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Effect of nitric oxide on mitochondrial activity of human synovial cells.

Cillero-Pastor B, Martin MA, Arenas J, López-Armada MJ, Blanco FJ - BMC Musculoskelet Disord (2011)

Bottom Line: The time course analyses of treatment with SNP at 0.5 mM demonstrated that treatment reliably and significantly reduced intracellular ATP production (68.34 ± 14.3% vs. basal = 100% at 6 hours; *p < 0.05).The analysis of the MRC at 48 hours showed that SNP at 0.5 mM increased the activity of complexes I (basal = 36.47 ± 3.92 mol/min/mg protein, SNP 0.5 mM = 58.08 ± 6.46 mol/min/mg protein; *p < 0.05) and III (basal = 63.87 ± 6.93 mol/min/mg protein, SNP 0.5 mM = 109.15 ± 30.37 mol/min/mg protein; *p < 0.05) but reduced CS activity (basal = 105.06 ± 10.72 mol/min/mg protein, SNP at 0.5 mM = 66.88 ± 6.08 mol/min/mg protein.; *p < 0.05), indicating a decrease in mitochondrial mass.This study suggests that NO reduces the survival of OA synoviocytes by regulating mitochondrial functionality, as well as the proteins controlling the cell cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Osteoarticular and Aging Research Unit, Biomedical Research Center, INIBIC, CH Universitario da Coruña, Xubias 84, 15006, A Coruña, Spain.

ABSTRACT

Background: Nitric oxide (NO) is a messenger implicated in the destruction and inflammation of joint tissues. Cartilage and synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) have high levels of NO. NO is known to modulate various cellular pathways and, thus, inhibit the activity of the mitochondrial respiratory chain (MRC) of chondrocytes and induce the generation of reactive oxygen species (ROS) and cell death in multiple cell types. For these reasons, and because of the importance of the synovial membrane in development of OA pathology, we investigated the effects of NO on survival, mitochondrial function, and activity of fibroblastic human OA synovial cells.

Methods: Human OA synovia were obtained from eight patients undergoing hip joint replacement. Sodium nitroprusside (SNP) was used as a NO donor compound and cell viability was evaluated by MTT assays. Mitochondrial function was evaluated by analyzing the mitochondrial membrane potential (Δψm) with flow cytometry using the fluorofore DePsipher. ATP levels were measured by luminescence assays, and the activities of the respiratory chain complexes (complex I: NADH CoQ₁ reductase, complex II: succinate dehydrogenase, complex III: ubiquinol-cytochrome c reductase, complex IV: cytochrome c oxidase) and citrate synthase (CS) were measured by enzymatic assay. Protein expression analyses were performed by western blot.

Results: SNP at a concentration of 0.5 mM induced cell death, shown by the MTT method at different time points. The percentages of viable cells at 24, 48 and 72 hours were 86.11 ± 4.9%, 74.31 ± 3.35%, and 43.88 ± 1.43%, respectively, compared to the basal level of 100% (*p < 0.05). SNP at 0.5 mM induced depolarization of the mitochondrial membrane at 12 hours with a decrease in the ratio of polarized cells (basal = 2.48 ± 0.28; SNP 0.5 mM = 1.57 ± 0.11; *p < 0.01). The time course analyses of treatment with SNP at 0.5 mM demonstrated that treatment reliably and significantly reduced intracellular ATP production (68.34 ± 14.3% vs. basal = 100% at 6 hours; *p < 0.05). The analysis of the MRC at 48 hours showed that SNP at 0.5 mM increased the activity of complexes I (basal = 36.47 ± 3.92 mol/min/mg protein, SNP 0.5 mM = 58.08 ± 6.46 mol/min/mg protein; *p < 0.05) and III (basal = 63.87 ± 6.93 mol/min/mg protein, SNP 0.5 mM = 109.15 ± 30.37 mol/min/mg protein; *p < 0.05) but reduced CS activity (basal = 105.06 ± 10.72 mol/min/mg protein, SNP at 0.5 mM = 66.88 ± 6.08 mol/min/mg protein.; *p < 0.05), indicating a decrease in mitochondrial mass. Finally, SNP regulated the expression of proteins related to the cellular cycle; the NO donor decreased bcl-2, mcl-1 and procaspase-3 protein expression.

Conclusions: This study suggests that NO reduces the survival of OA synoviocytes by regulating mitochondrial functionality, as well as the proteins controlling the cell cycle.

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Related in: MedlinePlus

ATP production of synoviocytes stimulated with sodium nitroprusside (SNP). Synoviocytes were cultured in basal conditions (only RPMI) or treated with SNP at 0.5 mM for 6, 24, 48 and 72 hours and collected for whole cell ATP measurement as described in the Methods section. ATP values are expressed as the mean ± standard error of eight different experiments performed in duplicate (*p < 0.05).
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Figure 3: ATP production of synoviocytes stimulated with sodium nitroprusside (SNP). Synoviocytes were cultured in basal conditions (only RPMI) or treated with SNP at 0.5 mM for 6, 24, 48 and 72 hours and collected for whole cell ATP measurement as described in the Methods section. ATP values are expressed as the mean ± standard error of eight different experiments performed in duplicate (*p < 0.05).

Mentions: We also assessed the effects of SNP on ATP production. Treatment with SNP at 0.5 mM for 6, 24, 48 and 72 hours significantly decreased ATP production [6 hours: 68.34 ± 14.3%; 24 hours: 75.04 ± 13.32%; 48 hours: 75.28 ± 12.94%; and 72 hours: 75.54 ± 16.3%, compared to the basal value of 100%, (n = 8) *p < 0.05)] (figure 3). These results indicate that SNP affects mitochondrial function, therefore, we evaluated the MRC activity of human OA synoviocytes treated with SNP at 0.5 mM for 48 hours. Figure 4 shows that the NO donor induced an increase in activity of complex I (basal: 36.47 ± 3.92 mol/min/mg prot.; SNP at 0.5 mM: 58.08 ± 6.46 mol/min/mg prot.) and of complex III (basal: 63.87 ± 6.93 mol/min/mg prot.; SNP at 0.5 mM: 109.15 ± 30.37 mol/min/mg prot.), (n = 6, *p < 0.05). However, SNP at 0.5 mM produced a decrease in the activity of CS [basal: 105.06 ± 10.72 mol/min/mg prot.; SNP at 0.5 mM: 66.88 ± 6.08 mol/min/mg prot., (n = 6, *p < 0.05)], which indicates a reduction in mitochondrial mass.


Effect of nitric oxide on mitochondrial activity of human synovial cells.

Cillero-Pastor B, Martin MA, Arenas J, López-Armada MJ, Blanco FJ - BMC Musculoskelet Disord (2011)

ATP production of synoviocytes stimulated with sodium nitroprusside (SNP). Synoviocytes were cultured in basal conditions (only RPMI) or treated with SNP at 0.5 mM for 6, 24, 48 and 72 hours and collected for whole cell ATP measurement as described in the Methods section. ATP values are expressed as the mean ± standard error of eight different experiments performed in duplicate (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045396&req=5

Figure 3: ATP production of synoviocytes stimulated with sodium nitroprusside (SNP). Synoviocytes were cultured in basal conditions (only RPMI) or treated with SNP at 0.5 mM for 6, 24, 48 and 72 hours and collected for whole cell ATP measurement as described in the Methods section. ATP values are expressed as the mean ± standard error of eight different experiments performed in duplicate (*p < 0.05).
Mentions: We also assessed the effects of SNP on ATP production. Treatment with SNP at 0.5 mM for 6, 24, 48 and 72 hours significantly decreased ATP production [6 hours: 68.34 ± 14.3%; 24 hours: 75.04 ± 13.32%; 48 hours: 75.28 ± 12.94%; and 72 hours: 75.54 ± 16.3%, compared to the basal value of 100%, (n = 8) *p < 0.05)] (figure 3). These results indicate that SNP affects mitochondrial function, therefore, we evaluated the MRC activity of human OA synoviocytes treated with SNP at 0.5 mM for 48 hours. Figure 4 shows that the NO donor induced an increase in activity of complex I (basal: 36.47 ± 3.92 mol/min/mg prot.; SNP at 0.5 mM: 58.08 ± 6.46 mol/min/mg prot.) and of complex III (basal: 63.87 ± 6.93 mol/min/mg prot.; SNP at 0.5 mM: 109.15 ± 30.37 mol/min/mg prot.), (n = 6, *p < 0.05). However, SNP at 0.5 mM produced a decrease in the activity of CS [basal: 105.06 ± 10.72 mol/min/mg prot.; SNP at 0.5 mM: 66.88 ± 6.08 mol/min/mg prot., (n = 6, *p < 0.05)], which indicates a reduction in mitochondrial mass.

Bottom Line: The time course analyses of treatment with SNP at 0.5 mM demonstrated that treatment reliably and significantly reduced intracellular ATP production (68.34 ± 14.3% vs. basal = 100% at 6 hours; *p < 0.05).The analysis of the MRC at 48 hours showed that SNP at 0.5 mM increased the activity of complexes I (basal = 36.47 ± 3.92 mol/min/mg protein, SNP 0.5 mM = 58.08 ± 6.46 mol/min/mg protein; *p < 0.05) and III (basal = 63.87 ± 6.93 mol/min/mg protein, SNP 0.5 mM = 109.15 ± 30.37 mol/min/mg protein; *p < 0.05) but reduced CS activity (basal = 105.06 ± 10.72 mol/min/mg protein, SNP at 0.5 mM = 66.88 ± 6.08 mol/min/mg protein.; *p < 0.05), indicating a decrease in mitochondrial mass.This study suggests that NO reduces the survival of OA synoviocytes by regulating mitochondrial functionality, as well as the proteins controlling the cell cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Osteoarticular and Aging Research Unit, Biomedical Research Center, INIBIC, CH Universitario da Coruña, Xubias 84, 15006, A Coruña, Spain.

ABSTRACT

Background: Nitric oxide (NO) is a messenger implicated in the destruction and inflammation of joint tissues. Cartilage and synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) have high levels of NO. NO is known to modulate various cellular pathways and, thus, inhibit the activity of the mitochondrial respiratory chain (MRC) of chondrocytes and induce the generation of reactive oxygen species (ROS) and cell death in multiple cell types. For these reasons, and because of the importance of the synovial membrane in development of OA pathology, we investigated the effects of NO on survival, mitochondrial function, and activity of fibroblastic human OA synovial cells.

Methods: Human OA synovia were obtained from eight patients undergoing hip joint replacement. Sodium nitroprusside (SNP) was used as a NO donor compound and cell viability was evaluated by MTT assays. Mitochondrial function was evaluated by analyzing the mitochondrial membrane potential (Δψm) with flow cytometry using the fluorofore DePsipher. ATP levels were measured by luminescence assays, and the activities of the respiratory chain complexes (complex I: NADH CoQ₁ reductase, complex II: succinate dehydrogenase, complex III: ubiquinol-cytochrome c reductase, complex IV: cytochrome c oxidase) and citrate synthase (CS) were measured by enzymatic assay. Protein expression analyses were performed by western blot.

Results: SNP at a concentration of 0.5 mM induced cell death, shown by the MTT method at different time points. The percentages of viable cells at 24, 48 and 72 hours were 86.11 ± 4.9%, 74.31 ± 3.35%, and 43.88 ± 1.43%, respectively, compared to the basal level of 100% (*p < 0.05). SNP at 0.5 mM induced depolarization of the mitochondrial membrane at 12 hours with a decrease in the ratio of polarized cells (basal = 2.48 ± 0.28; SNP 0.5 mM = 1.57 ± 0.11; *p < 0.01). The time course analyses of treatment with SNP at 0.5 mM demonstrated that treatment reliably and significantly reduced intracellular ATP production (68.34 ± 14.3% vs. basal = 100% at 6 hours; *p < 0.05). The analysis of the MRC at 48 hours showed that SNP at 0.5 mM increased the activity of complexes I (basal = 36.47 ± 3.92 mol/min/mg protein, SNP 0.5 mM = 58.08 ± 6.46 mol/min/mg protein; *p < 0.05) and III (basal = 63.87 ± 6.93 mol/min/mg protein, SNP 0.5 mM = 109.15 ± 30.37 mol/min/mg protein; *p < 0.05) but reduced CS activity (basal = 105.06 ± 10.72 mol/min/mg protein, SNP at 0.5 mM = 66.88 ± 6.08 mol/min/mg protein.; *p < 0.05), indicating a decrease in mitochondrial mass. Finally, SNP regulated the expression of proteins related to the cellular cycle; the NO donor decreased bcl-2, mcl-1 and procaspase-3 protein expression.

Conclusions: This study suggests that NO reduces the survival of OA synoviocytes by regulating mitochondrial functionality, as well as the proteins controlling the cell cycle.

Show MeSH
Related in: MedlinePlus