Communications - Epithelia & Membrane Transport PC 1 Regulation of Na + - H + exchange

The avascular nature of articular cartilage means that chondrocytes are dependent on the diffusion of O2 from the synovial fluid and subchondral bone. As a result, cartilage has a relatively low ambient O2 tension, estimated at between 5% and 7% O2. In addition, synovial, and cartilage, O2 tensions can fall to very low levels in inflammatory conditions. Low O2 tensions can affect ion homeostasis: cells acidify and Na+-H+ exchange (NHE) is inhibited (Milner et al. 2005). Reactive oxygen species (ROS) have been implicated in the response to altered O2 tension (Chandeel & Schumacker, 2000). In the present study, we investigated their possible role in O2 sensitivity of NHE in articular chondrocytes. Cartilage slices were obtained postmortem from bovine and equine metacarpophalangeal joints. Chondrocytes, isolated overnight by collagenase digestion at ambient levels of O2, were incubated for 3 h at 20% (termed normoxia) or 1% O2 (termed hypoxia). Intracellular pH (pHi) was then determined fluorimetrically using BCECF, using intracellular buffering to calculate proton efflux (JH in mmol H + min-1) following cell acidification (Wilkins & Hall, 1995). Production of reactive oxygen species (ROS), estimated using dichlorofluorescein fluorescence (Chandeel & Schumacker, 2000). O2 deprivation caused a significant acidification of steady-state pHi and also slowed recovery of pH, through inhibition of amiloride-sensitive NHE. Under these conditions, production of ROS was reduced by 41 ± 6% (n = 7) cf values at 20% O2 (p < 0.05). Following treatment with antimycin A (10-100 μM), ROS levels were increased from 59±9 to 84±7 (NS cf 20%). With antimycin A, JH increased from 1.90±0.15 mmol min -1 (p<0.05 cf 20%) to 2.51±0.17 (N.S. cf 20%), compared with 2.42±0.12 at 20% O2. Similar findings were observed following treatment with Co2+ (100 μM). NHE activity could therefore be correlated with ROS levels. Finally, when exposed to the protein phosphatase inhibitor calyculin A (100 nM), NHE activity (JH) was 3.29±0.22 at 1% O2, compared with 3.13±0.18 at 20% O2. Calyculin A had no effect on ROS levels. These findings suggest that NHE activity in articular chondrocytes is inhibited by low O2 tension, with the putative signal being a reduction in ROS. The signalling cascade coupling ROS levels with NHE activity appears to act via protein phosphorylation. The importance of these results to the chondrocyte in vivo is discussed.