The effect of O2 tension on pH homeostasis in equine articular chondrocytes

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 Na-22(+) 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 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. 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.