The effective control of cell volume is an essential property of animal cells for the maintenance of normal cell metabolism. Changes to cell volume can initiate regulatory responses involving the stimulation of membrane transporters/channels. The efficacy of the response is a determinant of subsequent metabolic events and cell survival (Okada et al. 2001). The metabolism of cartilage matrix is detrimentally influenced by altering chondrocyte volume (Urban et al. 1993), although transport pathways could limit this effect. An understanding of the systems involved and their regulation is thus of interest. Here we have studied RVD and [Ca²⁺]_i of avian chondrocytes following hypotonic challenge.

Avian articular chondrocytes were aseptically isolated from explants taken from the legs of ~40-day-old chickens obtained from the local supermarket, into Dulbecco’s modified Eagle’s medium (380 mosmol l^-1). The cells were incubated with fura-2AM (5 µM, 30 min, 37 °C) while attaching to coverslips. Cell volume and [Ca²⁺]_i were measured by recording the emitted light (510 nm) following excitation of the dye alternately at 358/380 nm (Kerrigan & Hall, 2000). To study RVD, experiments were performed in a saline comprising (mM): NaCl (190), KCl (5), Hepes (15), glucose (10), MgCl₂ (1) and CaCl₂ (1) (pH 7.4, 380 mosmol l^-1) with the hypotonic challenge (220 mosmol l^-1, using reduced NaCl with the other constituents at the same concentrations) delivered by perfusion. In addition, a Ca²⁺-free saline (0 mM Ca²⁺ with 2 mM EGTA) and gadolinium saline (Gd³⁺; 100 µM, a blocker of stretch-activated channels) was also used.

Following hypotonic challenge, cell volume increased within 2 min with some chondrocytes showing RVD. Rates of RVD were compared by regression analysis (all data had r ² > 0.91) and extrapolation of the data to the standardised initial volume. There was no difference in the final volume 10 min after the challenge for cells in EGTA or Gd³⁺ saline when compared with controls (P > 0.05, Student’s unpaired t test; n = 10 joints [249 cells]) with an average volume recovery of 66 ± 8 % (mean ± S.E.M.). Hypotonicity resulted in a rise in [Ca²⁺]_i in some cells that occurred with cell swelling and then returned to basal levels as RVD proceeded. This rise was attenuated in the presence of EGTA, but Gd³⁺ had no effect (P > 0.05; n = 3 [121]). For chondrocytes within explants, these dynamics of the [Ca²⁺]_i change were also observed following hypotonic challenge.

The RVD response of avian chondrocytes was similar to that observed for bovine cells (Kerrigan & Hall, 2000) despite their evolutionary separation. Some chondrocytes are capable of RVD, although it is not clear whether a change to [Ca²⁺]_i is necessary, or a coincident event as reported by others (e.g. Moran et al. 1997). The lack of effect of Gd³⁺ on RVD and the rise in [Ca²⁺]_i suggests that stretch-sensitive ion channels are not involved.

M.J.K. was supported by an MRC studentship.

Kerrigan, M.J. & Hall, A.C. (2000). J. Physiol. 527.P, 42P.

Moran, J., Morales-Mulia, S., Hernandez-Cruz, A. & Pasantes-Morales, H. (1997). J. Neurosci. Res. 47, 144-145.

Okada, Y., Maeno, E., Shimizu, T., Dezaki, K., Wang, J. & Morishima, S. (2001). J. Physiol. 532, 3-16. abstract

Urban, J.P.G., Hall, A.C. & Gehl, K.A. (1993). J. Cell. Physiol. 154, 262-270.