Control of chondrocyte regulatory volume decrease (RVD) by [Ca2+]i and cell shape.
OBJECTIVE
Optimal matrix metabolism by articular chondrocytes is controlled by the 'set-point' volume which is determined mainly by membrane transporters. The signal transduction pathway(s) for the key membrane transporter which responds to cell swelling ('osmolyte channel') and mediates regulatory volume decrease (RVD) is poorly understood, so here the role of Ca2+ and the effects of 2D culture have been clarified.


METHODS
Changes to the volume and intracellular calcium levels ([Ca2+]i) of freshly isolated and 2D cultured bovine articular chondrocytes subjected to hypotonic challenge using a 43% reduction in medium osmolarity were studied by single-cell fluorescence microscopy. The effects of ethylene glycol tetraacetic acid (EGTA), REV5901 and Gd(3+) were studied and the role of Ca2+ influx determined by Mn2+ quench.


RESULTS
In freshly isolated cells, approximately 50% of chondrocytes exhibited 'robust RVD' (6[120]). RVD was inhibited by REV 5901 (4+/-2% responding) (3[23]) and 2 mM EGTA (18+/-5% responding) (4[166]) whereas Gd3+ had no effect (3[89]). The hypotonic challenge resulted in a Gd3+-insensitive rise in [Ca2+]i that did not correlate with RVD in all cells. Following 2D culture, chondrocytes also demonstrated Gd3+-insensitive RVD, but in contrast, the [Ca2+]i rise was blocked by this agent.


CONCLUSIONS
The data suggested that in freshly isolated and 2D cultured chondrocytes, the rise in [Ca2+]i occurring during hypotonic challenge could be related to RVD, but only in some cells. However, with 2D culture, the Ca2+ response switched to being Gd3+-sensitive, suggesting that as a result of changes to chondrocyte shape, stretch-activated cation channels although present, do not appear to play a role in volume regulation.
Kerrigan
M J P
MJ
School of Biosciences, Department of Human and Health Sciences, University of Westminster, London, UK. M.J.Kerrigan@wmin.ac.uk
Hall
A C
AC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D002358:Cartilage, Articular / Q000166:cytology; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002468:Cell Physiological Phenomena / Q000187:drug effects*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D016257:Fura-2 / Q000494:pharmacology; D005682:Gadolinium / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009994:Osmolar Concentration; D011804:Quinolines / Q000494:pharmacology; D015398:Signal Transduction
D015220:Calcium Channels; D007038:Hypotonic Solutions; D011804:Quinolines; C049740:alpha-pentyl-3-(2-quinolinylmethoxy)benzenemethanol; D004533:Egtazic Acid; D005682:Gadolinium; D002118:Calcium; D016257:Fura-2