Effect of nitric oxide on mitochondrial activity of human synovial cells

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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 CoQ₁reductase, 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.</p> <p>Results</p> <p>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% (*<it>p </it>< 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; *<it>p </it>< 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; *<it>p </it>< 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; *<it>p </it>< 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; *<it>p </it>< 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.; *<it>p </it>< 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.</p> <p>Conclusions</p> <p>This study suggests that NO reduces the survival of OA synoviocytes by regulating mitochondrial functionality, as well as the proteins controlling the cell cycle.</p

Analysis of Transcription Complexes and Effects of Ligands by Microelectrospray Ionization Mass Spectrometry

The human vitamin D receptor (VDR) and retinoid X receptor-alpha (RXRalpha) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in the presence of their known endogenous ligands 1alpha,25-dihydroxyvitamin D3 (1, 25-[OH]2D3) and 9-cis-retinoic acid (9-c-RA). We used rapid buffer exchange gel filtration in conjunction with microelectrospray ionization mass spectrometry (microESI-MS) to study the binding of these receptors to the osteopontin vitamin D response element (OP VDRE). In the absence of DNA, both VDR and RXRalpha existed primarily as monomers, but in the presence of OP VDRE, homodimeric RXRalpha and heterodimeric RXRalpha-VDR complexes were shown to bind OP VDRE. Addition of 9-c-RA increased RXRalpha homodimer-OP VDRE complexes, and addition of 1,25-(OH) 2D3 resulted in formation of 1, 25-(OH)2D 3-VDR-RXRalpha-OP VDRE complexes. Addition of low-affinity binding ligands had no detectable effect on the VDR-RXRalpha-OP VDRE transcription complex. These results demonstrate the utility of microESI-MS in analyzing multimeric, high-molecular-weight protein-protein and protein-DNA complexes, and the effects of ligands on these transcriptional complexes