Blockade of acid-sensing ion channels protects articular chondrocytes from acid-induced apoptotic injury.

ABSTRACT

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.