In response to a hypo-osmotic challenge, cartilage cells (chondrocytes) regulate their volume primarily by the activation of an ‘osmolyte channel’ (Kerrigan & Hall, 2000). The sensor(s) involved are unclear, and therefore in the present study we examined whether there is a role for the actin cytoskeleton in mediating chondrocyte RVD.
Full-depth cartilage explants were excised aseptically from bovine metacarpal phalangeal joints (obtained from the local abattoir) and chondrocytes isolated as described into DMEM (380 mosmol; Hall et al. 1996). For measurements of cell volume and [Ca2+]i, chondrocytes were incubated with fura-2 AM and imaged using a PTI Imagemaster system (Kerrigan & Hall, 2000). The actin cytoskeleton was labelled with Phalloidin-FITC and DNase-Texas Red for F- and G-actin, respectively, and visualised using a Zeiss CLSM510 confocal microscope. To examine the role of the intact actin cytoskeleton in RVD, chondrocytes were incubated with latrunculin B (2-5 mM for up to 30 min; Wakatsuki et al. 2001). Data were collected for 2 min (380 mosmol) under constant perfusion and then a hypo-osmotic challenge (220 mosmol) applied. There was no significant difference (P > 0.05; Student’s unpaired t test) in either the fluorescence change (8.9 ± 1.5% 9.5 ± 0.8% data are means ± S.E.M.) or the time taken for maximal swelling between control and latrunculin B treated cells (n = 5 joints, 85 cells). In parallel, there was a transient rise in [Ca2+]i that was not inhibited by latrunculin. RVD proceeded linearly with no significant difference (P > 0.05) in either the rate (t 1/2 = 5.2 ± 1.6 and 5.2 ± 0.7 min, respectively) or % cells responding (61 ± 6 and 61 ± 12 %) with complete volume recovery within 10 min. Confocal imaging of the actin cytoskeleton showed that under control conditions the F-actin was arranged cortically, whereas latrunculin addition disrupted this arrangement. The addition of REV 5901 (50 mM), a potent inhibitor of RVD, did not disrupt the actin cytoskeleton.
Rearrangement of the actin cytoskeleton might be a step in activating chondrocyte RVD. However, the present work suggests that when the cytoskeleton is disrupted, RVD is unaffected and thus an intact actin cytoskeleton is not necessarily required.
This work was supported by the Arthritis Research Campaign (H0621) and the MRC.