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. 2005 Mar;64(3):388–395. doi: 10.1136/ard.2004.022152

Effect of nitric oxide on mitochondrial respiratory activity of human articular chondrocytes

E Maneiro 1, M Lopez-Armada 1, M C de Andres 1, B Carames 1, M Martin 1, A Bonilla 1, P del Hoyo 1, F Galdo 1, J Arenas 1, F Blanco 1
PMCID: PMC1755391  PMID: 15708893

Abstract

Objective: To investigate the effect of nitric oxide (NO) on mitochondrial activity and its relation with the apoptosis of human articular chondrocytes.

Materials and methods: Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities, and mitochondrial membrane potential (Δψm). The activities of the mitochondrial respiratory chain (MRC) complexes (complex I: NADH CoQ1 reductase, complex II: succinate dehydrogenase, complex III: ubiquinol cytochrome c reductase, complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from normal cartilage. The Δψm was measured by 5,5',6,6'-tetracholoro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide (JC-1) using flow cytometry. Apoptosis was analysed by flow cytometry. The mRNA expression of caspases was analysed by ribonuclease protection analysis and the detection of protein synthesis by western blotting. Sodium nitroprusside (SNP) was used as an NO compound donor.

Results: SNP at concentrations higher than 0.5 mmol/l for 24 hours induced cellular changes characteristic of apoptosis. SNP elicited mRNA expression of caspase-3 and caspase-7 and down regulated bcl-2 synthesis in a dose and time dependent manner. Furthermore, 0.5 mM SNP induced depolarisation of the mitochondrial membrane at 5, 12, and 24 hours. Analysis of the MRC showed that at 5 hours, 0.5 mM SNP reduced the activity of complex IV by 33%. The individual inhibition of mitochondrial complex IV with azide modified the Δψm and induced apoptosis.

Conclusions: This study suggests that the effect of NO on chondrocyte survival is mediated by its effect on complex IV of the MRC.

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Figure 1.

Figure 1

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 Kinetics of the NO effect (SNP) on chondrocyte apoptosis. Cells (5x105 normal human chondrocytes), treated with SNP at different concentrations for 12, 24, and 48 hours were fixed in 70% ethanol at 4°C, then washed and incubated with RNase and PI for 15 minutes at room temperature in the dark. Data are expressed as a percentage of apoptotic (hypodiploid) nuclei. The graph shows that SNP dose dependently induces chondrocyte apoptosis.

Figure 2.

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 Cellular changes induced by NO on normal human chondrocytes. 4',6-Dianidino-2-phenylindole dihydrochloride staining analysed by fluorescence microscopy (A–C) and by the combination of fluorescence and light microscopy (D). (A) Untreated cells (control). The normal morphology of a chondrocyte nucleus. (B, C) Cells treated with SNP (2 mmol/l) for 12 hours. Both panels show the typical morphology of an apoptotic nucleus, condensation (B) and fragmentation (C). (D) Cells treated with SNP (2 mmol/l) for 24 hours, showing simultaneous changes in the cytoplasmic membrane (bubbles) and in the nucleus margination of DNA.

Figure 3.

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 The time course of NO on mRNA expression of the caspases in normal human chondrocytes. (A) Confluent chondrocytic cells were incubated for the indicated time intervals (6, 12, and 48 hours), both in basal conditions or in the presence of SNP (0.5 mmol/l) or tumour necrosis factor α (TNFα, 10 ng/ml). After incubation, total RNA was isolated and the mRNA expression of the caspases was analysed by the RPA as reported in "Materials and methods". This autoradiograph represents a total of three experiments. (B) Densitometric analysis of the bands (at 6 hours of incubation) was conducted by computerised laser densitometry and normalised to the housekeeping L-32 mRNA level. Values are expressed as the percentage over control. TNFα was used as a positive control and as a comparator stimulus.

Figure 4.

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 Effect of NO on mitochondrial activity. (A) Fluorescence activated cell sorter analysis of mitochondrial membrane potential in human chondrocytes. Untreated and treated normal chondrocytes (5x105) with NO donor (SNP 0.5, 1, and 2 mmol/l for 5, 12, and 24 hours) were stained with 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazole carbocyanide iodide (JC-1) and analysed by flow cytometry. Photomultiplier settings were adjusted to detect JC-1 monomer fluorescence signals on the filter 1 (FL1) detector (green fluorescence) and JC-1 aggregate fluorescence signals on the FL2 detector (red fluorescence). The study showed that chondrocytes could be classified into four subgroups A-D as described in the text. Shown is an example of chondrocytes treated with 2 mM SNP for 5 hours, which shows that SNP increases the population of cells with depolarised mitochondria and decreases that of cells with normal polarisation. (B) Quantification of red and green fluorescence. Histograms represent the JC-1 fluorescence of normal cells (left) and those treated with 2 mM SNP for 5 hours (right). Green fluorescence (open graph) increases while red fluorescence (solid graph) decreases in the SNP treated chondrocytes, suggesting a reduction of the mitochondrial membrane potential and, therefore, a decrease in the red/green ratio. Shown is an example at 5 hours. Results are the mean (SD) from six different experiments. (C) Quantification of depolarised chondrocytes. Untreated cells and cells treated (5x105 normal chondrocytes) with SNP at different concentrations for 5, 12, and 24 hours were analysed by flow cytometry on a FACScan (Becton and Dickinson, Mountain View, CA). The proportion of cells with mitochondrial depolarisation (group D) is greater in SNP treated chondrocytes than in untreated cells. Furthermore, SNP reduced the proportion of cells with normal polarisation (group A). Shown are the results of six experiments at 5 hours. Bars show the mean (SD). *p⩽0.01 versus untreated chondrocytes.

Figure 5.

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 The effect of NO on mitochondrial depolarisation is reversible. Cells (5x105 normal human chondrocytes) were treated with 2 mM SNP for 24 hours. The medium was then removed and cells were washed, and new medium without SNP was added. After 24 hours, cells were analysed by flow cytometry on a FACScan (Becton and Dickinson, Mountain View, CA). The proportion of cells with mitochondrial depolarisation (group D) was quantified. The effect of SNP on Δψm was reversible. Shown are the results of six experiments at 5 hours. Bars show the mean (SD).

Figure 6.

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 Treatment with NO reduced the activation of the bcl-2 protein on human chondrocytic cells. (A) Confluent OA chondrocytes were incubated for the indicated times (24, 72, and 120 hours) in basal conditions or in the presence of SNP (0.5 mmol/l) or TNFα (10 ng/ml). Aliquots of total cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotting was performed using the anti-bcl-2 antibody as described in "Materials and methods". This autoradiograph is representative of two experiments. (B) Densitometric analyses of the bands were conducted by computerised laser densitometry and normalised to tubuline. Values are expressed as the percentage over control. TNFα was used as a positive control and a comparator stimulus.

Figure 7.

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 Effect of MRC inhibitor on Δψm. (A) Normal human chondrocytes (5x105) were incubated with NaN3 for 5, 12, and 24 hours. Cells were then analysed by flow cytometry to quantify the Δψm. Results are shown as the ratio of red/green fluorescence. Shown are the results of six experiments at 5 hours. Bars show the mean (SD). p = 0.05 treated v untreated chondrocytes. See "Materials and methods" for a description of the complexes. (B) Quantification of depolarised chondrocytes. Untreated and treated cells (5 x 105 normal human chondrocytes) with NaN3 at different concentrations for 5, 12, and 24 hours were analysed by flow cytometry on a FACScan. NaN3 reduced the proportion of cells with normal polarisation in chondrocytes (group A) cultured in medium with and without glucose. However, the proportion of cells with mitochondrial depolarisation (group D) was higher in chondrocytes stimulated with NaN3 and cultured in medium without glucose. Shown are the results of six experiments at 5 hours. Bars show the mean (SD). *p⩽0.05 treated v untreated chondrocytes.

Figure 8.

Figure 8

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 Effect of an MRC inhibitor on chondrocyte apoptosis. Normal human chondrocytes (5x105) were cultured in DMEM and glucose-free DMEM, and incubated with NaN3 (a mitochondrial inhibitor of complex IV) for 12, 24, and 48 hours. Cells were then analysed by flow cytometry to quantify the percentage of apoptosis. Shown are the results of six experiments at 24 hours. Bars show the mean (SD). *p = 0.05 treated v untreated chondrocytes. See "Materials and methods" for a description of the technique.

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