| Summary: | <p>Abstract</p> <p>Background</p> <p>Articular chondrocytes normally experience a lower O<sub>2 </sub>tension compared to that seen by many other tissues. This level may fall further in joint disease. Ionic homeostasis is essential for chondrocyte function but, at least in the case of H<sup>+ </sup>ions, it is sensitive to changes in O<sub>2 </sub>levels. Ca<sup>2+ </sup>homeostasis is also critical but the effect of changes in O<sub>2 </sub>tension has not been investigated on this parameter. Here we define the effect of hypoxia on Ca<sup>2+ </sup>homeostasis in bovine articular chondrocytes.</p> <p>Methods</p> <p>Chondrocytes from articular cartilage slices were isolated enzymatically using collagenase. Cytoplasmic Ca<sup>2+ </sup>levels ([Ca<sup>2+</sup>]<sub>i</sub>) were followed fluorimetrically using Fura-2 to determine the effect of changes in O<sub>2 </sub>tension. The effects of ion substitution (replacing extracellular Na<sup>+ </sup>with NMDG<sup>+ </sup>and chelating Ca<sup>2+ </sup>with EGTA) were tested. Levels of reactive oxygen species (ROS) and the mitochondrial membrane potential were measured and correlated with [Ca<sup>2+</sup>]<sub>i</sub>.</p> <p>Results</p> <p>A reduction in O<sub>2 </sub>tension from 20% to 1% for 16-18 h caused [Ca<sup>2+</sup>]<sub>i </sub>to approximately double, reaching 105 ± 23 nM (p < 0.001). Ion substitutions indicated that Na<sup>+</sup>/Ca<sup>2+ </sup>exchange activity was not inhibited at low O<sub>2 </sub>levels. At 1% O<sub>2</sub>, ROS levels fell and mitochondria depolarised. Restoring ROS levels (with an oxidant H<sub>2</sub>O<sub>2</sub>, a non-specific ROS generator Co<sup>2+ </sup>or the mitochondrial complex II inhibitor antimycin A) concomitantly reduced [Ca<sup>2+</sup>]<sub>i</sub>.</p> <p>Conclusions</p> <p>O<sub>2 </sub>tension exerts a significant effect on [Ca<sup>2+</sup>]<sub>i</sub>. The proposed mechanism involves ROS from mitochondria. Findings emphasise the importance of using realistic O<sub>2 </sub>tensions when studying the physiology and pathology of articular cartilage and the potential interactions between O<sub>2</sub>, ROS and Ca<sup>2+</sup>.</p>
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