OBJECTIVE: Acid-sensing ion channels (ASICs) are members of the degenerin/epithelial sodium channel (DEG/ENaC) protein superfamily and play a critical role in acid-induced cell injury. In this study, we examined whether drugs such as amiloride that block ASICs could attenuate acid-induced apoptotic injury to articular chondrocytes. METHODS: Articular chondrocytes were isolated from Sprague-Dawley rats, and their phenotype was determined by toluidine blue and immunocytochemical staining. Articular chondrocyte viability assay was performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Apoptosis of chondrocytes was observed by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling method as well as propidium iodide labeling methods. Intracellular calcium ([Ca(2+)](i)) was analyzed by a Ca(2+)-imaging method. In addition, the expression levels of calpain and calcineurin in articular chondrocytes were examined by real-time PCR and immunocytochemical staining. The activity of caspase-3 was evaluated by spectrophotometric assays. RESULTS: Positive staining for glycosaminoglycan and collagen II was seen in articular chondrocytes. Blocking acid-sensing ion channels significantly decreased the cell death percentage and increased cell viability following acid exposure. After pretreated with amiloride, acid-induced [Ca(2+)](i) rises were reduced. Amiloride also inhibited calpain and calcineurin expression levels in acid-induced chondrocytes, and inhibited caspase-3 activity. CONCLUSION: The data presented in this study provided some experimental evidence that blocking ASICs could protect acid-induced apoptotic injury to chondrocytes.
OBJECTIVE: Acid-sensing ion channels (ASICs) are members of the degenerin/epithelial sodium channel (DEG/ENaC) protein superfamily and play a critical role in acid-induced cell injury. In this study, we examined whether drugs such as amiloride that block ASICs could attenuate acid-induced apoptotic injury to articular chondrocytes. METHODS: Articular chondrocytes were isolated from Sprague-Dawley rats, and their phenotype was determined by toluidine blue and immunocytochemical staining. Articular chondrocyte viability assay was performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Apoptosis of chondrocytes was observed by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling method as well as propidium iodide labeling methods. Intracellular calcium ([Ca(2+)](i)) was analyzed by a Ca(2+)-imaging method. In addition, the expression levels of calpain and calcineurin in articular chondrocytes were examined by real-time PCR and immunocytochemical staining. The activity of caspase-3 was evaluated by spectrophotometric assays. RESULTS: Positive staining for glycosaminoglycan and collagen II was seen in articular chondrocytes. Blocking acid-sensing ion channels significantly decreased the cell death percentage and increased cell viability following acid exposure. After pretreated with amiloride, acid-induced [Ca(2+)](i) rises were reduced. Amiloride also inhibited calpain and calcineurin expression levels in acid-induced chondrocytes, and inhibited caspase-3 activity. CONCLUSION: The data presented in this study provided some experimental evidence that blocking ASICs could protect acid-induced apoptotic injury to chondrocytes.
Authors: H G Wang; N Pathan; I M Ethell; S Krajewski; Y Yamaguchi; F Shibasaki; F McKeon; T Bobo; T F Franke; J C Reed Journal: Science Date: 1999-04-09 Impact factor: 47.728
Authors: M J López-Armada; B Caramés; M Lires-Deán; B Cillero-Pastor; C Ruiz-Romero; F Galdo; F J Blanco Journal: Osteoarthritis Cartilage Date: 2006-02-21 Impact factor: 6.576
Authors: Xu Dong Liao; Ai Hui Tang; Quan Chen; Hai Jing Jin; Cai Hong Wu; Lan-Ying Chen; Shi Qiang Wang Journal: Biochem Biophys Res Commun Date: 2003-10-17 Impact factor: 3.575
Authors: Whasil Lee; Holly A Leddy; Yong Chen; Suk Hee Lee; Nicole A Zelenski; Amy L McNulty; Jason Wu; Kellie N Beicker; Jeffrey Coles; Stefan Zauscher; Jörg Grandl; Frederick Sachs; Farshid Guilak; Wolfgang B Liedtke Journal: Proc Natl Acad Sci U S A Date: 2014-11-10 Impact factor: 11.205