The effect of intracellular alkalinisation on intracellular Ca²⁺ homeostasis in a human chondrocyte cell line

Abstract.

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 [Ca²⁺] 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 [Ca²⁺]_i. The effect of pH_i changes on intracellular inositol 1,4,5-trisphosphate (IP₃) levels was also determined. The pH-sensitive properties of the Ca²⁺-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 Ca²⁺ ions. Intracellular alkalinisation elicited by treatment with either of the weak bases trimethylamine or ammonium chloride initiated a rise in [Ca²⁺]_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 Ca²⁺ ions. Prior depletion of intracellular Ca²⁺ stores by treatment with thapsigargin inhibited alkalinisation-induced increases in [Ca²⁺]_i and intracellular alkalinisation was also associated with increased levels of intracellular IP₃. These results confirm that alkaline pH_i changes associated with dynamic loading of cartilage also result in knock-on alterations to [Ca²⁺]_i. Given the sensitivity of cartilage matrix metabolism to [Ca²⁺]_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.