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.

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Articular chondrocytes experience low oxygen (O(2)) levels compared with many other tissues, and values fall further in disease states. Chondrocyte intracellular pH (pH(i)) is a powerful modulator of matrix synthesis and is principally regulated by Na(+)-H(+) exchange (NHE). In equine chondrocytes, NHE is inhibited when cells are incubated for 3 h at low O(2), leading to intracellular acidosis. O(2)-dependent changes in reactive oxygen species (ROS) levels appear to underlie this effect. The present study examines whether hypoxia can influence chondrocyte NHE activity and pH(i) over shorter timescales using the pH-sensitive fluoroprobe BCECF in cells isolated not only from equine cartilage but also from bovine tissue. O(2) levels in initially oxygenated solutions gassed with N(2) fell to approximately 1% within 2 h. A progressive fall in pH(i) and acid extrusion capacity was observed, with statistically significant effects (P < 0.05) apparent within 3 h. For equine and bovine cell populations subjected to step change in O(2) by resuspension in hypoxic (1%) solutions, a decline in acid extrusion and pH(i) was observed within 10 min and continued throughout the recording period. This effect represented inhibition of the NHE-mediated fraction of acid extrusion. Cells subjected to hypoxic solutions supplemented with CoCl(2) (100 microM) or antimycin A (100 microM) to raise levels of ROS did not acidify. The conserved nature and rapidity of the response to hypoxia has considerable implications for chondrocyte homeostasis and potentially for the maintenance of cartilage integrity.

Reasons for performing study: Ca2+ homeostasis in articular c h o n d rocytes affects synthesis and degradation of the cartilage matrix, as well as other cellular functions, thereby contributing to joint integrity. Although it will be affected by mechanical loading, the sensitivity of intracellular Ca 2+ concentration ([Ca 2+ ] i ) in equine articular chondrocytes to many stimuli remains unknown. Hypothesis: An improved understanding of Ca 2 + homeostasis in equine art i c u l a r c h o n d rocytes, and how it is a l t e red during joint loading and pathology, will be i m p o rtant in understanding how joints respond to mechanical loads. Methods: [Ca 2+ ] i was determined using the fluorophore fura-2. We examined the effects of hypotonic shock, a perturbation experienced in vivo during mechanical loading cycles. We used inhibitors of Ca 2+ transporters to ascertain the important factors in Ca 2+ homeostasis. R e s u l t s : U n d e r isotonic conditions, [Ca 2 + ] i was 148 ± 23 nmol/l, increasing by 216 ± 66 nmol/l in response to reduction in extracellular osmolality of 50%. Resting [ C a 2 + ] i , and the increase following hypotonic shock, were d e c reased by Ca 2 + removal; they were both elevated when e x t r a c e l l u l a r [ C a 2 + ] ([Ca 2 + ] o ) was raised or following Na + removal. The hypotonicity-induced rise in [Ca 2 + ] i w a s inhibited by exposure of cells to gadolinium (Gd 3 + ; 10 µmol/l), an inhibitor of mechanosensitive channels. [ C a 2 + ] i was also elevated following treatment of cells with thapsigargin (10 µmol/l), an inhibitor of the Ca 2 + pump of i n t r a c e l l u l a r s t o re s . Conclusions: A model is presented which interprets these findings in relation to Ca 2+ homeostasis in equine articular chondrocytes, including the presence of mechanosensitive channels allowing Ca 2+ entry, a Na + /Ca 2+ exchanger for removal of intracellular C a 2 + and intracellular s t o re s sensitive to thapsigargin. Potential relevance: A m o re complete understanding of Ca 2 + homeostasis in equine chondrocytes may allow development of future therapeutic regimes to ameliorate joint disease.