Low pH Attenuates Apoptosis by Suppressing the Volume-Sensitive Outwardly Rectifying (VSOR) Chloride Current in Chondrocytes

Figures

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  FIGURE 1. Extracellular acidification inhibits VSOR Cl− currents and impairs RVD in C28/I2 cells. (A) Mean Cl− current ± SEM measured over 15 min under pH 7.2 and pH 6.0 conditions. 24 h before Cl− currents were measured chondrocytes were preincubated in pH 7.4 medium (control, dark grey triangles), or in pH 6.0 medium (light grey circles). Each symbol represents the current normalized to cell membrane capacity (current density), which was obtained every 10 s at +100 mV (upper traces) and −100 mV (lower traces) in response to 500-ms voltage ramps applied every 10 s from a holding potential of 0 mV (n = 6–8). (B) Time course of mean Cl− current amplitudes under hypotonic pH 7.2 and pH 6.0 conditions. pH 6.0 solution was applied when Cl− currents reached a maximum under hypotonic conditions. Currents were normalized to peak values under hypotonic conditions. Each circle represents the current at +100 mV (upper traces) and −100 mV (lower traces) measured in response to 500-ms voltage ramps applied every 10 s from a holding potential of 0 mV (n = 6–7). (C) Representative current voltage relationship (500-s voltage ramps applied from −100 mV to +100 mV) recorded under hypotonic pH 7.2 and…

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  FIGURE 2. Pharmacological properties of VSOR Cl− channels in C28/I2 cells. (A) Mean peak VSOR Cl− current amplitudes ± SEM at +100 mV and −100 mV under hypotonic conditions in the absence and presence of different inhibitors. (B) VSOR Cl− current traces elicited by 500-ms voltage steps from −100 mV to +100 mV obtained at maximal VSOR Cl− current amplitudes under hypotonic conditions in the absence and presence of DCPIB (10 µM), niflumic acid (NFA; 500 µM), DIDS (100 µM), NPPB (100 µM) and Tamoxifen (10 µM) (n = 3).

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  FIGURE 3. Effect of acidic preconditioning on caspase 3/7 activity and cell viability. (A) Caspase 3/7 activity and viability assessed by (B) resazurin and (C) CellTiter-Glo assays based on the cells’ metabolic activity and ATP content and (D) CellTiter-Fluor assays based on constitutive protease activity in living cells. C28/I2 cells were kept for 8 h at different pH values in the absence or presence of staurosporine (st) (5 µM) (n = 3).

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  FIGURE 4. Effect of acidic preconditioning on annexin V/7-AAD staining. Annexin V/7-AAD staining of C28/I2 cells after 5 h incubation under neutral pH 7.4, acidic pH 5.5 and neutral pH 7.4 + DIDS (100 µM) conditions in the absence and presence of staurosporine (st) (1 and 5 µM). (A) Q1 annexin-V–/7-AAD+ (necrotic cells) (B) Q2 annexin-V+/7-AAD+ (late apoptotic cells) (C) Q4 annexin-V−/7-AAD− (living cells), (D) Q3 annexin-V+/7-AAD− (early apoptotic cells) (n = 4).

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  FIGURE 5. Exemplary annexin V/7-AAD staining scatter plots after 5 h of incubation in media of (A) pH 7.4, (B) pH 7.4 with staurosporine (st) (1 µM), (C) pH 5.5, (D) pH 5.5 with staurosporine (1 µM), (E) pH 7.4 in presence of DIDS (100 µM) and (F) pH 7.4 with staurosporine in presence of DIDS (100 µM).

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  FIGURE 6. Effect of different VSOR Cl− channel inhibitors on caspase 3/7 activity and cell viability. (A) Caspase 3/7 activity and (B) viability of C28/I2 cells treated for 6 h with pH 7.4, pH 5.5 and pH 7.4 + different VSOR Cl− current inhibitors in the absence and presence of staurosporine (st) (1 µM) (n = 3). (C) Caspase 3/7 activity and (D) viability of C28/I2 cells treated for 8 h with pH 7.4, pH 5.5 and pH 7.4 + DIDS (100 µM) in the absence or presence of staurosporine (st) (1 and 5 µM) (n = 4). (E) Caspase 3/7 activity and (F) viability of cells treated for 24 h with pH 7.4 and pH 6.0 with or without DCPIB (10 µM) in the absence or presence of staurosporine (st) (1 µM) (n = 2–3). Data are represented as mean ± SEM.

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  FIGURE 7. AVD in chondrocytes is impaired under acidic conditions and in the presence of VSOR Cl-channel inhibitors. (A, C, E) Mean cell volume (MCV) ± SEM in femtoliters (fl) measured (A) over 2 h under hypertonic (360 mOsm/kg) pH 7.4 conditions and under isotonic (300 mOsm/kg) conditions at pH 7.4 or pH 6.0 in the presence and absence of 5 µM staurosporine (st) (n = 4) and (C, E) MCV measured over 60 min under isotonic pH 7.4 conditions in the absence (control) or presence of the Cl− channel inhibitors NPPB (100 µM), Tamoxifen (Tamox) (10 µM), DCPIB (10 µM), niflumic acid (NFA; 500 µM) or DIDS (100 µM) ± staurosporine (1 µM) (n = 3). (B) Differences in MCV (ΔMCV in fl) calculated from data in (A) at the timepoints 60 and 120 min for matching pH conditions (pH 7.4 and 6.0) in the presence and absence of staurosporine. (D, F) ΔMCV at 60 min calculated from data in (C, E) for control (absence of blockers) and matching blocker conditions in the presence and absence of 1 µM staurosporine. (G) MCV ± SEM assessed by using flow cytometry after 5 h of incubation with pH 7.4, pH 7.4 + DIDS (100 µM) and pH 5.5 in the presence and absence of staurosporine (st) (1 µM or 5 µM) (n = 4).

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  FIGURE 8. Putative model of the antiapoptotic effect of acidosis in C28/I2 cells treated with staurosporine. Under pH 7.4 (left side) staurosporine (st) treatment leads to activation of volume-sensitive outwardly rectifying (VSOR) Cl− channels to induce cell shrinkage (AVD), which is an initial step during apoptosis, followed by phosphatidylserine (PS) exposure, caspase activation, DNA fragmentation, membrane blebbing and loss of membrane integrity evidenced by the 7-AAD uptake. Under acidic conditions (right side) VSOR channels are deactivated so that AVD is absent under isotonic conditions and consequently apoptosis is suppressed in the presence of staurosporine.