Contribution of the Epithelial Sodium Channel to Chondrocyte Regulatory Volume Increase

Abstract

Chondrocytes are the cells of articular cartilage, responsible for the production and maintenance of the extracellular milieu. They exist in a constantly changing osmotic environment and to survive such osmotic changes they must be able to quickly and effectively regulate their volume. Cells use regulatory volume increase (RVI) to oppose osmotic shrinkage. It has previously been shown that the epithelial sodium channel (ENaC) is important to this process in rat hepatocytes1. Here we investigate the possible contribution of ENaC to RVI mechanisms in canine chondrocytes.Chondrocytes were isolated from cartilage according to standard methods2. Amiloride sensitive single-channel activity reversed at a membrane potential of −1±5mV (n = 5), mean conductance was 9±0.4pS (n = 5) and kinetics were slow. The calculated ENa under these conditions was −6mV, which coupled to the very small conductance would be consistent with this channel being an ENaC. Channel open probability in control conditions was 0.3±0.06 and decreased by 97±2% after application of the ENaC inhibitor, amiloride (10μM; n = 3).Upon exposure to hypertonic solution, cell volume decreased significantly by 35±3% (n = 5; p<0.001). Within 20 minutes of reaching their smallest size, cells under control conditions had returned to 92±4% of their original volume, not significantly different to starting volume (p=0.07). When 100nM benzamil, a specific ENaC inhibitor, was added to the hypertonic solution, cells shrank by 41±3% (n = 5) and were unable to return to their original volume.These data suggest that ENaC contributes to RVI in canine chondrocytes.1. F. Wehner, C. Bohmer, H. Heinzinger et al., Cellular Physiology and Biochemistry 10 (5-6), 335 (2000).2. R. Lewis, K. Asplin, G. Bruce et al., J Cell Physiol 226 (8) (2011).