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


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.

  • CS, citrate synthase
  • DMEM, Dulbecco’s modified Eagle’s medium
  • Δψm, mitochondrial membrane potential
  • JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide
  • KP, phosphate buffer
  • MOPS, 4-morpholinepropane sulphonic acid
  • MRC, mitochondrial respiratory chain
  • NaN3, sodium azide
  • NO, nitric oxide
  • OA, osteoarthritis
  • PBS, phosphate buffered saline
  • PI, propidium iodide
  • RNS, reactive nitrogen species
  • ROS, reactive oxygen species
  • RPA, ribonuclease protection analysis
  • SDS, sodium dodecyl sulphate
  • SNP, sodium nitroprusside, TNFα, tumour necrosis factor α
  • chondrocytes
  • nitric oxide
  • apoptosis
  • mitochondria
  • osteoarthritis

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