Modeling of cell response to HYPO_NaCl when P_Cl is reduced.

<p>Time courses of V_t/V₀ (<b>A</b>), V_m/V_m0 (<b>B</b>), J_net (<b>C</b>) and V_m, Eq_Cl, Eq_K (<b>D</b>) simulated in cells exposed to HYPO_NaCl. Before the hypoosmotic shock, resting P_Cl was reduced a tenfold and remained constant throughout the entire simulation. At time = 0 extracellular osmolarity was reduced (ΔOsM = 100 mOsM) and after a delay of 20 s, P_K −but not P_Cl− was increased, according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057268#pone.0057268.e006" target="_blank">Equation 4</a>. A negative value of <i>J</i>_net indicates an outward flux.</p

Effects of extracellular media composition on RVD in MIO-M1 Cells.

<p>Representative kinetics of cell volume changes measured in BCECF-loaded MIO-M1 cells in response to hypoosmotic shock (ΔOsM = 100 mOsM) generated either by varying (HYPO_NaCl) or keeping constant extracellular ion composition (HYPO_Mannitol). Insert: Percentage of cell volume recovery at 10 minutes (% RVD₁₀) in both conditions. Values are mean ± SEM for 42–55 cells from 15 experiments, *p<0.05, HYPO_Mannitol vs. HYPO_NaCl.</p

V_m evolution after a hypoosmotic shock in MIO-M1 cells.

<p>V_m was monitored using DIBAC4₍₃₎ under different experimental conditions. <b>A–</b>V_m changes measured in response to a hypoosmotic shock (ΔOsM = 100 mOsM) generated either by varying (HYPO_NaCl) or keeping constant ion composition (HYPO_Mannitol). Effect of 10⁻³ M Ba²⁺ and 10⁻⁴ M NPPB on V_m changes under HYPO_Mannitol (<b>B</b>) or under HYPO_NaCl conditions (<b>C</b>). <b>D-</b> Bars indicating the difference between the peak maximum V_m and the V_m 30 minutes after being exposed to a hypoosmotic media (Vm_max−Vm_min) obtained after the hypoosmotic shock under each experimental condition. This value indicates the degree of repolarization after the initial swelling-induced depolarization. Values are mean ± SEM for 21–46 cells from 3–7 experiments, ###p<0.001, NaCl vs. Mannitol; ***p<0.001, Ba²⁺ vs. Control, **p<0.01, NPPB vs. Control.</p

Role of Ba²⁺- sensitive K⁺ channels on RVD in MIO-M1 Müller cells.

<p>Representative cell volume changes measured in BCECF-loaded MIO-M1 cells in response to a hypoosmotic shock (ΔOsM = 100 mOsM) generated either keeping constant (HYPO_Mannitol) (<b>A</b>) or varying ion composition (HYPO_NaCl) (<b>B</b>). In all the experiments 10⁻³ M Ba²⁺ or vehicle (water) was added to ISO_NaCl or ISO_Mannitol 10 minutes before the hypoosmotic shock and maintained during the entire experiment. <b>C-</b> % RVD₁₀ after the hypoosmotic challenge in vehicle or Ba²⁺ treated cells. Values are mean ± SEM for 21–80 cells from 5–9 experiments, ***p<0.001, Vehicle vs. Ba²⁺.</p

Values of parameters used in simulations.

<p>Data were obtained either from our own measurements in MIO-M1 cells or from literature, as follows: i- V_cell and A_c in isotonic conditions were estimated from confocal images as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057268#pone.0057268-DiGiusto1" target="_blank">[43]</a>; ii- P_f was obtained from the measured cell volume changes during a hypoosmotic challenge using a modification of the Fick’s law <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057268#pone.0057268-Ford1" target="_blank">[20]</a>; iii- V_m corresponds to the value recorded using sharp electrode patch clamp technique by Limb et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057268#pone.0057268-Limb1" target="_blank">[19]</a>; iv- intracellular Na⁺, K⁺ and Cl⁻ concentration are the typical values for most cell types; v- extracellular Na⁺, K⁺ and Cl⁻ concentrations are the same as in experimental solutions; vi- ion permeabilities were chosen in order to obtain the V_m value of MIO-M1 cells; vii- the initial parameters presented for the Mannitol condition were obtained by simulating an extracellular solution change, from ISO_NaCl to ISO_Mannitol (see Materials and Methods section).</p

Calibration of voltage sensitive dye DIBAC4₍₃₎ in MIO-M1 cells.

<p><b>A-</b> Representative experiment showing the response of cells previously loaded with 2.5 µM DIBAC4₍₃₎ for 15 minutes, exposed to different extracellular concentrations of NaCl. Points represent changes in fluorescence intensity relativized to the stationary values, in the absence of gramicidin (F_t/F₀ DIBAC4₍₃₎). When a stable signal was registered, control solution was replaced by a solution containing 5 µM gramicidin. Afterwards, extracellular NaCl concentration was replaced (0 mM, 70 mM and 126 mM). <b>B-</b> Relation between relative changes in fluorescence and membrane potential calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057268#pone.0057268.e004" target="_blank">Equation 3</a>.</p

Modeling of cell response to HYPO_NaCl when P_K is reduced.

<p>Time courses of V_t/V₀ (<b>A</b>), V_m/V_m0 (<b>B</b>), J_net (<b>C</b>) and V_m, Eq_Cl, Eq_K (<b>D</b>) simulated in cells exposed to HYPO_NaCl. Before the hypoosmotic shock, resting P_K was reduced by half and remained constant throughout the entire simulation. At time = 0 extracellular osmolarity was reduced (ΔOsM = 100 mOsM) and after a delay of 20 s, P_Cl −but not P_K− was increased, according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057268#pone.0057268.e006" target="_blank">Equation 4</a>. A negative value of <i>J</i>_net indicates an outward flux.</p

Modeling of cells exposed to different extracellular media compositions.

<p>Time courses of V_t/V₀ (<b>A</b>), V_m/V_m0 (<b>B</b>), J_net (<b>C</b>) and V_m, Eq_Cl, Eq_K (<b>D</b>) simulated in cells exposed to either HYPO_NaCl or to HYPO_Mannitol. At time = 0 extracellular osmolarity was reduced (ΔOsM = 100 mOsM) and after a delay of 20 s, P_K and P_Cl increased according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057268#pone.0057268.e006" target="_blank">Equation 4</a>. A negative value of <i>J</i>_net indicates an outward flux.</p

Ionic composition of experimental external solutions.

<p>Ionic composition of experimental external solutions.</p

Role of NPPB-sensitive Cl⁻ channels on RVD in MIO-M1 cells.

<p>Representative cell volume changes measured in BCECF-loaded MIO-M1 cells in response to a hypoosmotic shock (ΔOsM = 100 mOsM) generated either keeping constant (HYPO_Mannitol) (<b>A</b>) or varying ion composition (HYPO_NaCl) (<b>B</b>). In all the experiments 10⁻⁴ M NPPB or vehicle (DMSO) was added to ISO_NaCl or ISO_Mannitol 10 minutes before the hypoosmotic shock and maintained during the entire experiment. <b>C-</b> % RVD₁₀ after the hyposmotic challenge in DMSO or NPPB treated cells. Values are mean ± SEM for 28–76 cells from 5–13 experiments, *p<0.05, Vehicle vs. NPPB.</p