The role of mitochondrial reactive oxygen species in pH regulation in articular chondrocytes.
Milner PI., Wilkins RJ., Gibson JS.
OBJECTIVE: To examine the effect of O(2) and the role, and source, of reactive oxygen species (ROS) on pH regulation in articular chondrocytes. METHODS: Cartilage from equine metacarpo/tarsophalangeal joints was digested (collagenase) to isolate chondrocytes and loaded with 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein, a pH-sensitive fluorophore. O(2) tension was maintained using Eschweiler tonometers and a Wosthoff gas mixer. Cells were exposed to agents which alter ROS levels, mitochondrial inhibitors and/or inhibitors of protein phosphorylation. ROS levels were determined by dichlorofluorescein and mitochondrial membrane potential measured using JC-1. RESULTS: pH homeostasis was dependent on ROS. Na(+)/H(+) exchanger (NHE) activity was inhibited at low O(2) tension (acid efflux reducing from 2.30+/-0.05 to 1.27+/-0.11mMmin(-1) at 1%). NHE activity correlated with ROS levels (r(2)=0.65). ROS levels were increased by antimycin A (with levels at 1% O(2) tension increasing from 59+/-9% of the value at 20% to 87+/-7%), but reduced by rotenone, myxothiazol and diphenyleneiodonium. Hypoxia induced depolarisation of the mitochondrial membrane potential (with JC-1 red-green fluorescence ratio at 1% O(2) tension decreasing to 40+/-10% of the value at 20%). The response to changes in O(2) and to antimycin A was inhibited by staurosporine, wortmanin and calyculin A. CONCLUSION: The fall in ROS levels in hypoxia reduces the ability of articular chondrocytes to regulate pH, inhibiting NHE activity via changes in protein phosphorylation. The site of ROS generation is likely to be mitochondrial electron transport chain complex III. These effects are important to understanding normal chondrocyte function and response to altered O(2) tension.