The role of mitochondrial reactive oxygen species in pH regulation in articular chondrocytes  P.I. Milner, Ph.D., B.V.Sc., B.Sc. (Hons.), M.R.C.V.S., R.J. Wilkins, D.Phil., B.A. (Hons.), J.S. Gibson, Ph.D., M.A., B.A. (Hons.), Vet.M.B., M.R.C.V.S.  Osteoarthritis and Cartilage  Volume 15, Issue 7, Pages 735-742 (July 2007) DOI: 10.1016/j.joca.2007.01.008 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 (a) Effect of hydrogen peroxide and antimycin A on ROS levels and on acid efflux from isolated equine chondrocytes. ROS levels were measured as DCF fluorescence, and acid efflux (JH, mMmin−1) determined from the pH recovery following acid loading, converted to an acid equivalent flux using the intracellular buffering capacity. Cells were incubated at 20% or 1% O2 tension for 3h, in the presence or absence of H2O2 (100μM) or antimycin A (50μM). Values are given as percentages of the values measured in control cells at 20% O2. Histograms represent means±s.e.m., n=3–11. (b) Correlation between ROS levels and acid efflux from isolated equine chondrocytes. n=33, r2=0.65. Values are given as percentages of the values measured in control cells at 20% O2. Asterisks (*) indicate significant differences, P<0.05, with control values at 20%. Osteoarthritis and Cartilage 2007 15, 735-742DOI: (10.1016/j.joca.2007.01.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effect of rotenone on ROS levels and acid efflux from isolated equine chondrocytes. (a) ROS levels were measured as DCF fluorescence (given as percentages of the values measured in control cells at 20% O2), and (b) acid efflux (JH, mMmin−1) determined from the pH recovery following an acid load. Cells were incubated at 20% or 1% O2 tension for 3h, in the presence or absence of rotenone (1–100μM). Histograms represent means±s.e.m., n=3. For acid efflux, rotenone significantly reduced values compared to controls at 20%; for DCF, rotenone inhibition is significant at concentrations of 10μM and above at 20%, and at 100μM at 1%. Osteoarthritis and Cartilage 2007 15, 735-742DOI: (10.1016/j.joca.2007.01.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effect of myxothiazol on ROS levels and acid efflux from isolated equine chondrocytes. (a) ROS levels were measured as DCF fluorescence (given as percentages of the values measured in control cells at 20% O2), and (b) acid efflux (JH, mMmin−1) determined from the pH recovery following an acid load. Cells were incubated at 20% or 1% O2 tension for 3h, in the presence or absence of myxothiazol (0.1–10μM). Acid efflux was measured in the presence or absence of amiloride (100μM), allowing calculation of the amiloride-sensitive flux. Histograms represent means±s.e.m., n=3–5. Asterisks (*) indicate significant differences, P<0.05, comparing with 20% controls for DCF, and for amiloride-sensitive controls for acid efflux. Osteoarthritis and Cartilage 2007 15, 735-742DOI: (10.1016/j.joca.2007.01.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of DPI on ROS levels and acid efflux from isolated equine chondrocytes. (a) ROS levels were measured as DCF fluorescence (given as percentages of the values measured in control cells at 20% O2), and (b) acid efflux (JH, mMmin−1) determined from the pH recovery following an acid load. Cells were incubated at 20% or 1% O2 tension for 3h, in the presence or absence of DPI (10μM). Histograms represent means±s.e.m., n=4. Asterisks (*) indicate significant differences, P<0.05, compared to controls at 20%. Osteoarthritis and Cartilage 2007 15, 735-742DOI: (10.1016/j.joca.2007.01.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 (a) Effect of hypoxia and antimycin A on mitochondrial membrane potential. Mitochondrial membrane potential was measured using JC-1 fluorescence, expressed as the ratio of emission (EM 590nm/EM 525nm; EX 490nm) normalised to that at 20% O2 (the red/green emission ratio at 20% O2 tension was 47±13). Cells were incubated at 20% or 1% O2 and at 1% in the presence of antimycin A (50μM). Histograms represent means±s.e.m., n=3–5, given as percentages of the values measured in control cells at 20% O2. (b) Effect of inhibitors of protein kinases and phosphatases on ROS levels and acid efflux in isolated equine chondrocytes. ROS levels were measured as DCF fluorescence and acid efflux (JH, mMmin−1) determined from the pH recovery following acid loading. Cells were incubated at 20% or 1% O2 tension for 3h, in the presence or absence of calyculin A, staurosporine or wortmanin. Histograms represent means±s.e.m., n=3–6. Asterisks (*) indicate significant differences, P<0.05, compared to 20% controls. Osteoarthritis and Cartilage 2007 15, 735-742DOI: (10.1016/j.joca.2007.01.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions