The effect of O2 tension on pH homeostasis in equine articular chondrocytes
Version of Record online: 30 OCT 2006
Copyright © 2006 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 54, Issue 11, pages 3523–3532, November 2006
How to Cite
Milner, P. I., Fairfax, T. P. A., Browning, J. A., Wilkins, R. J. and Gibson, J. S. (2006), The effect of O2 tension on pH homeostasis in equine articular chondrocytes. Arthritis & Rheumatism, 54: 3523–3532. doi: 10.1002/art.22209
- Issue online: 30 OCT 2006
- Version of Record online: 30 OCT 2006
- Manuscript Accepted: 9 AUG 2006
- Manuscript Received: 23 MAY 2006
- Horserace Betting Levy Board
- Royal Society
- Sir Halley Stewart Trust
- Medical Research Council UK Studentship
To determine the effects of varying O2 on pH homeostasis, based on the hypothesis that the function of articular chondrocytes is best understood at realistic O2 tensions.
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 radioisotope22Na+ 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.
The pH homeostasis was unaffected when comparing tissue maintained at 20% O2 (the level in water-saturated air at 37°C) with that at 5% O2 (which approximates the normal level in healthy cartilage); however, an O2 tension of <5% caused a fall in intracellular pH (pHi) and slowed pHi 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 H2O2, Co2+, or antimycin A. Treatment with calyculin A also prevented hypoxic inhibition of NHE activity.
The ability of articular chondrocytes to carry out pH homeostasis is compromised when O2 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 O2, such as in chronic inflammation.