vertreter der SLC-Superfamilie potentiell am Mechanismus der hypotonen Volumenregulation beteiligt sind. Da SLC-Transporter als wichtige Transportsysteme fĂŒr Therapeutika angesehen werden und die Mechanismen der Volumenregulation bereits in zahlreichen biotechnologischen Anwendungen implementiert sind, könnte der hier aufgedeckte Zusammenhang einen Erkenntnisgewinn fĂŒr zahlreiche biomedizinische Forschungsgebiete darstellen.Cell volume homeostasis is critically important for the functional and structural integrity of mammalian cells. To counteract osmotically induced volume perturbations, cells possess efficient mechanisms that control the intracellular osmolyte composition. The volume regulatory mechanisms operating under hyper- and hypotonic conditions are known, respectively, as regulatory volume increase (RVI) and decrease (RVD). During both, RVI and RVD, cells adjust the cellular content of inorganic ions (most notably Na+, K+ and Cl-) and organic solutes in order to gain or lose osmotically obligated water. These mechanisms counteract osmotic cell damage and enable the adaptation of cells to a wide range of extracellular osmolarities. Despite decades of research in this field, many aspects of the mechanisms underlying RVD and RVI remain poorly understood.
In case of T lymphocytes, various cellular functions, including proliferation, migration and T cell activation are closely associated with the cell volume regulatory machinery. Among other mechanisms, all these processes are tightly linked by a network of potassium channels. The identification of this network is of great biomedical interest as it provides a key to pharmacological manipulation of the immune system. Current models of hypotonic volume regulation (RVD) in T-lymphocytes consider primarily the voltage-gated KV1.3 and the calcium-activated IKCa1 channel.
The first part of this thesis explores the potential role of two-pore domain (K2P) potassium channels in RVD in murine and human primary CD4+-T lymphocytes. Using a combined genetic and pharmacological approach, time-resolved cell volume analysis revealed an important role of the K2P channels TASK1, TASK2, TASK3 and TRESK in swelling activated K+ efflux from hypotonically swollen T cells. Based on the analysis carried out here, the voltage-gated TASK2 as well as the calcium-activated TRESK channel were found as the most important K2P channels involved in the RVD of both naĂŻve and stimulated T cells. The importance of TASK2 and TRESK in the RVD process was comparable to that of KV1.3. In summary, the data provide first evidence that hypotonic volume regulation of murine and human CD4+-T lymphocytes relies on K2P channels. With respect to the close relationship of T-cell function and volume regulatory mechanisms K2P channels may thus be considered as potential targets for immunomodulation.
In the second and major part of this thesis, the swelling-activated transport pathways for small organic osmolytes (SOOs) were investigated. Nearly all eukaryotic cells possess a considerable reservoir of SOOs, such as polyols (e.g. sorbitol, myo-inositol), methylamines (e.g. betaine, α-glycerophosphoryl choline) and small amino acids (e.g. α-/ÎČ- alanine, proline and the derivate taurine), which are synthesized within the cells or accumulated from the extracellular medium. Since SOOs do not interfere with the integrity of macromolecules and the membrane potential, cells tolerate great cytosolic fluctuations of these solutes without negative effects on cellular structure or function. Due to these properties, small organic osmolytes are important tools for cell volume regulatory mechanisms, by which the intracellular osmolarity can be adjusted independently of electrolytes. Although the importance of SOOs for hypotonic volume regulation has been known for long time, the molecular identity of participating membrane efflux pathways is far from being clear.
Regardless of the involved transporters, swelling-activated pathways have been reported to exhibit a size selective permeability for a wide range of sugars and related compounds. To gain a deeper insight into this issue, in a first step the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200â1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions was analyzed. Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane was found to remain impermeable to PEG300â1500, which allowed the cells to accomplish regulatory volume decrease. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600â1500, whereas 100 mOsm solutions of PEG300â400 inhibited RVD. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300â400, but not to PEG600â1500. Using the values of hydrodynamic radii Rh for PEGs, the observed size-selectivity of membrane permeation yielded an estimate of âŒ0.74 nm for the cut-off radius of the swelling-activated pathway for organic osmolytes. This result may be of interest for many biotechnological and biomedical applications, where swelling-activated SOO-pathways are widely used for cell-loading techniques.
As a second step, an attempt was made to elucidate the molecular identity of transporters for organic osmolytes potentially involved in RVD. Since it was not clear whether RVD-related efflux of SOOs is mediated by one common or several distinct transporter(s), at first, the plasma membrane permeability profiles for two structurally dissimilar SOOs, including the amino sulfonic acid taurine and the polyol myo-inositol were analyzed. The results of the time resolved volumetric measurements clearly showed that the membrane permeability to taurine was activated upon moderate cell swelling (by ~15%) in mildly hypotonic solutions (~230 mOsm). In sharp contrast, the membrane permeability to myo-inositol was activated after a much larger swelling (~50%) in strongly hypotonic media (<150 mOsm). Moreover, the swelling-activated permittivity to taurine during RVD in 100 mOsm medium persisted for about twice as long as that for myo-inositol (taurine ~95 min, myo-inositol ~40 min). These findings clearly showed that, taurine and myo-inositol utilized separate, apparently substrate-specific pathways, which were activated at different hypotonic thresholds.
Since many members of SLC-family proteins (Solute Carrier) are known for their substrate selectivity and also for their contribution to osmoregulatory mechanisms a participation of SLCs was investigated in the context of RVD. To this end, a combination of molecular biological (semiquantitative RT-PCR) and fluorescence microscopy techniques (confocal and super-resolution microscopy) was used. The semiquantitative RT-PCR data showed a transcriptional upregulation for the SLC proteins SLC5A3 (myo-inositol transporter; SMIT) and SLC6A6 (taurine transporter TauT) in hypotonically stressed Jurkat lymphocytes, HEK293, and HepG2 cells. In all three human cell lines strongly hypotonic solutions (100 mOsm) increased the mRNA level of the genes SLC5A3 and SLC6A6 between 20-60% and 30-100%, respectively, suggesting a potential participation of SLC transporters in RVD.
In addition, confocal microscopy images clearly showed the intracellular displacement of EGFP-tagged SLC5A3 expressed in HEK293 cells following strongly hypotonic stress (100 mOsm). Within 10 min the fluorescence of EGFP was shifted from intracellular regions towards the plasma membrane. Furthermore, super-resolution microscopy by means of dSTORM revealed a considerably increased membrane association of SLC5A3 in strongly hypotonic stressed (100 mOsm) HEK293 and Jurkat cells. This finding suggests that SLC5A3 is integrated into the plasma membrane by swelling-induced exocytosis. Taken together, the results of this investigation provided first evidence that transporters such as SLC5A3, SLC6A6 and probably other SLC-proteins participate in the mechanism of hypotonic volume regulation. Due to the relevance of SLC-proteins as potential drug delivery systems the possible role of these transporters might be of great interest for many biomedical research areas
Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy
Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100â275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200â275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of âŒ3 nm/s at 100â125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200â2000 localizations/ÎŒm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80â800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells
Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes
Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated K(v)1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K-2P) channels in the RVD of human CD4(+) T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K(2P)5.1 and K(2P)18.1 as the most important K-2P channels involved in the RVD of both naive and stimulated T cells. The impact of chemical inhibition of K(2P)5.1 and K(2P)18.1 on the RVD was comparable to that of K(v)1.3. K(2P)9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K-2P channels
<i>d</i>STORM imaging of immunolabeled SLC5A3 protein in the plasma membrane of HEK293 cells under isotonic and hypotonic conditions.
<p>Images of the same cells in transmitted light (TL) are also shown. From the <i>d</i>STORM images (reconstructed from 15,000 single frames), the surface membrane density of SLC5A3 localizations [loc/ÎŒm<sup>2</sup>] were identified in individual cells. The bar graph shows the impact of hypotonic stress on the surface membrane density of SLC5A3 protein localizations. The data are means (±SD) from 8â16 individual cells for each osmotic condition and hypotonic stress duration. The differences in the mean values between the isotonic control and the two hypotonic samples were statistically significant (as denoted by *; <i>P</i> < 0.05), according to the Mann-Whitney test conducted using the Software Origin 9 (Microcal, Northampton, MA). The difference between the two hypotonic samples (10 vs 20 min) was not significant (n.s.).</p
Hypotonic stress-induced upregulation of SLC5A3 at the mRNA and protein level in HEK293 cells revealed by semiquantitative RT-PCR and Western blot, respectively.
<p>Prior to RNA and protein extractions, the cells were incubated in 100-mOsm CGM for 10â30 min. Control cells were kept in isotonic CGM. The SLC5A3 mRNA level in isotonic sample was negligible, whereas hypotonicity induced substantial amounts of SLC5A3 mRNA. As with RT-PCR, Western blot analysis shows increased amounts of SLC5A3 protein (by up to âŒ40%, <i>see</i> text) in hypotonic samples. For RT-PCR, ÎČ-actin was used as a template loading control (<i>see</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#sec019" target="_blank">Supporting information</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.s002" target="_blank">S2 Fig.</a>, upper image). Prior to immunoblotting, reversible Ponceau-S protein staining has been used as a loading control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.s002" target="_blank">S2 Fig.</a>, lower image).</p
Impact of hypotonicity on the myo-inositol permeability <i>P</i><sub>ino</sub> in HEK293 cells.
<p>The <i>P</i><sub>ino</sub> values were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e004" target="_blank">Eq. 2</a> from the rates of secondary swelling, using the volumetric data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.g002" target="_blank">Fig. 2A</a>. The fit of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e007" target="_blank">Eq. 3</a> to the data yielded a <i>C</i><sub>50</sub> value of 144 ± 10 mOsm, i.e. the tonicity at which the myo-inositol permeability was half-activated. In the inset, the same <i>P</i><sub>ino</sub> data are plotted as function of the cell volume at the time point of <i>myo</i>-inositol application. Curve fitting (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e007" target="_blank">Eq. 3</a>) shows that <i>P</i><sub>ino</sub> was half-activated as cells swelled by about 26% (<i>v</i><sub>50</sub> = 1.26±0.02).</p
Volume changes of HEK293 cells in response to solutions of varying osmolality and composition.
<p>At time âŒ30 s, the cells were first transferred from isotonic growth medium to a sucrose-substituted solution having osmolality of 100, 125, ⊠250 or 275 mOsm. Thereafter, the hypotonic sucrose solutions were replaced at time âŒ5 min with myo-inositol solutions of the same osmolalities (<b><i>A</i></b>). In contrast, the cells were exposed for 20 min to sucrose solutions only (<b><i>B</i></b>). After the initial swelling in the presence of sucrose, the cells were capable of RVD over the entire hypotonicity range (<i>B</i>). A nearly complete RVD also occurred in slightly hypotonic solutions of myo-inositol (<i>A</i>, âŒ175â275 mOsm). Application of more diluted myo-inositol solutions (100â150 mOsm; <i>t</i> â 5â9min) considerably inhibited cell shrinkage via RVD. Thereafter (<i>t</i> â 9â20 min) the cells exhibited sustained secondary swelling (<b><i>A</i></b>), which is indicative of myo-inositol uptake by cells. For each tonicity, the rates of RVD Î<i>v</i>/Î<i>t</i><sub>RVD</sub> and secondary swelling Î<i>v</i>/Î<i>t</i><sub>ino</sub> were used to calculate the permeability coefficients for electrolytes <i>P</i><sub>el</sub> and inositol by applying Eqs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e004" target="_blank">2</a>, respectively.</p
Changes in the normalized volume <i>v</i> = V/V<sub>0</sub> of HEK293 cells in response to sequential application of sucrose and <i>myo</i>-inositol solutions of the same osmolality of 100 mOsm.
<p>The cells were bathed initially (<i>time</i> < âŒ30 s) in isotonic growth medium (300 mOsm) and then exposed to a 100-mOsm sucrose solution. The strongly hypotonic sucrose solution (<i>filled symbols</i>) caused fast cell swelling to a transient maximum volume <i>v</i><sub>max</sub> of âŒ1.6 within the first 2â3 min. After the initial swelling, the cells underwent RVD, i.e. they gradually shrank to reach the original isotonic volume (<i>v</i><sub>0</sub> â 1) within âŒ20 min in the presence of sucrose. The replacement of sucrose by an equiosmotic amount of <i>myo</i>-inositol (<i>arrows</i>) abolished RVD and caused secondary cell swelling (<i>empty symbols</i>). The rate of secondary swelling (Î<i>v</i>/Î<i>t</i><sub>ino</sub>, <i>red fitted lines</i>) decreased with time during and after RVD (7â35 min). The addition of <i>myo</i>-inositol 40 min after hypotonic shock did not cause any significant cell swelling. Each data point represents the mean ± SE of 25â42 individual cells measured in 2â3 independent experiments. For each time point of <i>myo</i>-inositol addition, the rates of RVD Î<i>v</i>/Î<i>t</i><sub>RVD</sub> (<i>blue lines</i>) and the rates of secondary swelling Î<i>v</i>/Î<i>t</i><sub>ino</sub> (<i>red lines</i>) were determined to calculate the permeability coefficients for myo-inositol <i>P</i><sub>ino</sub> by applying Eqs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.e004" target="_blank">2</a>. The inset illustrates the decay of <i>P</i><sub>ino</sub> with time during RVD.</p
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