Articular chondrocytes survive in an extracellular matrix rich in negatively charged proteoglycans, subject to substantial mechanical stress. This environment presents the challenge of high, fluctuating levels of hydrostatic pressure, osmolality, and [H⁺], and relative hypoxia (Wilkins et al. 2000). During galloping or jumping, the equine metacarpophalangeal (or fetlock) joint must withstand particularly high pressures. We report the first experiments to investigate the effect of some of these physicochemical variables on free [Ca²⁺]_i in chondrocytes from this joint.

Articular chondrocytes were isolated by collagenase digestion of cartilage slices taken aseptically from the equine fetlock joint (animal was killed humanely for other purposes, by barbiturate overdose). Cells were loaded with the Ca²⁺-sensitive fluorophore fura-2 (fura-2 AM, 5 mM). [Ca²⁺]_i was measured in cell suspensions incubated in a thermostatically regulated (37 °C) cuvette fluorimeter equipped with a magnetic stirrer (Ex. 340 nm/380 nm, Em. 510 nm; Browning & Wilkins, 2002). Following hypotonic shock (osmolality reduced from 290 to 145 mosmol kg^-1), [Ca²⁺]_i increased by 216 ± 66 nM (Fig. 1, trace a). The rise in free [Ca²⁺]_i was: (i) dependent on hypotonicity, trace b; (ii) greater when free [Ca²⁺]_o was increased from 2 to 5 mM, trace c; (iii) inhibited in Ca²⁺-free solutions (nominally 0 [Ca²⁺]_i plus 100 mM EGTA), trace d; and (iv) retarded by gadolinium (10 µM, an inhibitor of stretch-activated channels), trace e. In addition, thapsigargin (intracellular store Ca²⁺ pump inhibitor) and rises in pH_o both elevated free [Ca²⁺]_i.

These findings indicate that free [Ca²⁺]_i in equine chondrocytes responds to changes in osmolality, pH and [Ca²⁺]_o, in a similar way to that observed previously in human chondrocytes (Browning & Wilkins, 2002). The response appears to require entry through channels, although intracellular stores are also present and provide an additional source of Ca²⁺. Free [Ca²⁺]_i in chondrocytes is an important parameter, a change in whose magnitude affects many cell parameters including matrix synthesis. The equine preparation described here represents a valuable one in which to study further the mechanisms responsible.

This work was funded by the Arthritis Research Campaign, UK.

All procedures accord with current UK legislation.