The effect of prolonged hypotonicity on chondrocyte volume regulation and intracellular calcium signalling

Niwa Ali, Ala Qusous, Stephen Getting, Mark Kerrigan

University of Westminster, London, UK

Chondrocytes, present in articular cartilage, are exposed to anisotonic conditions in vivo through joint loading and articulation. Alterations in cell volume occur as a result of such changes and as optimal matrix metabolism is linked to cell volume, chondrocytes possess the capacity to rapidly re-equilibriate to their surrounding milieu by instigating the process of regulatory volume decrease (RVD). Intracellular calcium ([Ca2+]i) transients are proposed to act as mediators in initiating the activation of specific membrane transporters implicated in RVD (Kerrigan and Hall, 2008).

RT-PCR was used to determine the expression of the TRPV family of membrane channels, that have been associated with an [Ca2+]i influx in response to hypotonicity in many other cell types. Changes to volume and [Ca2+]i in a human chondrocytic cell line (C20/A4) were recorded via 4D multiple excitation fluorescent microscopy (MEFM) and volume regulation by confocal laser scanning microscopy (CLSM) in response to a 50% hypotonic challenge (Kerrigan et al., 2006). In distinct experiments, cells were plated at 1 x 106/well in 12 well plates and incubated under chronic hypotonic (96 hour; 140mOsm) and isotonic (280mOsm) conditions, respectively, to determine changes in [Ca2+]i via a fluorescent plate-reader in response to the pharmacological inhibitor REV5901 (50μM; a 5-lipoxygenase inhibitor and peptidoleukotriene antagonist) that disables short-term volume regulatory capacity of chondrocytes. Data are expressed as mean ± SD; *p <0.05 vs control, n = 4-6 at three determinations.

Following 96 hour chronic hypotonic challenge, RT-PCR revealed that hypotonic treated (HTT) cells displayed a ∼14-fold increase in TRPV4 expression (P < 0.05; n = 3). MEFM showed a sustained rise in [Ca2+]i that appeared to correlate with the hypotonic challenge rather than with the onset of volume recovery, suggesting [Ca2+]i may be involved in RVD. Chondrocyte volume recovery (t1/2) measured by CLSM was 9.90 ± 0.96 min. HTT cells were more sensitive to fluid-flow-induced [Ca2+]i mobilisation than were isotonic treated cells (p<0.001). The REV5901 induced a [Ca2+]i rise in isotonic cells (9.3%) and HTT cells (3.7%) was significantly attenuated in the presence of ruthenium red (RR; 75 μM; p <0.001 for both), an inhibitor of TRPV channels, suggesting TRPV-mediated [Ca2+]i influx. Upon the removal of [Ca2+]o and in the presence of EGTA (2 mM), 74.83 ± 0.25% (control cells) and 46 ± 0.54% (HTT cells) of the [Ca2+]i rise (p<0.001 for both) was significantly attenuated, suggesting a switch in the calcium signalling pathway following long-term hypotonicity.

Together these data suggest that REV 5901, an inhibitor of RVD, caused a rise in [Ca2+]i that differed in HTT cells suggesting that following long-term hypotonicity, volume recovery is initiated via an [Ca2+]i-dependant osmolyte flux mediated by TRPV4. As osteoarthritis is associated with an increased tissue hydration, these findings could have an impact on the treatment of this debilitating syndrome.

Kerrigan, M.J.P., Regulatory volume increase (RVI) by in situ and isolated bovine articular chondrocytes. Journal of Cellular Physiology, 2006. 209(2): p. 481-492.

Kerrigan, M.J.P. & Hall, A.C. Control of chondrocyte regulatory volume decrease (RVD) by [Ca2+](i) and cell shape. Osteoarthritis and Cartilage, 2008. 16(3): p. 312-322.