title: The effect of O2 tension on pH homeostasis in equine articular chondrocytes.
abstract: OBJECTIVE
  To determine the effects of varying O(2) on pH homeostasis, based on the hypothesis that the function of articular chondrocytes is best understood at realistic O(2) tensions.
  
  
  METHODS
  Cartilage from equine metacarpophalangeal/tarsophalangeal joints was digested with collagenase to isolate chondrocytes, and then loaded with the pH-sensitive fluorophore 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein. The radioisotope(22)Na(+) was used to determine the kinetics of Na(+)/H(+) exchange (NHE) and the activity of the Na(+)/K(+) pump, and ATP levels were assessed with luciferin assays. Levels of reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescein diacetate.
  
  
  RESULTS
  The pH homeostasis was unaffected when comparing tissue maintained at 20% O(2) (the level in water-saturated air at 37 degrees C) with that at 5% O(2) (which approximates the normal level in healthy cartilage); however, an O(2) tension of <5% caused a fall in intracellular pH (pH(i)) and slowed pH(i) recovery following acidification, an effect mediated via inhibition of NHE activity (likely through acid extrusion by NHE isoform 1). The Na(+)/K(+) pump activity and intracellular ATP concentration were unaffected by hypoxia, but the levels of ROS were reduced. Hypoxic inhibition of NHE activity and the reduction in ROS levels were reversed by treatment with H(2)O(2), Co(2+), or antimycin A. Treatment with calyculin A also prevented hypoxic inhibition of NHE activity.
  
  
  CONCLUSIONS
  The ability of articular chondrocytes to carry out pH homeostasis is compromised when O(2) tensions fall below those normally experienced, via inhibition of NHE. The putative signal is a reduction in levels of ROS derived from mitochondria, acting via altered protein phosphorylation. This effect is relevant to both physiologic and pathologic states of lowered O(2), such as in chronic inflammation.
authors.affiliation: University of Cambridge, Cambridge, UK.
authors.forename: P I
authors.initials: PI
authors.lastname: Milner
authors.forename: T P A
authors.initials: TP
authors.lastname: Fairfax
authors.forename: J A
authors.initials: JA
authors.lastname: Browning
authors.forename: R J
authors.initials: RJ
authors.lastname: Wilkins
authors.forename: J S
authors.initials: JS
authors.lastname: Gibson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology*; D015687:Cell Hypoxia / Q000502:physiology; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006706:Homeostasis / Q000187:drug effects / Q000502:physiology*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D017382:Reactive Oxygen Species; D017923:Sodium-Hydrogen Exchangers; C059041:calyculin A; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D010100:Oxygen