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 (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 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 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 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

<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/μm²] 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 8–16 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 10–30 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 ∼40%, <i>see</i> text) in hypotonic samples. For RT-PCR, β-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>_ino in HEK293 cells.

<p>The <i>P</i>_ino 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>₅₀ 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>_ino 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>_ino was half-activated as cells swelled by about 26% (<i>v</i>₅₀ = 1.26±0.02).</p

Changes in the normalized volume <i>v</i> = V/V₀ 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> < ∼30 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>_max of ∼1.6 within the first 2–3 min. After the initial swelling, the cells underwent RVD, i.e. they gradually shrank to reach the original isotonic volume (<i>v</i>₀ ≈ 1) within ∼20 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 (Δ<i>v</i>/Δ<i>t</i>_ino, <i>red fitted lines</i>) decreased with time during and after RVD (7–35 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 25–42 individual cells measured in 2–3 independent experiments. For each time point of <i>myo</i>-inositol addition, the rates of RVD Δ<i>v</i>/Δ<i>t</i>_RVD (<i>blue lines</i>) and the rates of secondary swelling Δ<i>v</i>/Δ<i>t</i>_ino (<i>red lines</i>) were determined to calculate the permeability coefficients for myo-inositol <i>P</i>_ino 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>_ino with time during RVD.</p

Volume changes of HEK293 cells in response to solutions of varying osmolality and composition.

<p>At time ∼30 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 ∼5 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>, ∼175–275 mOsm). Application of more diluted myo-inositol solutions (100–150 mOsm; <i>t</i> ≈ 5–9min) considerably inhibited cell shrinkage via RVD. Thereafter (<i>t</i> ≈ 9–20 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 Δ<i>v</i>/Δ<i>t</i>_RVD and secondary swelling Δ<i>v</i>/Δ<i>t</i>_ino were used to calculate the permeability coefficients for electrolytes <i>P</i>_el 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

A putative mechanism of SLC5A3-mediated myo-inositol release during RVD.

<p><b>A:</b> Under isotonic conditions the SLC5A3 protein is mainly localized in the cytosolic vesicles and its mRNA level is low. The plasma membrane permeability to SOOs and electrolytes is poor. <b>B:</b> Exposure of cells to a strongly hypotonic solution, e.g. 100-mOsm sucrose, causes rapid volume increase followed by cell shrinkage via RVD (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.g001" target="_blank">Fig. 1</a>). The swelling-associated increase in the plasma membrane area is achieved (i) by unfolding of microvilli and (ii) by exocytotic fusion of cytosolic vesicles [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.ref023" target="_blank">23</a>]. The fusion of vesicles carrying SLC5A3 protein leads to the incorporation of this transporter into the plasma membrane. The SLC5A3-mediated efflux of myo-inositol and related SOO contributes to cell shrinkage (RVD) and restoration of the original isotonic cell volume. <b>C:</b> During RVD, the cells recover the original membrane impermeability to myo-inositol (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.g001" target="_blank">Fig. 1</a>), presumably, via the endocytosis of excessive plasma membrane along with reinternalization and, possibly, lysosomal degradation of SLC5A3. The increased mRNA and protein expression of SLC5A3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119990#pone.0119990.g006" target="_blank">Fig. 6</a>) suggest that <i>de novo</i> synthesis of SLC5A3 occurs to restore the depleted cytoplasmic pool of the transporter.</p

Confocal fluorescence imaging of HEK293 cells overexpressing the fusion protein SLC5A3-EGFP.

<p>The images <i>A</i> and <i>B</i> were taken, respectively, under isotonic conditions and 10 min after application of a strongly hypotonic 100-mOsm myo-inositol-substituted solution. Hypotonic cell swelling is clearly seen in (<i>B</i>). The insets and the intensity diagrams (<i>C</i> and <i>D</i>) illustrate the impact of hypotonic stress on the intracellular distribution of the fusion protein. Comparison of the diagrams <i>C</i> and <i>D</i> reveals a marked hypotonicity-mediated depletion of the protein in the perinuclear regions along with its increase in the peripheral cytoplasm. Together, these findings suggest that hypotonic swelling caused translocation of a large portion of SLC5A3-EGFP towards the plasma membrane.</p

Approaches, strenghts, and limitations of avoidable mortality

Publication of recent papers such as the one by Schoenbaum and colleagues entitled 'Mortality Amenable to Health Care in the United States: The Roles of Demographics and Health Systems Performance' has stimulated this commentary. We discuss strengths and limitations of amenable and avoidable mortality in health-care systems' performance and their contribution to health inequalities. To illustrate, we present a case study of avoidable and amenable mortality in Spain over 27 years. We conclude that amenable mortality is not a good indicator of health-care systems' performance, or for determining whether it could give rise to health inequalities. To understand health problems and to assess the impact of interventions affecting health requires good, basic, and routine monitoring of health indicators and of socioeconomic determinants of health