The effect of intracellular alkalinisation on intracellular Ca 2+ 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 [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.

Regulation of human airway ciliary beat frequency by intracellular pH

“…Salathe & Bookman, 1995, 1999; Salathe et al 1997; Ma et al 2002; Zagoory et al 2002). It has been shown in several cell types that intracellular alkalization can increase, while intracellular acidification can decrease [Ca 2+ ] i via regulation of Ca 2+ release from intracellular stores (Thomas et al 1979; Browning & Wilkins, 2002). We investigated whether CBF regulation by pH i depends on [Ca 2+ ] i .…”

“…6 and ). To estimate [Ca 2+ ] i , constant K d (400 n m , Browning & Wilkins, 2002), R max , R min and β ‐values were used according to Grynkiewicz et al (1985) using in vitro calibration procedures as outlined in material and methods. However, the affinity of fura‐2 for Ca 2+ has been reported to increase with increasing pH (i.e.…”

“…K d decreases at alkaline pH). In addition, R max as well as β were found to be pH dependent (Lattanzio & Bartschat, 1991; Browning & Wilkins, 2002). Therefore, R max , R min and β were measured in vitro in the pH range of 7.0–8.0.…”

“…Fura‐2 ratios were computed every 10–20 s, after background subtraction. Calibration of the calcium signal was done with in vitro measurements as described (Salathe & Bookman, 1995) according to Grynkiewicz et al (1985); however, we re‐estimated [Ca 2+ ] i by adjusting fura‐2 K d values with changing pH (Lattanzio & Bartschat, 1991; Browning & Wilkins, 2002) as described below.…”

Oxygen and reactive oxygen species in articular cartilage: modulators of ionic homeostasis

“…Systems that import HCO À 3 ions play little or no role [37, 119], perhaps, because anions are to some extent excluded from the extracellular matrix, but most probably because levels of carbonic anhydrase expression are very low in chondrocytes [103]. As for other cells, Ca 2+ levels are determined by the balance of influx (leak through channels) and efflux (mediated in large part by Na + -Ca 2+ exchange, NCE) across the cell membrane, and by the exchange of Ca 2+ between the cytosol and intracellular stores [12,82,88,89,92].…”