Acid- and Volume-Sensitive Chloride Currents in Human Chondrocytes

Figures

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  FIGURE 1. Extracellular acidification induces an acid-sensitive outwardly rectifying (ASOR) current in C28/I2 cells and primary human chondrocytes. (A) Mean amplitudes ± standard error of the means (SEM) of ASOR currents measured at pH 7.2 (n = 20), 5.5 (n = 4), 5.0 (n = 9), and 4.5 (n = 7). Asterisks indicate significant differences between pH 5.0 and pH 7.2 and between pH 4.5 and pH 7.2 (p < 0.05). Wilcoxon rank sum test. Current–voltage (I–V) relationships of mean ASOR currents ± SEM analyzed at the beginning (I1—open triangles) and at the end (I2—closed circles) of voltage pulses (indicated by gray shadings in D and E) in C28/I2 cells (n = 8) (B) and primary chondrocytes (n = 7) (C). Asterisks indicate significant differences between I1 and I2 (p < 0.05). Paired t-tests. Examples of ASOR current traces elicited by 500-ms voltage steps from –100 to +100 mV in C28/I2 cells (D) and primary chondrocytes (E). (a) Close-up of the inward current peak at –100 mV indicated in (D).

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  FIGURE 2. ASOR current activation requires extracellular but not intracellular acidification. (A) Intracellular pH measurements in C28/I2 cells at different timepoints after extracellular acidification (pH 6.5, 6.0, 5.5, 5.0, and 4.5) versus control conditions (pH 7.4); means ± SEM (n = 6). (B) ASOR current time course in C28/I2 cells (single experiment) using an acidic intracellular (pipette) solution (pHi 4.5). Each circle represents the current at +100 mV (upper trace) and –100 mV (lower trace) measured in response to 500-ms voltage ramps applied every 10 s. The recording was performed in the ruptured patch clamp configuration. (C) Viability of C28/I2 cells after 2 and 4 h of exposure to pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4; means ± SEM (n = 3–4).

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  FIGURE 3. The volume-sensitive outwardly rectifying (VSOR) current in C28/I2 cells is affected by low pH. Examples of VSOR current traces elicited by 500-ms voltage steps from –100 to +100 mV obtained at half-maximal VSOR current amplitudes (VSOR Imax—pH 7.2) (A), at maximal VSOR current amplitudes (VSOR Imax—pH 7.2) (C), and at maximal VSOR current amplitudes immediately after reducing pHe to 4.5 (VSOR Imax—pH 4.5) (E). (B,D,F) I–V relationships of mean VSOR currents analyzed at the beginning (I1—closed circles) and at the end (I2—open triangles) of voltage pulses (indicated by gray shadings in A, C, and E). (B) VSOR Imax—pH 7.2 (n = 10). (D) VSOR Imax—pH 7.2 (n = 11). (F) VSOR Imax—pH 4.5 (n = 9). Asterisks indicate significant differences between I1 and I2 (p < 0.05). Paired t-tests. (G,H) I2/I1 ratios and mean current amplitudes, respectively, of ASOR currents and VSOR Imax—pH 7.2, VSOR Imax—pH 7.2, and VSOR Imax—pH 4.5 currents at +100 and –100 mV. Means ± SEM (n = 9–11). I2/I1 ratio > 1, time-dependent activation; I2/I1 ratio < 1, time-dependent inactivation.

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  FIGURE 4. The volume-sensitive outwardly rectifying (VSOR) current in primary chondrocytes is affected by low pH. VSOR current traces elicited by 500-ms voltage steps from –100 mV to +100 mV obtained at half-maximal VSOR current amplitudes (VSOR Imax—pH 7.2) (A), at maximal VSOR current amplitudes (VSOR Imax—pH 7.2) (C), and at maximal VSOR current amplitudes immediately after reducing pHe to 4.5 (VSOR Imax—pH 4.5) (E). (B,D,F) I–V relationships of mean VSOR currents analyzed at the beginning (I1—closed circles) and at the end (I2—open triangles) of voltage pulses (indicated by gray shadings in A, C, and E). (B) VSOR Imax—pH 7.2 (n = 5). (D) VSOR Imax—pH 7.2 (n = 6). (F) VSOR Imax—pH 4.5 (n = 3). Asterisks indicate significant differences between I1 and I2 (p < 0.05). Paired t-tests. (G,H) I2/I1 ratios and mean current amplitudes, respectively, of ASOR currents and VSOR Imax—pH 7.2, VSOR Imax—pH 7.2, and VSOR Imax—pH 4.5 currents at +100 and –100 mV. Means ± SEM. (G) n = 3–7 (I2/I1 ratio > 1, time-dependent activation; I2/I1 ratio < 1, time-dependent inactivation). (H) n = 2–3.

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  FIGURE 5. Volume- and acid-sensitive outwardly rectifying (VSOR and ASOR) current sensitivities to the Cl– channel blockers DCPIB and tamoxifen. (A,B) and (C,D) Representative current–voltage relationship obtained by 500-ms voltage ramps under acidic and hypotonic conditions in the absence and presence of DCPIB (10 μM) in C28/I2 cells and primary human chondrocytes, respectively. (E,F) Mean peak currents ± SEM under acidic and hypotonic conditions in the absence and presence of 10 μM DCPIB in C28/I2 (n = 2–4) and primary chondrocytes (n = 2–3), respectively. (G) Mean peak currents ± SEM under acidic and hypotonic conditions in the absence and presence of 10 μM tamoxifen in C28/I2 (n = 3). Asterisks in (E) Indicate significant differences between pH 5.0 and DCPIB and between hypotonic and DCPIB (p < 0.05). Paired t-tests.

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  FIGURE 6. Acidic and hypotonic conditions lead to depolarization of the cell membrane potential (Vmem). Vmem recordings of single C28/I2 chondrocytes at pH 7.4, pH 4.5, and pH 7.4 again (wash-out) (A), and under isotonic (iso), hypotonic (hypo), and isotonic (wash-out) (C). Each circle represents the mean Vmem obtained over 15 s. Insets in (A) show whole-cell Cl– currents at the indicated timepoints in response to 500-ms voltage ramps from −100 to +100 mV from membrane potentials prevailing at the respective timepoints. (B,D) Mean Vmem ± SEM under the conditions given in (A) and (C) (n = 3 and n = 3–6, respectively). Asterisks in (B) indicate significant differences between pH 7.4 and pH 4.5 and between pH 4.5 and wash-out (p < 0.05). In (D), the asterisk indicates a significant difference between isotonic and hypotonic conditions (p < 0.05). Paired t-tests.

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  FIGURE 7. Interplay between ASOR and VSOR currents in C28/I2 cells—effect of external acidification on VSOR currents. (A) Time course of a single experiment under isotonic (300 mosm/kg) and hypotonic (220 mosm/kg) conditions at pH 7.2 or 4.5 as indicated. Each circle represents the current at +100 mV (upper trace) and –100 mV (lower trace) measured in response to 500-ms voltage ramps applied every 10 s. (B) Mean current amplitudes ± SEM measured at the timepoints (1)–(10) as indicated in (A) (n = 5–11). *p < 0.05 as indicated. Paired t-tests. Representative current traces in response to 500-ms voltage steps to +100 and –100 mV recorded at the timepoints marked in (A) at +100 mV (C,D) and –100 mV (E,F). I2/I1 ratios ± SEM obtained at the timepoints indicated in (A) at +100 mV (n = 5–11) (G) and at –100 mV (n = 4–11) (H); I2/I1 ratio > 1, time-dependent activation; I2/I1 ratio < 1, time-dependent inactivation.

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  FIGURE 8. Interplay between ASOR and VSOR currents in primary human chondrocytes—effect of external acidification on VSOR currents. (A) Time course of a single experiment under isotonic (300 mosm/kg) and hypotonic (220 mosm/kg) conditions at pH 7.2 or 4.5 as indicated. Each circle represents the current at +100 mV (upper trace) and –100 mV (lower trace) measured in response to 500-ms voltage ramps applied every 10 s. (B) Mean current amplitudes ± SEM measured at the timepoints (1)–(8) as indicated in (A) (n = 3). Representative current traces in response to 500-ms voltage steps to +100 and –100 mV recorded at the timepoints marked in (A) at +100 mV (C,D) and –100 mV (E,F). I2/I1 ratios ± SEM obtained at the timepoints indicated in (A) at +100 mV (n = 2–3) (G) and at –100 mV (n = 2–3) (H); I2/I1 ratio > 1, time-dependent activation; I2/I1 ratio < 1, time-dependent inactivation.

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  FIGURE 9. Interplay between ASOR and VSOR currents—effect of hypotonicity on ASOR currents in C28/I2 cells. (A) Time course of a single experiment under isotonic (300 mosm/kg) and hypotonic (220 mosm/kg) conditions at pH 7.2 or 4.5 as indicated. Each circle represents the current at +100 mV (upper trace) and –100 mV (lower trace) measured in response to 500-ms voltage ramps applied every 10 s. (B) Mean current amplitudes ± SEM measured at the timepoints (1)–(6) as indicated in (A) (n = 3–8). *p < 0.05 as indicated. Paired t-test. Representative current traces in response to 500-ms voltage steps to +100 and –100 mV recorded at the timepoints marked in (A) at +100 mV (C,D) and at –100 mV (E,F). I2/I1 ratios ± SEM obtained at the timepoints as indicated in (A) at +100 mV (n = 5–11) (G) and at –100 mV (n = 4–11) (H); I2/I1 ratio > 1, time-dependent activation; I2/I1 ratio < 1, time-dependent inactivation.

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  FIGURE 10. Interrelation between ASOR and VSOR currents—effect of simultaneous application of acidic and hypotonic conditions in C28/I2 cells. (A) Time course of a single experiment under isotonic (300 mosm/kg) and hypotonic (220 mosm/kg) conditions at pH 7.2 or 4.5 as indicated. Each circle represents the current at +100 mV (upper trace) and –100 mV (lower trace) measured in response to 500-ms voltage ramps applied every 10 s. (B) Mean current amplitudes ± SEM measured at the timepoints (1)–(9) as indicated in (A) (n = 3–6). *p < 0.05 as indicated. Paired t-tests. Representative current–voltage relationship obtained by 500-ms voltage ramps recorded at the timepoints marked in (A) at +100 mV (C,D) and at –100 mV (E,F). I2/I1 ratios ± SEM obtained at the timepoints as indicated in (A) at +100 mV (n = 3–6) (G) and at –100 mV (n = 3–6) (H); I2/I1 ratio > 1, time-dependent activation; I2/I1 ratio < 1, time-dependent inactivation.

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  FIGURE 11. Cell volume regulation in C28/I2 cells is impaired under acidic conditions and in the presence of the Cl– channel blocker DCPIB. (A) Mean cell volume (MCV) ± SEM measured over 1 h under isotonic (300 mosm/kg; circles) or hypotonic (220 mosm/kg; diamonds) conditions at pH 7.4 (open symbols) or pH 4.5 (black symbols) (n = 4). (B) MCV ± SEM measured over 1 h under in isotonic solution pH 7.4 (open circles), hypotonic solution pH 7.4 (open diamonds) in the absence and presence of DCPIB (10 μM; gray diamonds), and under hypotonic conditions at pH 4.5 (black diamonds) (n = 4). (A) and (B) show the results of two independent experimental series.