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Logo of annrheumdAnnals of the Rheumatic DiseasesCurrent TOCInstructions for authors
Ann Rheum Dis. Mar 2005; 64(3): 388–395.
PMCID: PMC1755391

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

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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Selected References

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Figures and Tables

Figure 1
 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 ...
Figure 2
 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 ...
Figure 3
 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 ...
Figure 4
 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 ...
Figure 5
 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 ...
Figure 6
 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) ...
Figure 7
 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 ...
Figure 8
 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 ...

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