title: The progressive ankylosis gene product ANK regulates extracellular ATP levels in primary articular chondrocytes.
abstract: BACKGROUND
  Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process.
  
  
  METHODS
  We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release.
  
  
  RESULTS
  eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels.
  
  
  CONCLUSIONS
  These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.
authors.forename: Ann K
authors.initials: AK
authors.lastname: Rosenthal
authors.forename: Claudia M
authors.initials: CM
authors.lastname: Gohr
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mitton-Fitzgerald
authors.forename: Megan K
authors.initials: MK
authors.lastname: Lutz
authors.forename: George R
authors.initials: GR
authors.lastname: Dubyak
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Ryan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000844:Ankylosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D028061:Phosphate Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011339:Probenecid / Q000494:pharmacology; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D058848:Receptors, Purinergic P2X4 / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013552:Swine; D050916:TRPV Cation Channels / Q000819:agonists
chemical_list: C530602:N-(1-((4-(2-(((2,4-dichlorophenyl)sulfonyl)amino)-3-hydroxypropanoyl)-1-piperazinyl)carbonyl)-3-methylbutyl)-1-benzothiophene-2-carboxamide; D028061:Phosphate Transport Proteins; D058920:Purinergic P2X Receptor Antagonists; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; D013449:Sulfonamides; D050916:TRPV Cation Channels; D000255:Adenosine Triphosphate; D007930:Leucine; D011339:Probenecid; D002118:Calcium