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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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Measurement of mitochondrial membrane depolarization following sodium nitroprusside (SNP) treatment of human osteoarthritic (OA) synoviocytes. A) Cells were incubated for 12 hours in medium alone (control), with 0.5 mM SNP, or with 1 μM valinomycin (positive control). The mitochondrial membrane potential was determined by flow cytometry using DePsipher. Data acquisition was performed using a FACScan flow cytometer as detailed in the Methods section. A representative density plot for each treatment group is shown. The numbers represent the percentage of the mean ± standard error of each population from eight different experiments performed in duplicate (*p < 0.01). B) Histograms represent DePsipher fluorescence of SNP-stimulated synoviocytes. Relative to the control, green fluorescence (light line) increases while red fluorescence (dark line) decreases in SNP- or valinomycin-stimulated synoviocytes. The numbers represent the mean ± standard error of red/green fluorescence ratio values of eight different experiments performed in duplicate (*p < 0.01).
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Figure 2: Measurement of mitochondrial membrane depolarization following sodium nitroprusside (SNP) treatment of human osteoarthritic (OA) synoviocytes. A) Cells were incubated for 12 hours in medium alone (control), with 0.5 mM SNP, or with 1 μM valinomycin (positive control). The mitochondrial membrane potential was determined by flow cytometry using DePsipher. Data acquisition was performed using a FACScan flow cytometer as detailed in the Methods section. A representative density plot for each treatment group is shown. The numbers represent the percentage of the mean ± standard error of each population from eight different experiments performed in duplicate (*p < 0.01). B) Histograms represent DePsipher fluorescence of SNP-stimulated synoviocytes. Relative to the control, green fluorescence (light line) increases while red fluorescence (dark line) decreases in SNP- or valinomycin-stimulated synoviocytes. The numbers represent the mean ± standard error of red/green fluorescence ratio values of eight different experiments performed in duplicate (*p < 0.01).

Mentions: A second set of experiments examined the effects of SNP on mitochondrial function by determining the Δψm, ATP synthesis and activities of the respiratory chain complexes I-IV and CS. To assess the effect of SNP on the Δψm of synoviocytes, the fluoresecent probe DePsipher was used. The staining pattern of DePsipher for basal synoviocytes was established as the standard. The total cell population was divided into two subsets: one with normal Δψm (red fluorescence or high levels of FL-2), as the left upper panels represent in figure 2A, and another with lower Δψm (green fluorescence or high levels of FL-1), as the right lower panels represent in figure 2A. The treatment of synoviocytes with SNP at 0.5 mM for 12 hours reduced the percentage of cells with normal mitochondrial polarization [basal: 48.08 ± 3.59%; SNP at 0.5 mM: 36.04 ± 2.77%; and valinomycin at 1 μM: 17.04 ± 2.38% (n = 8, *p < 0.01)] (Figure 2A) and increased the number of cells with mitochondrial depolarization [basal: 6.76 ± 2.14%; SNP at 0.5 mM: 9.96 ± 1.45%; and valinomycin at 1 μM: 26.87 ± 2.92%, (n = 8, *p < 0.01)] (Figure 2A). SNP treatment at 0.5 mM caused a significant decrease in the red/green fluorescence ratio (basal: 2.48 ± 0.28; SNP at 0.5 mM: 1.57 ± 0.11; and valinomycin at 1 μM: 0.93 ± 0.06, (n = 8, *p < 0.01)] (figure 2B).


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)

Measurement of mitochondrial membrane depolarization following sodium nitroprusside (SNP) treatment of human osteoarthritic (OA) synoviocytes. A) Cells were incubated for 12 hours in medium alone (control), with 0.5 mM SNP, or with 1 μM valinomycin (positive control). The mitochondrial membrane potential was determined by flow cytometry using DePsipher. Data acquisition was performed using a FACScan flow cytometer as detailed in the Methods section. A representative density plot for each treatment group is shown. The numbers represent the percentage of the mean ± standard error of each population from eight different experiments performed in duplicate (*p < 0.01). B) Histograms represent DePsipher fluorescence of SNP-stimulated synoviocytes. Relative to the control, green fluorescence (light line) increases while red fluorescence (dark line) decreases in SNP- or valinomycin-stimulated synoviocytes. The numbers represent the mean ± standard error of red/green fluorescence ratio values of eight different experiments performed in duplicate (*p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Measurement of mitochondrial membrane depolarization following sodium nitroprusside (SNP) treatment of human osteoarthritic (OA) synoviocytes. A) Cells were incubated for 12 hours in medium alone (control), with 0.5 mM SNP, or with 1 μM valinomycin (positive control). The mitochondrial membrane potential was determined by flow cytometry using DePsipher. Data acquisition was performed using a FACScan flow cytometer as detailed in the Methods section. A representative density plot for each treatment group is shown. The numbers represent the percentage of the mean ± standard error of each population from eight different experiments performed in duplicate (*p < 0.01). B) Histograms represent DePsipher fluorescence of SNP-stimulated synoviocytes. Relative to the control, green fluorescence (light line) increases while red fluorescence (dark line) decreases in SNP- or valinomycin-stimulated synoviocytes. The numbers represent the mean ± standard error of red/green fluorescence ratio values of eight different experiments performed in duplicate (*p < 0.01).
Mentions: A second set of experiments examined the effects of SNP on mitochondrial function by determining the Δψm, ATP synthesis and activities of the respiratory chain complexes I-IV and CS. To assess the effect of SNP on the Δψm of synoviocytes, the fluoresecent probe DePsipher was used. The staining pattern of DePsipher for basal synoviocytes was established as the standard. The total cell population was divided into two subsets: one with normal Δψm (red fluorescence or high levels of FL-2), as the left upper panels represent in figure 2A, and another with lower Δψm (green fluorescence or high levels of FL-1), as the right lower panels represent in figure 2A. The treatment of synoviocytes with SNP at 0.5 mM for 12 hours reduced the percentage of cells with normal mitochondrial polarization [basal: 48.08 ± 3.59%; SNP at 0.5 mM: 36.04 ± 2.77%; and valinomycin at 1 μM: 17.04 ± 2.38% (n = 8, *p < 0.01)] (Figure 2A) and increased the number of cells with mitochondrial depolarization [basal: 6.76 ± 2.14%; SNP at 0.5 mM: 9.96 ± 1.45%; and valinomycin at 1 μM: 26.87 ± 2.92%, (n = 8, *p < 0.01)] (Figure 2A). SNP treatment at 0.5 mM caused a significant decrease in the red/green fluorescence ratio (basal: 2.48 ± 0.28; SNP at 0.5 mM: 1.57 ± 0.11; and valinomycin at 1 μM: 0.93 ± 0.06, (n = 8, *p < 0.01)] (figure 2B).

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