The effect of intracellular alkalinisation on intracellular Ca(2+) homeostasis in a human chondrocyte cell line

Intracellular pH (pH(i)) is a well-established determinant of cartilage matrix metabolism. Changes to chondrocyte pH(i), and therefore matrix turnover rates, arise following joint loading. It is not yet clear whether pH changes exert their effects on matrix metabolism directly, or by changing the concentration of another, as yet unidentified, intracellular factor. In this study the effect of intracellular alkalinisation on intracellular [Ca(2+)] has been examined using the human chondrocyte C-20/A4 cell line. pH(i) was manipulated by the addition of weak bases to suspensions of chondrocytes and fluorimetric techniques were employed to measure pH(i) and [Ca(2+)](i). The effect of pH(i) changes on intracellular inositol 1,4,5-trisphosphate (IP(3)) levels was also determined. The pH-sensitive properties of the Ca(2+)-sensitive fluoroprobe employed in this study, Fura-2, were investigated such that artefactual effects of pH changes upon the dye could be discounted. It was demonstrated that, for dye loaded into cells, alkalinisation resulted in a small increase in the affinity of the dye for Ca(2+) ions. Intracellular alkalinisation elicited by treatment with either of the weak bases trimethylamine or ammonium chloride initiated a rise in [Ca(2+)](i). This effect was too large to be explicable by the effects of pH changes on Fura-2 and was not dependent on the presence of extracellular Ca(2+) ions. Prior depletion of intracellular Ca(2+) stores by treatment with thapsigargin inhibited alkalinisation-induced increases in [Ca(2+)](i) and intracellular alkalinisation was also associated with increased levels of intracellular IP(3). These results confirm that alkaline pH(i) changes associated with dynamic loading of cartilage also result in knock-on alterations to [Ca(2+)](i). Given the sensitivity of cartilage matrix metabolism to [Ca(2+)](i) it is likely that this signalling cascade forms an important part of the mechanotransduction pathway that determines the response of chondrocytes to applied load.