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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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Effect of sodium nitroprusside (SNP) on the cell viability of human osteoarthritic (OA) synoviocytes. Human OA synoviocytes were incubated under basal conditions (RPMI only) or with SNP at concentrations of 0.5, 1 and 2 mM for 24, 48 and 72 hours. Cell viability was evaluated using a colorimetric analysis based on the MTT assay as detailed in the Methods section. The data are expressed as percentages of those of the control conditions (100%), and represent the mean ± standard error of eight different experiments performed in duplicate (*p < 0.05).
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Figure 1: Effect of sodium nitroprusside (SNP) on the cell viability of human osteoarthritic (OA) synoviocytes. Human OA synoviocytes were incubated under basal conditions (RPMI only) or with SNP at concentrations of 0.5, 1 and 2 mM for 24, 48 and 72 hours. Cell viability was evaluated using a colorimetric analysis based on the MTT assay as detailed in the Methods section. The data are expressed as percentages of those of the control conditions (100%), and represent the mean ± standard error of eight different experiments performed in duplicate (*p < 0.05).

Mentions: Analyses of viability using MTT assay methods to measure the cellular reduction power showed that the NO donor, SNP, reduced synoviocyte viability with a time-dependent decrease in the percentages (figure 1). At 24, 48 and 72 hours of incubation, respectively: SNP at 0.5 mM: 86.11 ± 4.9%, 74.31 ± 3.35%, 43.88 ± 1.43% vs. 100% basal; SNP at 1 mM: 85.52 ± 5.91%, 11.91 ± 0.72%, 7.1 ± 0.41% vs. 100% basal; and SNP at 2 mM: 77.85 ± 6.46%, 15 ± 0.52%, 10.85 ± 0.56% vs. basal 100%, ([n = 8]; *p < 0.05).


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)

Effect of sodium nitroprusside (SNP) on the cell viability of human osteoarthritic (OA) synoviocytes. Human OA synoviocytes were incubated under basal conditions (RPMI only) or with SNP at concentrations of 0.5, 1 and 2 mM for 24, 48 and 72 hours. Cell viability was evaluated using a colorimetric analysis based on the MTT assay as detailed in the Methods section. The data are expressed as percentages of those of the control conditions (100%), and represent 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 1: Effect of sodium nitroprusside (SNP) on the cell viability of human osteoarthritic (OA) synoviocytes. Human OA synoviocytes were incubated under basal conditions (RPMI only) or with SNP at concentrations of 0.5, 1 and 2 mM for 24, 48 and 72 hours. Cell viability was evaluated using a colorimetric analysis based on the MTT assay as detailed in the Methods section. The data are expressed as percentages of those of the control conditions (100%), and represent the mean ± standard error of eight different experiments performed in duplicate (*p < 0.05).
Mentions: Analyses of viability using MTT assay methods to measure the cellular reduction power showed that the NO donor, SNP, reduced synoviocyte viability with a time-dependent decrease in the percentages (figure 1). At 24, 48 and 72 hours of incubation, respectively: SNP at 0.5 mM: 86.11 ± 4.9%, 74.31 ± 3.35%, 43.88 ± 1.43% vs. 100% basal; SNP at 1 mM: 85.52 ± 5.91%, 11.91 ± 0.72%, 7.1 ± 0.41% vs. 100% basal; and SNP at 2 mM: 77.85 ± 6.46%, 15 ± 0.52%, 10.85 ± 0.56% vs. basal 100%, ([n = 8]; *p < 0.05).

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