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 (Dym). The activities of the mitochondrial respiratory chain (MRC) complexes (complex I: NADH CoQ 1 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 Dym was measured by 5,59,6,69-tetracholoro-1,19,3,39-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 Dym 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.

Mitochondrial dysfunction activates cyclooxygenase 2 expression in cultured normal human chondrocytes

“…We recently demonstrated that OA chondrocytes, as compared with normal chondrocytes, display a significant decrease in activity of the MRC complexes II and III and a reduction in mitochondrial membrane potential (10). We also found that various key mediators involved in the pathogenesis of OA, such as the cytokines TNF␣ and IL-1␤ or the reactive nitrogen intermediate nitric oxide (NO), modulate the activity of different complexes of the MRC in cultured chondrocytes (24,25). However, the activity of the MRC is low in articular chondrocytes, due to the absence of vascularization and a hypoxic environment, as compared with that in vascularized tissue, and most of the ATP molecules arise from the glycolysis pathway.…”

“…To examine the effect of mitochondrial dysfunction on COX-2 protein expression and PGE 2 synthesis, normal human chondrocytes were treated with several concentrations of different inhibitors of mitochondrial respiration commonly utilized to induce inhibition of the MRC. We selected rotenone, NPA, antimycin A, sodium azide, and oligomycin as inhibitors of complexes I, II, III, IV, and V, respectively (10,11,(24)(25)(26)(27).…”

“…Our laboratory and other investigators have used inhibitors of the MRC to mimic degenerative disorders (11,12,(24)(25)(26)(27). Specifically, we have previously shown that 1) antimycin A induces a high percentage of depolarized cells in cultured chondrocytes (11), 2) the individual inhibition of mitochondrial complex IV with sodium azide modifies the mitochondrial membrane potential and induces apoptosis (25), and 3) rotenone (an inhibitor of complex I) causes a significant reduction in the content of proteoglycans in the extracellular matrix of normal cartilage explants (24). Johnson et al have also demonstrated, in cultured articular chondrocytes, that antimycin A and oligomycin attenuate collagen and proteoglycan synthesis and block transforming growth factor ␤ from increasing the levels of intracellular ATP (12).…”

“…The cells were treated with rotenone (10 g/ml, 25 g/ml, and 50 g/ml), 3-nitropropionic acid (NPA; 0.5 mM, 2 mM, and 10 mM), antimycin A (20 g/ml, 40 g/ml, and 60 g/ml), sodium azide (2 mM, 10 mM, and 25 mM), or oligomycin (5 g/ml, 10 g/ml, and 50 g/ml; all from SigmaAldrich) for various times, to inhibit mitochondrial complexes I, II, III, IV, and V, respectively (10,11,(24)(25)(26)(27). IL-1␤ (5 ng/ml; Sigma-Aldrich) and H 2 O 2 (100 M; Sigma-Aldrich) were used as positive controls.…”

Protective effect of Shenmai injection on knee articular cartilage of osteoarthritic rabbits and IL-1β-stimulated human chondrocytes

“…Excessive production of oxidants such as NO may occur due to the IL-1β-induced expression of iNOS (43)(44)(45). In addition, IL-1β stimulates COX-2 expression to increase synthesis of PGE2, which is responsible for joint pain in KOA (46,47).…”