Rapid Effects of Hypoxia on H+ Homeostasis in Articular Chondrocytes

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.