Regulatory volume increase (RVI) by in situ and isolated bovine articular chondrocytes

Metabolism of the matrix by chondrocytes is sensitive to alterations in cell volume that occur, for example, during static loading and osteoarthritis. The ability of chondrocytes to respond to changes in volume could be important, and this study was aimed attesting the hypothesis that chondrocytes can regulate their volume following cell shrinking by regulatory volume increase (RVI). We used single cell fluorescence imaging of in situ bovine articular chondrocytes, cells freshly isolated into 280 or 380 mOsm, or 2-D cultured chondrocytes loaded with calcein or fura-2, to investigate RVI and changes to [Ca2+](i) during shrinkage. Following a 42% hyperosmotic challenge, chondrocytes rapidly shrunk, however, only similar to 6% of the in situ or freshly isolated chondrocytes demonstrated RVI. This contrasted with 2D-cultured chondrocytes where similar to 54% of the cells exhibited RVI. The rate of RVI was the same for all preparations. During the 'post-RVD/RVI protocol', similar to 60% of the in situ and freshly isolated chondrocytes demonstrated RVD but only similar to 5% showed RVI. There was no relationship between [Ca2+] i and RVI either during hyperosmotic challenge, or during RVD suggesting that changes to [Ca2+] i were not required for RVI. Depolymerisation of the actin cytoskeleton by latrunculin, increased RVI by freshly isolated chondrocytes, in a bumetanide-sensitive manner. The results showed that in situ and freshly isolated articular chondrocytes have only limited RVI capacity. However, RVI was stimulated by treating freshly isolated chondrocytes with latrunculin B and following 2D culture of chondrocytes, suggesting that cytoskeletal integrity plays a role in regulating RVI activity which appears to be mediated principally by the Na+-K+-2Cl(-) cotransporter.