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The COX-2 selective blocker etodolac inhibits TNFα-induced apoptosis in isolated rabbit articular chondrocytes.

Kumagai K, Kubo M, Imai S, Toyoda F, Maeda T, Okumura N, Matsuura H, Matsusue Y - Int J Mol Sci (2013)

Bottom Line: Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD) and elevation of caspase-3/7 activity.In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα.Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan. kumamp@belle.shiga-med.ac.jp.

ABSTRACT
Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis (OA). Recently, we reported that activation of volume-sensitive Cl- current (ICl,vol) mediates cell shrinkage, triggering apoptosis in rabbit articular chondrocytes. A cyclooxygenase (COX) blocker is frequently used for the treatment of OA. In the present study, we examined in vitro effects of selective blockers of COX on the TNFα-induced activation of ICl,vol in rabbit chondrocytes using the patch-clamp technique. Exposure of isolated chondrocytes to TNFα resulted in an obvious increase in membrane Cl- conductance. The TNFα-evoked Cl- current exhibited electrophysiological and pharmacological properties similar to those of ICl,vol. Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD) and elevation of caspase-3/7 activity. In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα. Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

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Activation of ICl,vol by an apoptosis inducer, TNFα. (A) Chart recording of the whole-cell current in response to voltage ramps (dV/dt = ±0.25 V/s, applied every 6 s) before and during application of TNFα; (B) The TNFα-evoked Cl− current exhibited a prominent inactivation at larger positive potential than +50 mV; (C) An outward rectification of the I–V relationship with a reversal potential close to the Nernst ECl (−18.4 mV).
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f2-ijms-14-19705: Activation of ICl,vol by an apoptosis inducer, TNFα. (A) Chart recording of the whole-cell current in response to voltage ramps (dV/dt = ±0.25 V/s, applied every 6 s) before and during application of TNFα; (B) The TNFα-evoked Cl− current exhibited a prominent inactivation at larger positive potential than +50 mV; (C) An outward rectification of the I–V relationship with a reversal potential close to the Nernst ECl (−18.4 mV).

Mentions: Figure 2 shows a representative experiment examining the effect of bath application of TNFα (1 μg/mL) on membrane currents in rabbit articular chondrocytes. Whole-cell currents were recorded under conditions designed to minimize Na+, K+ and Ca2+ currents and electrogenic Na+/K+ pump current. The Gd3+-sensitive stretch-activated channels were also blocked by adding 30 μM GdCl3 to the bath. During superfusion with control isosmotic solution, membrane currents elicited during square steps applied from a holding potential of −30 mV to test potentials between +80 and −100 mV were of small amplitude and practically time-independent (Figure 2A(a), B(a)). Bath application of 1 μg/mL TNFα under isosmotic conditions (360 mosmol/L) gradually activated the membrane current, which reached a steady level in about 10 min after drug application (Figure 2A). This TNFα-induced current, obtained by digital subtraction of membrane currents recorded before and during exposure to TNFα using the square-step protocol (Figure 2B), exhibited a marked inactivation at potentials positive to +50 mV (Figure 2B(b)) and an outward rectification with a reversal potential of −18.7 ± 0.3 mV (n = 5, N = 5; Figure 2C(b)), close to the equilibrium potential for Cl− (ECl = −18.4 mV) under the present experimental conditions. This increase in membrane current was not accompanied by an appreciable change in cell size (a, diameter, 13.8 ± 0.2 μm; b, 13.8 ± 0.2 μm; n = 5, N = 5), as assessed by measuring the cross-sectional area of microscopic cell images.


The COX-2 selective blocker etodolac inhibits TNFα-induced apoptosis in isolated rabbit articular chondrocytes.

Kumagai K, Kubo M, Imai S, Toyoda F, Maeda T, Okumura N, Matsuura H, Matsusue Y - Int J Mol Sci (2013)

Activation of ICl,vol by an apoptosis inducer, TNFα. (A) Chart recording of the whole-cell current in response to voltage ramps (dV/dt = ±0.25 V/s, applied every 6 s) before and during application of TNFα; (B) The TNFα-evoked Cl− current exhibited a prominent inactivation at larger positive potential than +50 mV; (C) An outward rectification of the I–V relationship with a reversal potential close to the Nernst ECl (−18.4 mV).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3821581&req=5

f2-ijms-14-19705: Activation of ICl,vol by an apoptosis inducer, TNFα. (A) Chart recording of the whole-cell current in response to voltage ramps (dV/dt = ±0.25 V/s, applied every 6 s) before and during application of TNFα; (B) The TNFα-evoked Cl− current exhibited a prominent inactivation at larger positive potential than +50 mV; (C) An outward rectification of the I–V relationship with a reversal potential close to the Nernst ECl (−18.4 mV).
Mentions: Figure 2 shows a representative experiment examining the effect of bath application of TNFα (1 μg/mL) on membrane currents in rabbit articular chondrocytes. Whole-cell currents were recorded under conditions designed to minimize Na+, K+ and Ca2+ currents and electrogenic Na+/K+ pump current. The Gd3+-sensitive stretch-activated channels were also blocked by adding 30 μM GdCl3 to the bath. During superfusion with control isosmotic solution, membrane currents elicited during square steps applied from a holding potential of −30 mV to test potentials between +80 and −100 mV were of small amplitude and practically time-independent (Figure 2A(a), B(a)). Bath application of 1 μg/mL TNFα under isosmotic conditions (360 mosmol/L) gradually activated the membrane current, which reached a steady level in about 10 min after drug application (Figure 2A). This TNFα-induced current, obtained by digital subtraction of membrane currents recorded before and during exposure to TNFα using the square-step protocol (Figure 2B), exhibited a marked inactivation at potentials positive to +50 mV (Figure 2B(b)) and an outward rectification with a reversal potential of −18.7 ± 0.3 mV (n = 5, N = 5; Figure 2C(b)), close to the equilibrium potential for Cl− (ECl = −18.4 mV) under the present experimental conditions. This increase in membrane current was not accompanied by an appreciable change in cell size (a, diameter, 13.8 ± 0.2 μm; b, 13.8 ± 0.2 μm; n = 5, N = 5), as assessed by measuring the cross-sectional area of microscopic cell images.

Bottom Line: Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD) and elevation of caspase-3/7 activity.In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα.Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan. kumamp@belle.shiga-med.ac.jp.

ABSTRACT
Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis (OA). Recently, we reported that activation of volume-sensitive Cl- current (ICl,vol) mediates cell shrinkage, triggering apoptosis in rabbit articular chondrocytes. A cyclooxygenase (COX) blocker is frequently used for the treatment of OA. In the present study, we examined in vitro effects of selective blockers of COX on the TNFα-induced activation of ICl,vol in rabbit chondrocytes using the patch-clamp technique. Exposure of isolated chondrocytes to TNFα resulted in an obvious increase in membrane Cl- conductance. The TNFα-evoked Cl- current exhibited electrophysiological and pharmacological properties similar to those of ICl,vol. Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD) and elevation of caspase-3/7 activity. In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα. Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

Show MeSH
Related in: MedlinePlus