A hyperosmotic stimulus regulates intracellular pH, calcium, and S-100 protein levels in avian chondrocytes

Cartilage is exposed to mechanical loads, generating at the level of single chondrocytes a hyperosmotic stimulus (HOS). The direct effect of HOS on second messenger pathways in avian chondrocytes was evaluated by fluorimetric and image analysis techniques. HOS caused an immediate intracellular acidification of 0.07 ± 0.02 pH units (n = 7), followed by an initial pH recovery rate of 0.033 ± 0.04 pH units/min towards the pre-stimulus baseline values. Concomitantly, the intracellular calcium ([Ca²⁺](i)) responded with a transient rise from baseline value of 84.7 ± 7.4 nM to peak level of 403.1 ± 51.0 nM (n = 16, p < 0.001). The calcium response was abolished by two calmodulin inhibitors chlorpromazine and W-7, Since these inhibitors are known to be specific ligands of a S-100 protein, its intracellular staining was determined following HOS. The amount of immunodetectable S-100 protein was significantly increased following exposure to HOS (p < 0.05), and did not require an increase of [Ca²⁺](i). It appears that compression of cartilage is transduced into HOS of chondrocytes, and further elicits its effect through transient intracellular elevation of protons and calcium ions accompanied by increased staining of S-100 protein.