The regulation of pH_i by isolated articular chondrocytes is almost exclusively by the Na⁺-H⁺ antiporter (Wilkins & Hall, 1995). There are, however, significant variations in the environment of in situ chondrocytes with cartilage depth from synovial surface to bone. Here, we have investigated whether there are differences in the ability of chondrocytes within three cartilage zones (superficial, mid, deep; SZ, MZ, DZ) to recover their pH_i following an intracellular acid load.

Explants of bovine articular cartilage were obtained from metacarpophalangeal joints of animals freshly slaughtered at the local abattoir and cultured in Dulbecco’s modified Eagle’s medium (DMEM, 280 mosmol, pH 7.4). When required, explants were incubated with BCECF-AM (10 mM, 30 min, 37 °C) and chondrocytes within the zones imaged (Ex. 490/439 nm; Em. 535 nm) using a Photon Technology International ImageMaster system (Hall & Bush, 2001). pH_i was altered using the ammonium pre-pulse technique (Thomas, 1984) and recovery in fluorescence ratio studied in the presence of NaHCO₃ (23 mM) or its absence (NaCl substituted; both pH 7.4). There was no significant difference (ANOVA) to the change of fluorescence ratio following ammonium pre-pulse of cells within the three zones in the presence or absence of HCO₃ (P = 0.68; n = 14). In HCO₃-free media, recovery of pH_i (in units of change in fluorescence ratio s^-1 X 10⁵) of cells within the MZ and DZ was rapid (32 ± 2 and 56 ± 2, respectively) and completely inhibited by ethylisopropyl amiloride (EIPA, 100 mM; -2 ± 6 and -1 ± 1, respectively). In contrast, cells within the SZ demonstrated no significant (P > 0.05; Student’s unpaired t test) recovery of pH_i over the same time period in the absence of HCO₃, although in its presence recovery was markedly stimulated (49 ± 1; data are means ± S.E.M., n = 3).

These data suggest that chondrocytes within the SZ of bovine articular cartilage utilise a HCO₃-dependent system for pH_i recovery from acid load, whereas cells within the MZ and DZ of cartilage largely utilise the Na⁺-H⁺ antiporter. These differences in the mechanisms of chondrocyte pH_i regulation may relate to the environment experienced by the cells within the three cartilage zones. It is possible that this property of SZ chondrocytes has not previously been observed in isolated cell preparations, because they represent a relatively small (< 10 %) proportion of the total cell population.

This work was supported by the Arthritis Research Campaign (H0621), The Wellcome Trust (045925/Z/95/A) and the MRC.

All procedures accord with current UK legislation.