articleThe effect of oxygen tension on calcium homeostasis in bovine articular chondrocytes

Background: Articular chondrocytes normally experience a lower O2 tension compared to that seen by many other tissues. This level may fall further in joint disease. Ionic homeostasis is essential for chondrocyte function but, at least in the case of H+ ions, it is sensitive to changes in O2 levels. Ca2+ homeostasis is also critical but the effect of changes in O2 tension has not been investigated on this parameter. Here we define the effect of hypoxia on Ca2+ homeostasis in bovine articular chondrocytes. Methods: Chondrocytes from articular cartilage slices were isolated enzymatically using collagenase. Cytoplasmic Ca2+ levels ([Ca]i) were followed fluorimetrically using Fura-2 to determine the effect of changes in O2 tension. The effects of ion substitution (replacing extracellular Na+ with NMDG+ and chelating Ca2+ with EGTA) were tested. Levels of reactive oxygen species (ROS) and the mitochondrial membrane potential were measured and correlated with [Ca]i. Results: A reduction in O2 tension from 20% to 1% for 16-18 h caused [Ca]i to approximately double, reaching 105 ± 23 nM (p < 0.001). Ion substitutions indicated that Na+/Ca2+ exchange activity was not inhibited at low O2 levels. At 1% O2, ROS levels fell and mitochondria depolarised. Restoring ROS levels (with an oxidant H2O2, a non-specific ROS generator Co2+ or the mitochondrial complex II inhibitor antimycin A) concomitantly reduced [Ca]i. Conclusions: O2 tension exerts a significant effect on [Ca]i. The proposed mechanism involves ROS from mitochondria. Findings emphasise the importance of using realistic O2 tensions when studying the physiology and pathology of articular cartilage and the potential interactions between O2, ROS and Ca2+. Background Due to the avascularity of its matrix, articular cartilage is hypoxic compared to other tissue types [1]. O2 tension is uncertain, but most cells probably experience 5-7% O2 [2]. Perhaps as a consequence, articular chondrocytes have few mitochondria and metabolism is largely anaerobic. Notwithstanding, chondrocytes consume O2 and are adversely affected if maintained in an anoxic environment [3,4]. Lowered O2 levels can occur in vivo in various disease conditions [2]. It is becoming increasingly evident that O2 tension is a critical parameter in modulating chondrocyte function [5]. At low O2 tension, glycolysis is inhibited, glucose uptake is reduced, and ATP and lactic acid production fall, the apparently paradoxical "negative Pasteur effect" [3]. Other responses include changes in production of growth factors, proinflammatory mediators and matrix components [5]. In other tissues, change in O2 tension is an important signal leading to modulation of ionic permeability and alteration of ionic homeostasis, thereby impacting upon cell function [6]. Similarly, pH homeostasis in articular chondrocytes is perturbed by alteration in O2 levels [7,8]. When O2 is reduced from 20% to 1%, the main H+ efflux pathway, the Na+/H+ exchanger [9], is inhibited leading to acidification of the cells. A reduction in reactive oxygen species (ROS) acting, via alterations in protein phosphorylation, appears to constitute the link between hypoxia and reduction in NHE activity [7]. Intracellular Ca2+ levels are also critical [10]. Changes in Ca2+ will affect matrix synthesis, as well as other func* Correspondence: jsg1001@cam.ac.uk 1 Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 OES, UK Full list of author information is available at the end of the article BioMed Central © 2010 White and Gibson; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. White and Gibson Journal of Orthopaedic Surgery and Research 2010, 5:27 http://www.josr-online.com/content/5/1/27 Page 2 of 7 tions. Low O2 tension has been shown previously to cause a rise in Ca2+ in cultured embryonal chick chondrocytes, acting to slow ageing processes [11]. An interaction between O2 and Ca2+ is therefore anticipated in articular chondrocytes but has not been described hitherto. Our overall aim therefore was to elucidate whether Ca2+ levels are sensitive to O2. Because reduction in O2 tension from 20% to 1% has been shown to have important effects on pH homeostasis, we concentrated on these values for this study. Cytoplasmic Ca2+ levels, ROS and the mitochondrial membrane pd were measured fluorimetrically. Results show that Ca2+ levels are increased during hypoxia, with a transduction path involving mitochondrial depolarization and ROS.


Background
Due to the avascularity of its matrix, articular cartilage is hypoxic compared to other tissue types [1]. O 2 tension is uncertain, but most cells probably experience 5-7% O 2 [2]. Perhaps as a consequence, articular chondrocytes have few mitochondria and metabolism is largely anaerobic. Notwithstanding, chondrocytes consume O 2 and are adversely affected if maintained in an anoxic environment [3,4]. Lowered O 2 levels can occur in vivo in various disease conditions [2].
It is becoming increasingly evident that O 2 tension is a critical parameter in modulating chondrocyte function [5]. At low O 2 tension, glycolysis is inhibited, glucose uptake is reduced, and ATP and lactic acid production fall, the apparently paradoxical "negative Pasteur effect" [3]. Other responses include changes in production of growth factors, proinflammatory mediators and matrix components [5]. In other tissues, change in O 2 tension is an important signal leading to modulation of ionic permeability and alteration of ionic homeostasis, thereby impacting upon cell function [6]. Similarly, pH homeostasis in articular chondrocytes is perturbed by alteration in O 2 levels [7,8]. When O 2 is reduced from 20% to 1%, the main H + efflux pathway, the Na + /H + exchanger [9], is inhibited leading to acidification of the cells. A reduction in reactive oxygen species (ROS) acting, via alterations in protein phosphorylation, appears to constitute the link between hypoxia and reduction in NHE activity [7].
Intracellular Ca 2+ levels are also critical [10]. Changes in Ca 2+ will affect matrix synthesis, as well as other func-tions. Low O 2 tension has been shown previously to cause a rise in Ca 2+ in cultured embryonal chick chondrocytes, acting to slow ageing processes [11]. An interaction between O 2 and Ca 2+ is therefore anticipated in articular chondrocytes but has not been described hitherto. Our overall aim therefore was to elucidate whether Ca 2+ levels are sensitive to O 2 . Because reduction in O 2 tension from 20% to 1% has been shown to have important effects on pH homeostasis, we concentrated on these values for this study. Cytoplasmic Ca 2+ levels, ROS and the mitochondrial membrane pd were measured fluorimetrically. Results show that Ca 2+ levels are increased during hypoxia, with a transduction path involving mitochondrial depolarization and ROS.

Chondrocytes
Bovine feet from animals aged between 18 and 36 months were obtained following abattoir slaughter. Full depth hyaline cartilage shavings from the proximal metacarpophalangeal joint were taken at ambient O 2 tension, then placed in DMEM containing penicillin (100 IU.ml -1 ), streptomycin (0.1 μg.ml -1 ) and fungizone (2.5 μg.ml -1 ) and incubated at 37°C, 5% CO 2 for 16-18 h at 20% or 1% O 2 whilst matrix was digested with 0.1% (w/v) collagenase type I. Isolated chondrocytes were resuspended in saline (at the required O 2 tension) at a final dilution of 10 6 cells.ml -1 . Cell viability was determined by the Trypan Blue exclusion test, at >95%. See [12] for further details.

Maintenance of O 2 tension
During longer term incubations (>3 hours), cells were maintained at the correct O 2 tension in a variable O 2 /CO 2 incubator (Galaxy R, RS Biotech, Irvine, UK). For shorter term incubations, cells were placed in Eschweiler tonometers (Kiel, Germany) and flushed with appropriate gas mixtures using a Wösthoff gas mixing pump (Bochum, Germany). Similarly, solutions were pre-equilibrated to the required O 2 tension in Eschweiler tonometers before being applied to cells.

Measurement of Ca 2+
Cytoplasmic Ca 2+ levels ([Ca 2+ ] i ) were measured using Fura-2 (see [12]). Cells were loaded with 5 μM fura-2-AM for 30 min at room temperature followed by 15 min at 37°C. Fluorescence was measured in a thermostatically regulated fluorimeter (F-2000 Fluorescence Spectrophotometer, Hitachi). Fura-2 was alternately excited at 340 nm and 380 nm, with emission intensity was measured at 510 nm. In most cases, the 340:380 nm fluorescence ratio (R) was converted to Ca 2+ values, as described previously [12]. When reagents were added to alter ROS levels, however, Ca 2+ levels are presented as raw R values. In these cases, exact [Ca 2+ ] i could not be calculated because, after digitonin treatment, on exposure to the high concentrations of the reagents found extracellularly, Fura-2 was partially quenched.

Measurement of reactive oxygen species (ROS)
Chondrocytes were loaded with DCF-DA (10 μM) at 37°C for 45 min [7]. In the presence of ROS, DCF is converted to dichlorofluorescin, resulting in a change in fluorescence. DCF was excited at 488 nm and emission intensity measured at 530 nm.

Measurement of the mitochondrial pd
Chondrocytes were loaded with 5 μM JC-1 for 20 min at 37°C [8]. JC-1 was then excited at 490 nm and the emission intensity monitored at 525 nm (green) and 590 nm (red). The dye is sequestered inside mitochondria at negative pds. Membrane depolarization is indicated by a shift in the emission fluorescence from red to green, as dye is released into the cytosol and the formation of red fluorescent J-aggregates causing a fall in the red/green fluorescence intensity ratio.

Statistics
Student's paired or Independent t-test were used to determine statistical significance (p < 0.05) between results. Data are given as means ± S.E.M. for n replicates, where each replicate indicates a separate individual animal.

Effect of hypoxia on Ca 2+ homeostasis
Previously published reports on the effects of hypoxia on pH homeostasis in equine articular chondrocytes demonstrated effects within 3 hours when O 2 was reduced from 20% to 1% [7]. Evidence for a similar effect was therefore tested on Ca 2+ levels. Bovine articular chondrocytes were isolated at 20% O 2 and the effect of maintain-

Hypoxia, Ca 2+ and ion substitutions
Ion substitution experiments were carried out to determine the source of the extra Ca 2+ . Chondrocytes were again isolated, and then maintained for 16-18 hours, at either 20% or 1% O 2 in standard Ca 2+ -and Na + -containing saline. Ca 2+ levels were then measured in this standard saline and also following transfer to Ca 2+ -free or Na + -free saline (Figures 1 and 2). In Ca 2+ -free conditions (Figure 1) (Figure 2), but again remained higher at 1% O 2 compared to 20% O 2 . In fact, the difference in Ca 2+ comparing cells maintained at 20% and 1% O 2 was greater in Na + -free conditions.

Interaction of reactive oxygen species and Ca 2+ homeostasis
Levels of reactive oxygen species (ROS) in equine articular chondrocytes decrease when O 2 tension is reduced from 20% to 1% [7]. This finding was confirmed in the present work for bovine chondrocytes held at different O 2 levels for 16-18 hours. ROS levels at 1% fell to 60 ± 6% (mean ± S.E.M., n = 3) of the value at 20% O 2 . Three different protocols were carried out to elevate ROS levels: treatment with the oxidant H 2 O 2 (100 μM), the non-specific ROS generator Co 2+ (100 μM) or the mitochondrial complex III inhibitor antimycin A (50 μM). In each case, ROS levels recorded in treated cells incubated at 1% O 2 were restored to those observed at 20%, (eg for Co 2+ levels reached 96 ± 8% values at 20%, N.S.). Using Fura-2 340 nm:380 nm emission ratio (R) as a measure of [Ca 2+ ] i , in cells incubated at 1% but treated to raise ROS levels, it was found that R decreased by a similar amount, reaching values similar to those observed at 20%. For example, R at 1% following addition of H 2 O 2 fell from 1.41 ± 0.001 to 1.06 ± 0.001 (n = 15). For all three protocols, therefore, at 1% O 2 when ROS levels were restored, so was [Ca 2+ ] i .

Hypoxia and mitochondria
The effect of changes in O 2 and treatment with antimycin A on mitochondrial pd was then investigated. Chondrocytes were isolated at 20% O 2 and then incubated at either 20% O 2 or 1% O 2 for 16-18 hours prior to loading with JC-1. They were also treated with antimycin A (50 μM) at both O 2 tensions (Figure 3). It can be seen that the red/ green ratio was reduced at 1% O 2 indicative of mitochon-

The effect of O 2 tension on steady state Ca 2+
The present findings are the first to demonstrate an effect of changes in O 2 tension on Ca 2+ homeostasis in articular chondrocytes. We show here that Ca 2+ homeostasis is maintained in response to shorter term (3 hours) reduction in O 2 tension from 20% to 1%. Longer exposure to 1% O 2 , however, caused significant elevation in [Ca 2+ ] i with levels approximately doubling, sufficient to perturb cell function. These effects were associated with both mitochondrial depolarization and a fall in levels of reactive oxygen species (ROS). Since NCE activity requires a functional ATP-driven Na + /K + pump, it is unlikely that ATP was limiting (as shown previously [7]). In addition, because inhibition of the mitochondrial electron transport chain with antimycin A reduces [Ca 2+ ] i , any Ca 2+ release from mitrochondrial stores following their hypoxia-induced depolarization, would likely to be insufficient on its own to raise [Ca 2+ ] i . In this context, it is important to note that mitochondria in articular chondrocytes occupy a relatively small volume (1-2% cytoplasm) [13] compared to that seen in other tissues (typically 15-20%, eg liver). There is also some reduction in mitochondrial volume with depth and age [14,15]. They may also lack a functional electron transport chain [16], relying on glycolysis for metabolic energy [3]. Taken together, these findings are consistent with hypoxic release of Ca 2+ into the cytoplasm from intracellular nonmitochondrial stores, probably endoplasmic reticulum.

Oxygen and chondrocyte function
As noted above, it is unlikely that articular chondrocytes require O 2 for energy, at least directly. Nevertheless, O 2 tension is a critical parameter in modulating chondrocyte function. Changes in O 2 level affect ATP production [3], growth factors [17], proinflammatory mediators [18] and matrix components [19]. Dedifferentiation of chondrocytes occurs when they are maintained at abnormally high O 2 . This includes restoration of the ability to carry out oxidative phosphorylation [20]. Standard chondrocyte markers, such as collagen type II and aggrecan, are affected [19]. In effect, low O 2 tensions (c.5%), which are normal for articular cartilage but hypoxic for other cell types, promote a chondrocyte phenotype [21][22][23]. In addition, however, a pathological role for O 2 has also received considerable attention. Thus abnormally high or low O 2 levels with concomitant alterations in levels of ROS, may be important in disease states such as osteoarthritis [24][25][26]. O 2 also affects acid-base balance in articular chondrocytes [7,8]. The present findings extend the action of O 2 to include modulation of an additional important ion, ie Ca 2+ , with low O 2 causing intracellular [Ca 2+ ] to rise. The O 2 tension at which perturbation of Ca 2+ requires further definition, it being particularly important to study the likely physiological levels of between 10% and 1%.

Calcium and chondrocyte function
Intracellular Ca 2+ in chondrocytes, as in other cell types, also has numerous physiological and probably pathological roles [27]. Of particular relevance to chondrocytes is the observation that perturbation of normal Ca 2+ levels  [10]. It also affects both chondrocyte differentiation [28] and ageing [11]. Ca 2+ signalling has been implicated in a range of other chondrocyte functions including mechanotransduction [29][30][31][32], volume regulation [33][34][35][36][37][38][39] and response to electrical stimulation [40]. It may therefore play a critical role in how joint loading and unloading promotes cartilage health. Intracellular Ca 2+ elevations, for example, induce chondrogenesis via a calcineurin/NF-AT pathway [41]. Extracellular levels of Ca 2+ are also important in the longer term, when they too may be involved in alteration of matrix production including proteoglycan synthesis and expression of collagen [42][43][44] -extracellular Ca 2+ receptors are present. Ca 2+ is also implicated in the action of proinflammatory cytokines such as IL-1 and, again therefore, has received attention in the context of joint disease such as osteoarthritis [45].

Crosstalk between oxygen, reactive oxygen species and Ca 2+
The elevation of intracellular Ca 2+ at low O 2 reported here was associated with a fall in ROS and also mitochondrial depolarization. In most cell types, though probably not articular chondrocytes, mitochondria are critical for oxdative phosphorylation and hence central to energy production. They are also involved in Ca 2+ regulation, acting as a sink of, or sometimes a source for, cytoplasmic Ca 2+ -Ca 2+ being released via the mitochondrial permeability transition pore (PTP) [46][47][48]. ROS are generated during mitochondrial respiration [49,50], as well as at other cellular sites. ROS, of course, can be harmful but have also been implicated in intracellular signalling, regulating redox sensitive enzymes and also ion channels. By these means, ROS may modulate intracellular Ca 2+ , eg acting via modulation of ryanodine receptors, IP3 receptors, Ca 2+ pumps and NCE [51][52][53]. Ca 2+ uptake by mitochondria may itself alter ROS generation -both reduction of ROS (through dissipation of the negative mitochondrial pd) or their elevation have been reported [54,55]. To a certain extent, the direction of change depends on tissue type and respiratory rate. Another obvious signal is represented by hypoxia-inducible factor (HIF). Stabilization of HIF1α occurs during hypoxia (eg [6,56,57]) and may affect [Ca 2+ ] i through effects calcium channel gene expression and activity [58,59]. There is thus considerable scope for cross-talk between O 2 , ROS and Ca 2+ , together with the role of mitochondria [51,53,55] but the exact coupling in chondrocytes awaits description.

Reactive oxygen species, mitochondria and regulation of Ca 2+
We show here that a fall in ROS during hypoxia correlated with elevation of Ca 2+ , whilst restoration of ROS levels to those seen at 20% by three disparate reagents (H 2 O 2 , Co 2+ or antimycin A) all resulted in decreased Ca 2+ . Hypoxia also induced depolarization of mitochondria, indicative of a reduction in electron flow through the mitochondrial electron transport chain, and hence ROS production. Addition of antimycin A also blocks electron transport to the terminal complexes, acting at the Q i site of complex III to increase ROS output [8], as also observed in the present work. It is thus likely that reduced production of ROS from mitochondria is involved in the rise in Ca 2+ , as proposed for O 2 -induced changes in NHE activity and intracellular pH [8]. In the case of H + , however, perturbed homeostasis on change in O 2 tension is observed rapidly, within a few minutes [60]. Effects on Ca 2+ appear to occur over a much longer time course, despite sharing sensitivity to ROS levels. The reason for this is not immediately apparent. It may be that Ca 2+ homeostasis, as a more critical modulator of chondrocyte function, is better protected than pH. Alternatively, it may be that the mechanism involves genomic effects, such as though involving HIF. In addition, a link between Ca 2+ and pH in chondrocytes has been shown previously, with alkalinisation causing a rise in Ca 2+ [61].
Since chondrocytes acidify in response to low O 2 , however, rather than increasing their pH, the hypoxiainduced rise in Ca 2+ cannot be secondary to changes in pH.

Conclusion
O 2 tension exerts a significant effect on cytoplasmic Ca 2+ levels of articular chondrocytes, with the proposed mechanism involving ROS from mitochondria. Results emphasise the importance of O 2 to chondrocyte function and that of using realistic O 2 tensions when studying the pathophysiology of articular cartilage.