Mechanisms contributing to intracellular pH homeostasis in an immortalised human chondrocyte cell line

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Abstract

The maintenance of chondrocyte pH is an important parameter controlling cartilage matrix turnover rates. Previous studies have shown that, to varying degrees, chondrocytes rely on Na+/H+ exchange to regulate pH. HCO3-dependent buffering and HCO3-dependent acid-extrusion systems seem to play relatively minor roles. This situation may reflect minimal carbonic anhydrase activity in cartilage cells. In the present study, the pH regulation of the human chondrocyte cell line, C-20/A4 has been characterised. Intracellular pH (pHi) was measured using the H+-sensitive fluoroprobe BCECF. In solutions lacking HCO3/CO2, pHi was approximately 7.5, and the recovery from intracellular acidification was predominantly mediated by a Na+-dependent, amiloride- and HOE 694-sensitive process. A small additional component which was sensitive to chloro-7-nitrobenz-2-oxa-1,3-diazole, an inhibitor of the V-type H+-ATPase, was also apparent. In solutions containing HCO3/CO2, pHi was approximately 7.2. Comparison of buffering capacity in the two conditions showed that this variable was not significantly augmented in HCO3/CO2-containing media. The recovery from intracellular acidification was more rapid in the presence of HCO3/CO2, although under these conditions it was again largely dependent on Na+ ions and inhibited by amiloride and HOE 694. A small component was inhibited by SITS, although this effect did not reach the level of statistical significance. These findings indicate that HCO3-dependent processes play only a minimal role in pH regulation in C-20/A4 chondrocytes. pH regulation instead relies heavily on the Na+/H+ exchanger together with a H+-ATPase. The absence of extrinsic (HCO3/CO2) buffering is likely to reflect the low levels of carbonic anhydrase in these cells. In addition to providing fundamental information about a widely-used cell line, these findings support the contention that the unusual nature of pH regulation in chondrocytes reflects the paucity of carbonic anhydrase activity in these cells.

Introduction

Chondrocytes, the sole cell type normally resident within articular cartilage, synthesise and maintain the extracellular matrix (ECM) in response to external loading patterns (Stockwell, 1991, Bjelle, 1975, Slowman and Brandt, 1986). They exist in an avascular, hypoxic environment and, consequently, their metabolism is predominantly by means of anaerobic pathways producing large amounts of lactate (Marcus, 1973, Otte, 1991). This, coupled with the prevalence of large negatively charged molecules within the ECM (proteoglycans; PGs) which, via Donnan equilibration, concentrate small mobile cations (including H+; Maroudas, 1979), results in the extracellular pH of cartilage being lower than that commonly reported for other tissue types. Values as low as 6.9 have been reported for healthy ECM (Urban, 1994).

In this context, intracellular pH (pHi) regulation by chondrocytes is significant for two reasons. First, the acidic nature of cartilage exposes chondrocytes to the constant threat of intracellular acidosis. Chondrocytes, like all cell types, must counter this threat by maintaining pHi at levels appropriate to their normal physiology (for example Thomas, 1984). Second, it is well established that cartilage turnover is sensitive to changes to pHi (Wilkins and Hall, 1995). Indeed, controlled modulations to chondrocyte pHi, brought about by load-induced alterations to the normal pHi regulatory mechanisms, represent a significant signalling pathway through which chondrocytes modify the synthesis and degradation of ECM (for example Browning et al., 1999, Yamazaki et al., 2000).

The mechanisms regulating chondrocyte pHi are therefore critical, both as a defence against intracellular acidosis and as a transduction pathway allowing chondrocytes to modulate cartilage turnover in response to physical load. These factors have prompted the investigation of mechanisms regulating pHi in chondrocytes (Wilkins and Hall, 1992, Dascalu et al., 1993). Studies on bovine cells have established that chondrocyte pHi is regulated primarily by the action of HCO3-independent proton buffering systems and acid extrusion by Na+/H+ exchange; it appears from preliminary findings that HCO3-dependent transporters play little role (Wilkins and Hall, 1992, Wilkins and Hall, 1993), aside from chondrocytes from the most superficial areas of cartilage, where a HCO3-dependent component is reported (O'Neill et al., 2002). The paucity of HCO3-dependent processes, whilst unusual in the context of other cell types, is appropriate given the low concentration of HCO3 in cartilage matrix. Furthermore, we have shown in a previous study that bovine chondrocytes possess only very low levels of the enzyme carbonic anhydrase (Swietach et al., 2002). This necessarily implies that the supply of HCO3 ions in this cell type is limited and precludes them from playing a role in proton buffering systems or from being transported in large quantities across the cell membrane.

Nevertheless, studies on other types of cartilage cells suggest that this picture may not be uniform. For example, cultured avian chondrocytes demonstrate a HCO3-dependent component to recovery from acidification (Dascalu et al., 1993) while acid extrusion from bovine intervertebral disc cells is also increased in the presence of HCO3 ions (Razaq et al., 2000). It is unclear to what extent these findings represent real differences between chondrocytes, or are simply a consequence of, for example, the conditions employed for chondrocyte isolation, preparation and culture.

In the present study, the pHi-regulating properties of chondrocytes from an alternative source have been investigated. C-20/A4 cells, a transformed human chondrocyte cell line routinely used in investigations of cartilage cell biology (Moulton et al., 1997, Goldring et al., 1994), have been employed. We have previously shown that these cells, like freshly isolated bovine chondrocytes, express only low levels of carbonic anhydrase (Swietach et al., 2002). It is possible that for chondrocytes with low levels of carbonic anhydrase activity HCO3-dependent pH regulation is employed to a limited degree. Recovery of pHi following ammonium-induced acidification has been recorded in HCO3-free and HCO3-containing media. A combination of ion-substitution and inhibitor studies has been used to dissect the identity of the mechanisms contributing to proton-fluxes across the cell membrane. Aside from the obvious benefit of obtaining fundamental information about the properties of a commonly-used cell line, this study supports the contention that there is a relation between the unusual pH regulation exhibited by articular chondrocytes and the low levels of carbonic anhydrase activity which these cells possess.

Section snippets

Materials and media

Unless otherwise stated, all reagents used were purchased from Sigma-Aldrich Company Limited, Poole, UK.

Solutions were prepared as follows. HEPES buffered solution (HBS): 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 10 mM HEPES, 10 mM d-glucose, pH adjusted to 7.40 with 5 M NaOH. Na+-free HEPES buffered solution (Na+-free HBS): 145 mM N-methyl-d-glucamine (NMDG)-Cl, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 10 mM HEPES, 10 mM d-glucose, pH adjusted to 7.40 with 5 M HCl. Bicarbonate buffered solution

Resting intracellular pH of C-20/A4 cells in HBS and BBS

Chondrocyte steady-state pHi was measured using BCECF fluorescence for cells equilibrated for at least 10 min in either HBS or BBS. Steady-state pHi was significantly more alkaline for cells suspended in HBS than for cells in BBS (HBS 7.52±0.03 (n=17) vs. BBS 7.22±0.03 (n=22), P<0.05).

Measurement of C-20/A4 buffering capacity

BCECF-loaded C-20/A4 cells were suspended in Na+-free-HBS and acidified by ammonium prepulse. The concentration of NH4Cl used and the duration of the ammonium prepulse were varied (1–25 mM NH4Cl; 2–15 min) such

Discussion

The experiments described in this study were undertaken to characterise the pHi regulating properties of a cultured human chondrocyte cell line (C-20/A4). Similar studies using bovine chondrocytes have previously identified pHi as an important regulator of cartilage metabolism in response to joint loading (Wilkins and Hall, 1992, Wilkins and Hall, 1995). The more acidic the extracellular environment, the lower the rate of matrix synthesis, an effect mediated in part by changes to cellular pH.

Acknowledgements

This work was funded by the Arthritis Research Campaign, The Halley Stewart Trust, The EPA Cephalosporin Fund and Christ Church, Oxford. We thank Professor Mary Goldring for her gift of C-20/A4 cell samples.

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