Glutamine protects articular chondrocytes from heat stress and NO-induced apoptosis with HSP70 expression.
OBJECTIVE
To investigate the effect of l-glutamine (Gln) on stress responses of chondrocytes exposed to heat stress or nitric oxide (NO).


METHODS
Cultures of articular chondrocytes were established from rabbit joints, and treated for 12h with various concentrations of Gln (0-20 mM). In some experiments, cells were also treated with quercetin (Que), a heat shock protein 70 (HSP70) inhibitor. Heat stress (43 degrees C) was applied to the cells for 0-120 min. Apoptosis was induced by 0.5mM sodium nitroprusside (SNP) dihydrate that produces NO. After stress loading, HSP70 expression was detected by Western blot analysis. Cell viability was assessed by lactate dehydrogenase (LDH) release and tetrazolium salt-based assays, while apoptosis was evaluated by Hoechst 33342 staining, TUNEL methods and active caspase-3 determination.


RESULTS
Gln demonstrated dose-dependent enhancing effect on stress-mediated induction of HSP70, while in the absence of any stress HSP70 was not induced by Gln alone. After heating or SNP loading, chondrocytes showed severe reduction in viability, while the cytotoxic outcome was almost completely abrogated by conditioning with Gln. The protective effect of Gln was significantly blocked by Que that effectively suppressed stress-induced HSP70 expression in chondrocytes. The Gln also rendered chondrocytes unsusceptible to NO-induced apoptosis that was frequently seen in SNP-treated culture.


CONCLUSIONS
This study demonstrated that the treatment of chondrocytes with Gln protected the cells from heat stress and NO-induced apoptosis. These chondroprotective effects of Gln may be mediated by HSP70.
Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kyoto 602-8566, Japan.
H
H
Tonomura
K A
KA
Takahashi
O
O
Mazda
Y
Y
Arai
A
A
Inoue
R
R
Terauchi
M
M
Shin-Ya
T
T
Kishida
J
J
Imanishi
T
T
Kubo
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002358:Cartilage, Articular / Q000187:drug effects* / Q000473:pathology; D053148:Caspase 3; D020169:Caspases / Q000032:analysis; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000502:physiology; D053938:DNA Fragmentation / Q000187:drug effects; D005973:Glutamine / Q000494:pharmacology*; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors; D006358:Hot Temperature; D008297:Male; D009569:Nitric Oxide / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D011794:Quercetin / Q000494:pharmacology; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000494:pharmacology; D013312:Stress, Physiological / Q000503:physiopathology
D000893:Anti-Inflammatory Agents; D018840:HSP70 Heat-Shock Proteins; D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; D005973:Glutamine; D009599:Nitroprusside; D009569:Nitric Oxide; D011794:Quercetin; D053148:Caspase 3; D020169:Caspases