Effect of nitric oxide on mitochondrial respiratory activity of human articular chondrocytes.
OBJECTIVE
To investigate the effect of nitric oxide (NO) on mitochondrial activity and its relation with the apoptosis of human articular chondrocytes.


METHODS
Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities, and mitochondrial membrane potential (Delta psi m). The activities of the mitochondrial respiratory chain (MRC) complexes (complex I: NADH CoQ(1) reductase, complex II: succinate dehydrogenase, complex III: ubiquinol cytochrome c reductase, complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from normal cartilage. The Delta psi m was measured by 5,5',6,6'-tetracholoro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide (JC-1) using flow cytometry. Apoptosis was analysed by flow cytometry. The mRNA expression of caspases was analysed by ribonuclease protection analysis and the detection of protein synthesis by western blotting. Sodium nitroprusside (SNP) was used as an NO compound donor.


RESULTS
SNP at concentrations higher than 0.5 mmol/l for 24 hours induced cellular changes characteristic of apoptosis. SNP elicited mRNA expression of caspase-3 and caspase-7 and down regulated bcl-2 synthesis in a dose and time dependent manner. Furthermore, 0.5 mM SNP induced depolarisation of the mitochondrial membrane at 5, 12, and 24 hours. Analysis of the MRC showed that at 5 hours, 0.5 mM SNP reduced the activity of complex IV by 33%. The individual inhibition of mitochondrial complex IV with azide modified the Delta psi m and induced apoptosis.


CONCLUSIONS
This study suggests that the effect of NO on chondrocyte survival is mediated by its effect on complex IV of the MRC.
Maneiro
E
E
Laboratory of Investigation, Rheumatology Division, Juan Canalejo Hospital, C/Xubias 84, 15006-A Coruña, Spain.
López-Armada
M J
MJ
de Andres
M C
MC
Caramés
B
B
Martín
M A
MA
Bonilla
A
A
Del Hoyo
P
P
Galdo
F
F
Arenas
J
J
Blanco
F J
FJ
D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000187:drug effects; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019069:Cell Respiration / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport / Q000187:drug effects; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology
D020030:Nitric Oxide Donors; D009599:Nitroprusside; D009569:Nitric Oxide