Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism

Abstract

Introduction: The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way.

Methods: Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (Δψm) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[(3)H]glucose (2-[(3)H]DG). Statistical analysis was performed using the Mann-Whitney U test.

Results: NOC-12 liberates approximately ten times more NO2- than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ΔΨm and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[(3)H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[(3)H]DG than healthy cells.

Conclusions: These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO.

Figure 1

Nitric oxide (NO) donors differ from the amount of NO₂^- released. NO₂^- accumulation in supernatants of normal chondrocytes treated with different NO donors for 24 hours in standard culture conditions. The lines show the dose-dependent increase of NO accumulation in all cases. Values are the mean ± SD; n = 5. SNP, sodium nitroprusside; NOC-12, N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine.

Figure 2

Nitric oxide (NO) effect on chondrocyte apoptosis. (A) Cell death levels in chondrocytes treated with different NO donors was determined by the iodide propidium (PI) method. Control chondrocytes were treated with 1 mM sodium nitroprusside (SNP) and 1 mM N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Data are expressed as a percentage of apoptotic (hypodiploid) nuclei. Values are the mean ± SD; n = 5. *P ≤ 0.05 versus untreated chondrocytes (control). (B) Cellular changes induced by NO on normal human chondrocytes were analysed by 49,6-diamino-2-phenylindole dihydrochloride (DAPI staining) and fluorescence microscopy. Shown is a representative example of five experiments.

Figure 3

Effect of nitric oxide (NO) donors on mitochondrial membrane potential. (A) Fluorescence-activated cell sorter analysis of mitochondrial membrane potential in human chondrocytes. Untreated and treated normal chondrocytes with NO donors were stained with 5,5',6,6'- tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide (JC-1) and analysed by flow cytometry. Photomultiplier settings were adjusted to detect JC-1 monomer fluorescence signals on the filter 1 (FL1) detector (green fluorescence) and JC-1 aggregate fluorescence signals on the FL2 detector (red fluorescence). Shown is an example of chondrocytes treated with 1 mM SNP and 2 mM N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. (B) Quantification of red and green fluorescence. Histograms represent the JC-1 fluorescence of normal cells and those treated with NO donors. Green fluorescence (open graph) increases, whereas red fluorescence (solid graph) decreases in the NOC-12 and sodium nitroprusside (SNP)-treated chondrocytes, suggesting a reduction of the mitochondrial membrane potential, and therefore, a decrease in the red/green ratio. Shown is an example at 24 hours. Results are the mean ± SD; n = 5. *P ≤ 0.05 versus untreated chondrocytes (control).

Figure 4

ATP generation by normal chondrocytes treated with different nitric oxide (NO) donors. Articular chondrocytes were treated with 1 mM sodium nitroprusside (SNP) and 2 mM N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Then the ATP assay was performed as described in Materials and methods. Values are the mean ± SD; n = 7. *P ≤ 0.05 versus untreated chondrocytes (control).

Figure 5

Nitric oxide (NO) influence on 2-deoxy-(³H) glucose uptake by normal chondrocytes. Effect of the NO donors sodium nitroprusside (SNP) and N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) on the uptake of 2-deoxy-(³H) glucose by normal chondrocytes for 15 minutes. The detailed procedure is described in Materials and methods. Values are the mean ± SD; n = 7. *P ≤ 0.05 versus untreated chondrocytes (control).

Figure 6

The effect of nitric oxide (NO) on chondrocyte viability depends on glucose levels. Articular chondrocytes were cultured in medium with crescent glucose concentrations and treated with 1 mM sodium nitroprusside (SNP) and 1 mM N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Data are expressed as a percentage of apoptotic (hypodiploid) nuclei. Values are the mean ± SD; n = 3.

Figure 7

Osteoarthritis (OA) chondrocytes are efficient cells in the uptake of glucose. Differences between human articular normal and OA chondrocytes in the uptake of 2-deoxy-(³H)glucose for 1 hour. The detailed procedure is described in Materials and methods. Values are the mean ± SD; n = 12. *P ≤ 0.05 versus normal chondrocytes.