Articular cartilage faces continuous fluctuations in load, which alter the physicochemical environment, including the osmolarity, of the matrix surrounding chondrocytes. Studies have shown that the turnover of cartilage can be modified by components of load, the effects being mediated by changes to the concentration of intracellular cations (Wilkins et al. 2000). Here we determine the effects of hypotonic shock (HTS) – as experienced by chondrocytes upon relaxation of static load – on [Ca²⁺]_i.

Bovine articular chondrocytes were isolated from metacarpophalangeal cartilage obtained at abattoir from 18- to 36-month-old cattle, using standard enzymatic digestion (Wilkins & Hall, 1995). [Ca²⁺]_i was measured using fura-2-loaded cells (fura-2 AM, 5 µM), in a thermostatically regulated (37 °C) fluorimeter (EX = 340 nm/380 nm, EM = 510 nm) in solutions of pH 7.4. The effects of HTS (osmolality reduced from 290 to 145 mosmol kg^-1) on [Ca²⁺]_i were recorded for 300 s, and the maximal change determined (mean ± S.E.M., n ▓ge│ 3). Significant differences were determined by Student’s unpaired t test.

HTS increased [Ca²⁺]_i to a sustained plateau within 100 s, with a maximal increase of 250 ± 15 % recorded. In Ca²⁺-free media (nominally 0 [Ca²⁺]_i plus 1 mM EGTA) there was a partial but significant inhibition of the rise (147 ± 7.50 %, P < 0.01); the same occurred when the cells were treated with thapsigargin (1 µM, 30 min pre-incubation; 143 ± 10 %, P < 0.01), to deplete intracellular stores, or with neomycin (10 mM), a phospholipase C inhibitor (125 ± 7.5 %, P < 0.01). A variety of inhibitors of potential Ca²⁺ influx pathways was tested (Fig. 1). NiCl₂ (2 mM), a non-specific inhibitor of Ca²⁺ channels, inhibited the response, as did GdCl₃ (10 µM), an inhibitor of stretch-activated channels (SACs). Ruthenium Red (100 µM), a specific inhibitor of the epithelial Ca²⁺ channel (ECaC) also attenuated the rise. Verapamil (100 µM), an inhibitor of L-type voltage-activated Ca²⁺ channels, and KBR7943, a specific inhibitor of sodium-calcium exchange reverse mode, had no effects (data not shown).

In summary, the hypotonic-induced rise in [Ca²⁺]_i depends on both extracellular and intracellular sources of Ca²⁺. Influx of Ca²⁺ ions is mediated by SACs and ECaC; IP₃-initiated mobilisation from intracellular stores is also implicated. Changes in [Ca²⁺]_i in response to HTS may play a role in the modulation of matrix turnover by mechanical stresses associated with joint movements.

This work was supported by the Arthritis Research Campaign, UK (W0604, W0616). J.C.S. was supported by Universidad Tecnologica de Pereira, Pereira, Colombia.