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°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. Conclusion.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.

Inhibition of Hypoxia-inducible Factor-targeting Prolyl Hydroxylase Domain-containing Protein 2 (PHD2) Enhances Matrix Synthesis by Human Chondrocytes

“…The tissue is therefore dependent on diffusion of oxygen from the synovial fluid on one side and the vascularized underlying bone on the other. As a result, articular cartilage (at least in larger animals and man) is maintained in a low oxygen environment (1-5% oxygen tension) throughout life (3)(4)(5). There is now significant evidence that hypoxia is a critical parameter in promoting the chondrocyte phenotype.…”

Osteopontin inhibits HIF-2α mRNA expression in osteoarthritic chondrocytes

“…To the best of our knowledge, the current study is the first to report this association. Articular cartilage is an avascular connective tissue in which the availability of oxygen and glucose is significantly lower compared with that in the synovial fluid and plasma (11). Oxygen and nutrient maintenance is critical to cell fate, senescence and apoptosis (21).…”

“…Thus, articular cartilage is maintained in a low oxygen environment in the body (11). Chondrocytes are therefore adapted to these hypoxic conditions.…”

Cellular Physiology of Articular Cartilage in Health and Disease

“…Since extracellular pH has a potent influence on cellular function (Das et al, 2010) any effect on the ability of the cell to regulate intracellular pH is likely to result in alteration in chondrocyte function, including matrix synthesis. Very low levels of oxygen, likely to be experienced in joint disease, reduce the activity of NHE resulting in intracellular acidosis in articular chondrocytes (Milner et al, 2006).…”