TRPV4 Contributes to Resting Membrane Potential in Retinal Müller Cells: Implications in Cell Volume Regulation

Neural activity alters osmotic gradients favoring cell swelling in retinal Müller cells. This swelling is followed by a regulatory volume decrease (RVD), partially mediated by an efflux of KCl and water. The transient receptor potential channel 4 (TRPV4), a nonselective calcium channel, has been proposed as a candidate for mediating intracellular Ca2+ elevation induced by swelling. We previously demonstrated in a human Müller cell line (MIO-M1) that RVD strongly depends on ion channel activation and, consequently, on membrane potential (Vm ). The aim of this study was to investigate if Ca2+ influx via TRPV4 contributes to RVD by modifying intracellular Ca2+ concentration and/or modulating Vm in MIO-M1 cells. Cell volume, intracellular Ca2+ levels, and Vm changes were evaluated using fluorescent probes. Results showed that MIO-M1 cells express functional TRPV4 which determines the resting Vmassociated with K+ channels. Swelling-induced increases in Ca2+ levels was due to both Ca2+ release from intracellular stores and Ca2+ influx by a pathway alternative to TRPV4. TRPV4 blockage affected swelling-induced biphasic response (depolarization-repolarization), suggesting its participation in modulating Vm changes during RVD. Agonist stimulation of Ca2+ influx via TRPV4 activated K+ channels hyperpolarizing Vm and accelerating RVD. We propose that TRPV4 forms a signaling complex with Ca2+ and/or voltage-dependent K+ channels to define resting Vm and Vm changes during RVD. TRPV4 involvement in RVD depends on the type of stimuli and/or degree of channel activation, leading to a maximum RVD response when Ca2+ influx overcomes a threshold and activates further signaling pathways in cell volume regulation.Fil: Netti, Vanina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Fernández, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Kalstein, Maia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Pizzoni, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Di Giusto, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Rivarola, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ford, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Capurro, Claudia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentin

Cell volume regulation in cultured human retinal Müller cells is associated with changes in transmembrane potential.

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

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

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

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

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

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

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

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