Table 1.
Ionic composition of experimental external solutions.
Figure 1.
Calibration of voltage sensitive dye DIBAC4(3) in MIO-M1 cells.
A- 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 (Ft/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- Relation between relative changes in fluorescence and membrane potential calculated from Equation 3.
Table 2.
Values of parameters used in simulations.
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 (ΔOsM = 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 ± SEM for 42–55 cells from 15 experiments, *p<0.05, HYPOMannitol vs. HYPONaCl.
Figure 3.
Role of Ba2+- sensitive K+ channels on RVD in MIO-M1 Müller cells.
Representative cell volume changes measured in BCECF-loaded MIO-M1 cells in response to a hypoosmotic shock (ΔOsM = 100 mOsM) generated either keeping constant (HYPOMannitol) (A) or varying ion composition (HYPONaCl) (B). In all the experiments 10−3 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 Ba2+ treated cells. Values are mean ± SEM for 21–80 cells from 5–9 experiments, ***p<0.001, Vehicle vs. Ba2+.
Figure 4.
Role of NPPB-sensitive Cl− channels on RVD in MIO-M1 cells.
Representative cell volume changes measured in BCECF-loaded MIO-M1 cells in response to a hypoosmotic shock (ΔOsM = 100 mOsM) generated either keeping constant (HYPOMannitol) (A) or varying ion composition (HYPONaCl) (B). In all the experiments 10−4 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 ± SEM for 28–76 cells from 5–13 experiments, *p<0.05, Vehicle vs. NPPB.
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 (ΔOsM = 100 mOsM) generated either by varying (HYPONaCl) or keeping constant ion composition (HYPOMannitol). Effect of 10−3 M Ba2+ and 10−4 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 (Vmmax−Vmmin) 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.
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 (ΔOsM = 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.
Figure 7.
Modeling of cell response to HYPONaCl when PK is reduced.
Time courses of Vt/V0 (A), Vm/Vm0 (B), Jnet (C) and Vm, EqCl, EqK (D) simulated in cells exposed to HYPONaCl. Before the hypoosmotic shock, resting PK 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, PCl −but not PK− was increased, according to Equation 4. A negative value of Jnet indicates an outward flux.
Figure 8.
Modeling of cell response to HYPONaCl when PCl is reduced.
Time courses of Vt/V0 (A), Vm/Vm0 (B), Jnet (C) and Vm, EqCl, EqK (D) simulated in cells exposed to HYPONaCl. Before the hypoosmotic shock, resting PCl 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, PK −but not PCl− was increased, according to Equation 4. A negative value of Jnet indicates an outward flux.