Cell Volume Regulation in Cultured Human Retinal Müller Cells Is Associated with Changes in Transmembrane Potential

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Abstract

Müller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K+ and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (Vm), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in Vm. Cell volume and Vm changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by Vm depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K+ or Cl- channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations -all leading to changes in Vm- define the success of RVD. © 2013 Fernández et al.

Figures

  • Table 1. Ionic composition of experimental external solutions.
  • Figure 1. Calibration of voltage sensitive dye DIBAC4(3) in MIOM1 cells. A- Representative experiment showing the response of cells previously loaded with 2.5 mM 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 (Ft/F0 DIBAC4(3)). When a stable signal was registered, control solution was replaced by a solution containing 5 mM gramicidin. Afterwards, extracellular NaCl concentration was replaced (0 mM, 70 mM and 126 mM). B- Relation between relative changes in fluorescence and membrane potential calculated from Equation 3. doi:10.1371/journal.pone.0057268.g001
  • Figure 2. Effects of extracellular media composition on RVD in MIO-M1 Cells. Representative kinetics of cell volume changes measured in BCECF-loaded MIO-M1 cells in response to hypoosmotic shock (DOsM = 100 mOsM) generated either by varying (HYPONaCl) or keeping constant extracellular ion composition (HYPOMannitol). Insert: Percentage of cell volume recovery at 10 minutes (% RVD10) in both conditions. Values are mean 6 SEM for 42–55 cells from 15 experiments, *p,0.05, HYPOMannitol vs. HYPONaCl. doi:10.1371/journal.pone.0057268.g002
  • Table 2. Values of parameters used in simulations.
  • Figure 4. Role of NPPB-sensitive Cl2 channels on RVD in MIOM1 cells. Representative cell volume changes measured in BCECFloaded MIO-M1 cells in response to a hypoosmotic shock (DOsM = 100 - mOsM) generated either keeping constant (HYPOMannitol) (A) or varying ion composition (HYPONaCl) (B). In all the experiments 10 24 M NPPB or vehicle (DMSO) was added to ISONaCl or ISOMannitol 10 minutes before the hypoosmotic shock and maintained during the entire experiment. C- % RVD10 after the hyposmotic challenge in DMSO or NPPB treated cells. Values are mean 6 SEM for 28–76 cells from 5–13 experiments, *p,0.05, Vehicle vs. NPPB. doi:10.1371/journal.pone.0057268.g004
  • Figure 3. Role of Ba2+- sensitive K+ channels on RVD in MIO-M1 Müller cells. Representative cell volume changes measured in BCECFloaded MIO-M1 cells in response to a hypoosmotic shock (DOsM = 100 - mOsM) generated either keeping constant (HYPOMannitol) (A) or varying ion composition (HYPONaCl) (B). In all the experiments 10 23 M Ba2+ or vehicle (water) was added to ISONaCl or ISOMannitol 10 minutes before the hypoosmotic shock and maintained during the entire experiment. C- % RVD10 after the hypoosmotic challenge in vehicle or Ba 2+ treated cells. Values are mean 6 SEM for 21–80 cells from 5–9 experiments, ***p,0.001, Vehicle vs. Ba2+. doi:10.1371/journal.pone.0057268.g003
  • Figure 5. Vm evolution after a hypoosmotic shock in MIO-M1 cells. Vm was monitored using DIBAC4(3) under different experimental conditions. A–Vm changes measured in response to a hypoosmotic shock (DOsM = 100 mOsM) generated either by varying (HYPONaCl) or keeping constant ion composition (HYPOMannitol). Effect of 10 23 M Ba2+ and 1024 M NPPB on Vm changes under HYPOMannitol (B) or under HYPONaCl conditions (C). D- Bars indicating the difference between the peak maximum Vm and the Vm 30 minutes after being exposed to a hypoosmotic media (Vmmax2Vmmin) 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 6 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. doi:10.1371/journal.pone.0057268.g005
  • Figure 6. Modeling of cells exposed to different extracellular media compositions. Time courses of Vt/V0 (A), Vm/Vm0 (B), Jnet (C) and Vm, EqCl, EqK (D) simulated in cells exposed to either HYPONaCl or to HYPOMannitol. At time = 0 extracellular osmolarity was reduced (DOsM = 100 mOsM) and after a delay of 20 s, PK and PCl increased according to Equation 4. A negative value of Jnet indicates an outward flux. doi:10.1371/journal.pone.0057268.g006

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Fernández, J. M., Di Giusto, G., Kalstein, M., Melamud, L., Rivarola, V., Ford, P., & Capurro, C. (2013). Cell Volume Regulation in Cultured Human Retinal Müller Cells Is Associated with Changes in Transmembrane Potential. PLoS ONE, 8(2). https://doi.org/10.1371/journal.pone.0057268

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