title: 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.
  
  
  CONCLUSIONS
  The data presented in this study provided some experimental evidence that blocking ASICs could protect acid-induced apoptotic injury to chondrocytes.
authors.affiliation: College of Pharmacy, Anhui Medical University, 81 Meishan Road, Hefei, 230032 Anhui, People's Republic of China. huweiyf@163.com
authors.forename: Wei
authors.initials: W
authors.lastname: Hu
authors.forename: Fei-Hu
authors.initials: FH
authors.lastname: Chen
authors.forename: Feng-Lai
authors.initials: FL
authors.lastname: Yuan
authors.forename: Teng-Yue
authors.initials: TY
authors.lastname: Zhang
authors.forename: Fan-Rong
authors.initials: FR
authors.lastname: Wu
authors.forename: Chao
authors.initials: C
authors.lastname: Rong
authors.forename: Sheng
authors.initials: S
authors.lastname: Jiang
authors.forename: Jie
authors.initials: J
authors.lastname: Tang
authors.forename: Cheng-Cheng
authors.initials: CC
authors.lastname: Zhang
authors.forename: Mei-Ying
authors.initials: MY
authors.lastname: Lin
mesh_terms: D062565:Acid Sensing Ion Channels; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D006851:Hydrochloric Acid; D008297:Male; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels
chemical_list: D062565:Acid Sensing Ion Channels; D009419:Nerve Tissue Proteins; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D000584:Amiloride; D019703:Calcineurin; D002154:Calpain; D053148:Caspase 3; D006851:Hydrochloric Acid; D002118:Calcium