SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Articular cartilage chondrocytes consume remarkably little O2 in comparison with most other animal cells; glycolysis forms the principal source of ATP in this cartilage. Although not lethal for many days, imposition of anoxia immediately lowers intracellular ATP, inhibits rates of glycolysis, and prevents articular chondrocytes from producing extracellular matrix macromolecules. This study was undertaken to investigate the role of O2 in articular chondrocyte metabolism.

Methods

We examined the effects of oxygen and of several other classes of exogenous oxidants, i.e., 1) the dyes methylene blue and 2,6-dichlorophenol-indophenol, 2) the iron (III) complex ferricyanide, and 3) the keto-acids oxaloacetate and pyruvate (and phosphoenolpyruvate, a metabolic precursor of pyruvate), on rates of glycolysis and of sulfate incorporation by bovine articular cartilage in vitro.

Results

Lactate production was lowest under conditions of anoxia and was stimulated severalfold by addition of O2 (air-saturated medium). Under strict anoxia, other oxidants restored lactate production to rates at least comparable with those seen in aerobic controls; under aerobic conditions, they had little effect. Oxygen and all of the other oxidants examined stimulated sulfate incorporation more strongly than lactate production. The compounds that promoted glycolysis and hence sulfate incorporation in cartilage under anoxia were themselves reduced; that is, they functioned as oxidants in lieu of O2.

Conclusion

For normal function, articular cartilage appears to require exogenous oxidants to stimulate glycolysis and produce ATP and extracellular matrix. Under physiologic conditions, oxygen acts as this oxidant, but its role can be adequately assumed by other agents.

The chondrocytes of articular cartilage use remarkably little O2 compared with most other animal cells. According to results summarized by Stockwell (1), articular cartilage consumes only 2–5% as much O2 per cell as liver or kidney, for example. There is a metabolic explanation for this striking difference. Whereas the majority of animal cells, including the chondrocytes of avian growth cartilage (2), derive their energy by using oxygen for mitochondrial oxidative phosphorylation, mammalian articular chondrocytes do not appear to use this pathway. Instead, carbohydrate breakdown in articular cartilage is dominated by a near-quantitative conversion of glucose to lactate by the Embden-Meyerhof-Parnas (E-M-P) pathway of glycolysis (3–5), a sequence of reactions in which no O2 is consumed.

There are several lines of evidence that support this finding. Although malate dehydrogenase, isocitrate dehydrogenase, and succinate dehydrogenase have been detected in articular or articular epiphyseal cartilage by extraction (6) or cytochemically (7, 8), experiments with 14C-labeled glucose suggest that little pyruvate enters the Krebs cycle (9, 10), and release of CO2 from articular cartilage is small (11). Moreover, mitochondria are sparse in articular chondrocytes, occupying only 1–2% of the intracellular volume (12), compared with ∼15–20% in more typical animal cells (for example, liver [13]), and in situ, they appear to lack certain cytochromes (14). Cytochrome oxidase has been found to be absent or barely detectable in cells of the surface or midzone of rabbit articular cartilage (7). Oxidative metabolism by articular chondrocytes has been observed, but only after several days of culture in monolayer (3, 15), possibly because dedifferentiation appears linked to an increase in oxidative metabolism (14).

Based on the above evidence, the current view is that the substrate-level phosphorylations of the E-M-P pathway form the principal source of energy (ATP) in articular cartilage (15) and that mitochondrial oxidative phosphorylation makes only a minor contribution to energy supply (15–17). A positive correlation between glycolytic rate and the concentration of ATP in bovine articular cartilage has been noted (5).

Even so, oxygen (O2) plays a role in chondrocyte physiology which is not well understood. Oxygen concentration may influence inflammation associated with cartilage injury and disease (18), and despite the low specific O2 consumption noted above, many studies have shown that articular cartilage responds to O2 in its surroundings. Although not lethal for many days, imposition of anoxia immediately prevents articular chondrocytes from carrying out one of their principal functions, namely, the production of extracellular matrix macromolecules. Thus, at low O2 concentrations, rates of incorporation of sulfate into the large aggregating proteoglycan aggrecan fall steeply (19–21), and assembly of aggrecan into the matrix is affected (22). Hyperoxia has been shown to be as deleterious for matrix synthesis, and indeed for cell survival, as severe hypoxia (20, 21, 23). But the picture obtained from culture experiments is not entirely clear-cut, and there are reports of increased glucose breakdown and lactate production by rabbit articular chondrocytes or cartilage explants after 6–7 days under low O2 concentrations (3, 24). Moreover, in contrast with tissues in which a positive Pasteur effect occurs, in articular cartilage glycolysis decreases at O2 concentrations less than ∼5% in the gas phase, and intracellular levels of ATP are lower (5).

In short-term (4.5-hour) experiments with bovine articular cartilage, we found that rates of glucose utilization and lactate production were markedly greater under aerobic conditions than under anoxia (5), indicating that carbon flux through glycolysis was stimulated by the presence of O2. The effect of O2 on 35S-sulfate incorporation into matrix proteoglycans was even more pronounced (5). Given that glycolysis uses no oxygen (as mentioned above), this result was surprising, and its mechanism unclear. To further examine the connection between O2 supply and glycolysis in articular cartilage, we assumed that O2 functions as an oxidant (electron acceptor) here as it does elsewhere and asked the question: can other oxidants reproduce the effect of O2 and stimulate glycolysis in this tissue? Accordingly, we investigated the effects of reducible substrates on the rate of glycolysis in bovine articular cartilage under anoxic and/or aerobic conditions. If the added substrate is reduced by the cartilage, it functions as an oxidant under the conditions of the test.

The ability of cartilage to reduce externally supplied substrates other than O2 was first reported 70 years ago. Kuwabara (25), in 1932, observed that excised cartilage from several sources (rabbit epiphysis, chick sternum, calf and rabbit costal cartilage) decolorized methylene blue (MEB) under anoxic conditions. Lutwak-Mann (26) noted a “slow, thermolabile reduction of methylene blue” by calf articular cartilage in anaerobic medium, and Bywaters (27) mentioned a similar reaction in equine articular cartilage; however, since then the phenomenon has attracted little attention. Taking these early experiments as a starting point, we examined the response of bovine articular cartilage to a range of reducible compounds (electron acceptors, hence oxidants) including the dyes MEB and 2,6-dichlorophenol-indophenol (DCIP), the iron (III) complex ferricyanide (FECY), and the keto-acids oxaloacetate and pyruvate. We measured lactate production as the primary index of glycolytic flux, a procedure whose validity for this tissue has been critically considered elsewhere (5). In some experiments glucose uptake was recorded at the same time as was lactate production. Incorporation of 35S-sulfate into proteoglycans of the cartilage matrix was also measured as an indication of the broader metabolic and biosynthetic activity of the chondrocytes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Preparation and incubation of cartilage.

Procedures for cartilage preparation were generally the same as those previously described in detail (5). Briefly, articular cartilage was excised in small slices (typically 0.5 mm thickness) to full depth from the metacarpophalangeal joints of 1.5–2-year-old cattle within 2 hours of slaughter. Detailed examination of cartilage from different zones and depths was not an objective of this study. Accordingly, for each experiment the cartilage (usually from 2 feet) was pooled, mixed very thoroughly, and washed 6 times in a filter-sterilized medium having the same mineral salts composition as Dulbecco's modified Eagle's medium (28) but without amino acids, vitamins, ferric nitrate, or Phenol Red. Glucose (1 gm/liter, or 0.3 gm/liter in some experiments), 25 mM HEPES buffer, and an additional 30 mM NaCl were included in the medium, which had an osmolality of 300 mOsm/kg water after adjustment to pH 7.40 in air. The cartilage was then distributed into the treatment tubes under blinded conditions.

Cartilage was incubated in the above medium with the compound(s) to be tested. Where anoxic conditions were required, O2 was excluded by bubbling the medium continuously with O2-free N2 in sealed polypropylene tubes (5), the excess gas escaping to the atmosphere through a fine (25G) needle. Otherwise the tubes were loosely capped and the medium was air-saturated (aerobic). Except in the case of oxaloacetate, reduction of substrates was observed spectrophotometrically (see below). Glucose uptake was calculated from measurements of depletion, and 35S-sulfate incorporation by liquid scintillation counting of papain digests (5). Problems related to measuring lactate in media containing air-reactive metabolites are discussed below. Further details relating to individual experiments are shown in the table and figures. At the end of each experiment, dry weights of cartilage were recorded after drying for 3 days at 65°C. Full-depth bovine articular cartilage like that used in our studies contained, on average, 28.4 × 106 cells/gm fresh weight (29), or ∼120 × 106 cells/gm dry weight, since the fresh cartilage contained 77 ± 1% water (mean ± SD; n = 9).

Measurement of reduction of MEB, DCIP, and FECY.

Time courses of MEB and DCIP reduction were monitored for up to 2 hours by recording the decrease in absorbance at 665 nm and 600 nm, respectively (30). FECY reduction was observed for 3–4 hours at 420 nm (30).

To sustain reduction of MEB, DCIP, or FECY at steady rates over longer periods (e.g., 4.5 hours) without substrate depletion halting the reaction, cartilage (40–80 mg dry weight per replicate) was incubated aerobically or under anoxia in 2.4 ml of medium which contained initially MEB or DCIP at concentrations of 0.01–0.1 mM, or FECY at concentrations of 0.025–0.3 mM (0.5 mM in one experiment). As soon as the medium became colorless, 20-μl aliquots of concentrated MEB, DCIP, or FECY solutions were injected without opening the tubes; the amount of these compounds added was just sufficient to restore the original concentration. Depending on the rate of reduction, this cycle was repeated several times during the experiment, always finishing with a colorless or near-colorless solution. By preparing the concentrated solutions appropriately, the cartilage was made to reduce greater or smaller quantities of MEB, DCIP, or FECY during a 4.5-hour period. This approach provided some indication of whether there was an optimum rate of supply of each of these compounds. The total quantity of MEB, DCIP, or FECY reduced during an experiment was calculated from the amount initially present in the incubation medium plus the amount injected.

Measurement of lactate.

Lactate in the medium at the end of the incubation period was measured using a blood lactate analyzer (model 23L; Yellow Springs Instruments, Yellow Springs, OH). Tests with fresh, air-saturated solutions of MEB, DCIP, FECY, oxaloacetate, pyruvate, phosphoenolpyruvate (PEP), and several other compounds at appropriate concentrations showed no interference with the detection of lactate and no spurious readings in its absence.

The Yellow Springs lactate analyzer depends for its operation on H2O2 production by lactate oxidase, and the instrument responded readily to externally supplied H2O2. It was important to establish that formation of H2O2 (if any) during oxidation of the reduced forms of MEB or DCIP by air at the end of the experiment (“auto-oxidation” [30]) did not lead to an overestimate of lactate production. In tests we found that complete reoxidation of decolorized DCIP in used incubation medium during 2 days of storage at −20°C caused no change in the apparent concentration of lactate; in used medium containing decolorized MEB, the lactate measurements were actually 7% lower after reoxidation had occurred than before. These findings ruled out serious error due to H2O2 in the MEB and DCIP experiments. Likewise, when samples of used medium from aerobic or anoxic incubations without dyes were treated with immobilized catalase before being analyzed, there was no change in the apparent concentration of lactate beyond that due to calculated dilution. Thus, our measurements of lactate formation in aerobic or anoxic cartilage were not distorted by the presence of H2O2 in the incubation medium.

Analysis of organic acids.

Organic acids in used incubation medium were separated by descending chromatography on Whatman no. 1 paper (23 × 18 cm), using the upper layer of a mixture of butan-1-ol/formic acid/water (4/1/5 by volume) as solvent (31). Before analysis the acid anions were converted to free acids by passage through 0.2-ml columns of Dowex 50X8-400 ion-exchange resin in the H+ form; the eluates were freeze-dried and redissolved in 20 μl water for application to the paper. After chromatography, the paper was dried very thoroughly in warm air, then sprayed with a 0.01% ethanolic solution of Phenol Red. Acids appeared as yellow spots on a pink background.

Reagents.

Chemicals were obtained from Sigma (St. Louis, MO), and 35S-sulfate was from Amersham Pharmacia (Little Chalfont, UK).

Statistical analysis.

The statistical significance of the effects of MEB, DCIP, FECY, ferrocyanide, and O2 on lactate production was tested by one-way analysis of variance.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Reduction of MEB, DCIP, FECY, and oxaloacetate levels by bovine articular cartilage.

All experiments with MEB or DCIP were performed under anoxic (N2-bubbled) conditions to prevent reoxidation of the reduced forms of the dyes by atmospheric O2 (see Materials and Methods). Well-washed bovine articular cartilage readily reduced anoxic solutions of MEB or DCIP. Time courses in representative experiments are shown in Figures 1A and B. Dye reduction depended on the presence of living cells: anoxic solutions of MEB or DCIP remained unchanged in the absence of cartilage, and were not reduced by cartilage that had been boiled for 15 minutes. Lactate is a major product of cartilage metabolism (1), but neither MEB nor DCIP was decolorized by an anoxic solution of L-lactate (10 mM, pH 7.4) without cartilage.

thumbnail image

Figure 1. Time course of reduction of A, methylene blue (MEB), B, 2,6-dichlorophenol-indophenol (DCIP), and C, ferricyanide (FECY) by bovine articular cartilage. In A and B, reduction was measured in a 5 × 1.5–cm plastic tube with a 1 cm–square plastic cuvette glued to it near the top so as to form a side-arm. The tube cap had inlet and outlet needles for gases. Cartilage was incubated in this apparatus for up to 2 hours at 37°C, in 3 ml of standard medium with A, 30 μM MEB or B, 30 μM DCIP, bubbled continuously with N2. At intervals, the N2 flow was stopped briefly and the medium was decanted away from the cartilage into the cuvette side-arm, enabling absorbance (at 665 nm or 600 nm, respectively) to be measured without opening the tube. Results of representative experiments are shown. Values in A are the mean ± SD of 3 replicates (each with 31–33 mg dry weight cartilage) in 1 experiment. Values in B are 2 replicates (with 10 mg and 15 mg dry weight cartilage, respectively) in 1 experiment. In C, cartilage (mean 90 mg dry weight per replicate) was incubated for 3–4 hours at 37°C in 12 ml of standard medium with 0.3 mM potassium FECY, bubbled continuously with air or N2. At intervals, 1-ml samples were withdrawn using a syringe (to avoid opening the tubes) and transferred to a cuvette. Absorbance (at 420 nm) was quickly measured, and the samples were immediately returned to the incubation tubes from which they came. Values in graph 1 are from studies under aerobic conditions (mean ± SD of 3 replicates in 1 experiment); values in graph 2 are from studies under anoxic conditions (mean ± SD of 3 replicates in 1 experiment); values in graph 3 are from a study under aerobic conditions in which the cartilage had been boiled for 30 minutes and then rinsed (anoxic-condition results were similar); values in graph 4 are from a study under aerobic conditions, without cartilage (anoxic-condition results were similar).

Download figure to PowerPoint

Bovine articular cartilage also reduced FECY levels (Figure 1C). The colorless product, ferrocyanide, does not react with atmospheric O2 (30), so it was unnecessary to impose anoxic conditions in order to be able to observe the reaction. Indeed, rates of FECY reduction were similar (P > 0.7) in air-bubbled and N2-bubbled media (Figure 1C, graphs 1 and 2). Minimal changes occurred in controls without cartilage or containing boiled cartilage (Figure 1C, graphs 3 and 4), and as with MEB and DCIP, FECY was not reduced by L-lactate (10 mM). Decolorization of FECY-containing medium was not due to large-scale absorption of FECY into or onto the cartilage, because the used medium without cartilage was restored to its original yellow color (at 97–101% of the original intensity) upon treatment with an oxidizing agent (H2O2 in 100-fold excess) that converts ferrocyanide to FECY.

The α–keto-acid oxaloacetate was tested as an exogenous substrate for well-washed bovine articular cartilage. Paper chromatography showed that an acidic compound whose Rf closely matched that of malic acid (0.52) was present in medium in which the cartilage had been incubated with 20 mM oxaloacetate under anoxic conditions (Figure 2, lanes 2 and 4). The putative malic acid was not detected in used medium following anoxic incubation without oxaloacetate (Figure 2, lanes 1 and 3). A compound corresponding in Rf to lactic acid (0.72) was formed with both treatments. Incubation with oxaloacetate also yielded traces of a more lipophilic acid with an Rf of ∼0.85 (for comparison, a fumaric acid standard ran with Rf 0.86).

thumbnail image

Figure 2. Chromatographic analysis of medium in which bovine articular cartilage had been incubated. Cartilage was incubated under anoxic conditions for 4.5 hours in the standard medium alone, or in standard medium with 20 mM oxaloacetate (OAA). Organic acids in the used incubation medium were analyzed by paper chromatography (see Materials and Methods). Shown are tracings of the chromatograms from 2 separate experiments. The origin line is marked. Treatments applied to the cartilage during incubation were as follows: anoxia alone (lanes 1 and 3), and anoxia with OAA (lanes 2 and 4). MAL LAC = mixture of malate (M) and lactate (L), used as standard (not incubated with cartilage). Approximate Rf values of standards were lactate 0.72, malate 0.52, oxaloacetate 0.35 and 0.70 (perhaps indicating some breakdown during analysis).

Download figure to PowerPoint

Effect of MEB, DCIP, and FECY on glycolysis and 35S-sulfate incorporation.

In initial experiments designed to maximize the effects of MEB, DCIP, or FECY on cartilage metabolism, we supplied and maintained these compounds at high availability, at nominal concentrations of 0.1 mM MEB or DCIP and 0.3–0.5 mM FECY. Anoxic cartilage reduced (decolorized) approximately the same amount of each of these compounds during the experiment; the median amounts reduced (μmoles/gm dry weight cartilage in 4.5 hours) were as follows: MEB ∼26 (results from 5 experiments), DCIP ∼33 (4 experiments), and FECY ∼38 (3 experiments). Under these conditions, anoxic lactate formation was increased by ratios of 2.5 (with MEB), 1.9 (with DCIP), and 1.4 (with FECY), relative to anoxia-only controls (Figure 3) (P < 0.01 in each of 3 separate experiments with each compound). In contrast, the iron (II) complex ferrocyanide (0.3 mM) in anoxic medium did not stimulate lactate production (P > 0.2, combined results of 2 separate experiments). Iron (II) in ferrocyanide is incapable of undergoing further reduction, and ferrocyanide therefore does not function as an oxidant.

thumbnail image

Figure 3. Effect of abundant supplies of A, MEB or DCIP or B, FECY on lactate production by bovine articular cartilage. Cartilage was incubated for 4.5 hours under aerobic or anoxic conditions in 2.4 ml of standard medium in which concentrations of MEB or DCIP near 0.1 mM or of FECY near 0.3–0.5 mM were maintained by periodic addition of concentrated stock solutions as described in Materials and Methods. Values are the mean and SEM of 3 experiments, each with 5 replicates per treatment. See Figure 1 for definitions.

Download figure to PowerPoint

Under aerobic conditions, FECY decreased lactate formation significantly (Figure 3B) (P < 0.01 in each of 3 experiments) compared with aerobic-only controls; this was not due to accumulation of ferrocyanide in the medium because aerobic lactate formation in the presence of ferrocyanide but no FECY was the same as in the aerobic-only controls. Like FECY, MEB in its nonreduced (blue) form was deleterious to aerobic cartilage: 0.05 mM MEB decreased lactate production by 25% in aerobic medium (data not shown).

By way of comparison with artificial oxidants, it should be noted that O2 itself (i.e., air-saturated medium) increased lactate production by a ratio of 2.6 in the experiments with MEB and DCIP, and by a ratio of 1.8 in the experiments with FECY (P < 0.01 in each of 3 experiments with each compound) (Figure 3).

Even submaximum rates of dye or FECY reduction brought about a marked increase in lactate production and 35S-sulfate incorporation by anoxic cartilage (Figure 4). DCIP and FECY were particularly effective substitutes for O2, as judged by their stimulation of lactate formation which, compared with anoxia-only controls, was approximately doubled when 3.7 μmoles DCIP or 5.8 μmoles FECY per gm dry weight of anoxic cartilage was being reduced over a 4.5-hour period (Figure 4).

thumbnail image

Figure 4. Effect of restricted supplies of MEB, DCIP, or FECY on A, lactate production and B, sulfate incorporation by bovine articular cartilage. The procedure was as described in Figure 3 except that smaller quantities of MEB, DCIP, and FECY were used (see Materials and Methods). The amount of MEB, DCIP, or FECY reduced during each experiment (μmoles/gm dry weight cartilage in 4.5 hours) is shown in parentheses. Values are the mean and SD of 5 separate experiments, each with 5 replicates. See Figure 1 for definitions.

Download figure to PowerPoint

Glucose was depleted more rapidly from anoxic medium containing 0.1 mM MEB or DCIP than from anoxic medium alone. However, because of difficulties in measuring small differences in glucose concentration colorimetrically in the presence of dyes, absolute rates of glucose uptake under the latter conditions were somewhat uncertain. In 2 experiments, anoxic-only cartilage and aerobic cartilage took up (respectively) 9–16 and ∼45 μmoles glucose/gm dry weight cartilage in 4.5 hours, from medium containing 1 gm glucose/liter. In the same experiments, when 0.1 mM DCIP was present under anoxic conditions, glucose uptake was approximately midway between the anoxia-only and aerobic-condition rates (above); with 0.1 mM MEB under anoxia, glucose uptake exceeded the aerobic-condition rate. Whatever the exact values of the uptake rates may have been, their rank order was the same in both experiments.

Effect of oxaloacetate, pyruvate, and PEP on glycolysis and 35S-sulfate incorporation.

The keto-acids oxaloacetate and pyruvate, and the glycolytic precursor of pyruvate (PEP), counteracted the effect of anoxia and increased both lactate production and 35S-sulfate incorporation to levels comparable with or greater than those observed in studies with aerobic controls. Results from many separate experiments with each compound, performed on different occasions, are summarized in Figures 5 and 6. In medium containing oxaloacetate, pyruvate, or PEP at 20 mM, anoxic lactate production was typically stimulated by a ratio of ∼3–4.5 (Figure 5A) (P ≤ 0.05 for treatment means in 3–11 experiments, relative to anoxia-only mean). Likewise, anoxic 35S-sulfate incorporation was stimulated by a ratio of >6 (Figure 5B) (P < 0.1 to P < 0.05 for treatment means in 3–9 experiments, relative to anoxia-only mean).

thumbnail image

Figure 5. Effect of oxaloacetate (OAA), pyruvate (PYR), and phosphoenolpyruvate (PEP) on A, lactate production and B, sulfate incorporation by bovine articular cartilage. Cartilage was incubated for 4.5 hours at 37°C under aerobic or anoxic conditions in 2 ml of standard medium with the addition of 20 mM OAA, PYR, or PEP, as their sodium salts, or in the same medium with the addition of equivalent amounts of sodium as NaCl. For each treatment condition, lactate production and sulfate incorporation were expressed as ratios of their values in anoxia-only controls. Values are the mean and SEM of this ratio in 3–23 separate experiments, each with 4–5 replicates per treatment; numbers of experiments are shown in parentheses. Anoxia-only cartilage produced 24 ± 9 μmoles lactate and incorporated 14 ± 9 nmoles sulfate/gm dry weight cartilage in 4.5 hours (mean ± SD of results from 23 and 21 experiments, respectively).

Download figure to PowerPoint

thumbnail image

Figure 6. Effect of oxaloacetate (OAA) or pyruvate (PYR) (“keto-acids”) on lactate production by bovine articular cartilage: inhibition by 2-deoxyglucose (DOG) or iodoacetamide (IOD). The procedure was as described in Figure 5 except that, where indicated, DOG (50 mM) or IOD (10 mM) was added to the incubation medium. Lactate production was expressed as a ratio of its value in anoxia-only controls. For certain treatments (with error bars), values are the mean and SEM of this ratio measured in 3 separate experiments; otherwise the ratio was obtained from 1 experiment. In each experiment there were 5 replicates per treatment. Anoxia-only cartilage produced 22 ± 9 μmoles lactate/gm dry weight cartilage in 4.5 hours (mean ± SD of results from 3 experiments).

Download figure to PowerPoint

Oxaloacetate or pyruvate (or PEP) also increased external glucose uptake under anoxia. Thus, the cartilage took up 5 ± 2 μmoles glucose/gm dry weight in 4.5 hours from anoxic medium containing 0.3 gm glucose/liter (mean ± SEM from 8 experiments). With addition of 20 mM pyruvate or oxaloacetate to the anoxic medium, glucose uptake increased to 23 ± 5 μmoles/gm dry weight cartilage and 39 ± 3 μmoles/gm dry weight cartilage, respectively, in 4.5 hours (mean ± SEM from 4 experiments with each compound). Even so, oxaloacetate and pyruvate were able to stimulate anoxic lactate production in the absence of external glucose. Starting from a lower baseline (anoxic lactate production without glucose was 12 μmoles and 14 μmoles/gm dry weight in 4.5 hours in 2 experiments; compare with Figures 5 and 6), the addition of 20 mM oxaloacetate or 20 mM pyruvate under anoxia increased lactate production by ratios of 2.9 and 2.2, respectively. When glucose uptake and lactate production were measured concurrently under a variety of conditions, the molar ratio of additional lactate production:additional glucose consumption was ∼2:1 (Table 1).

Table 1. Comparison of additional glucose uptake and additional lactate production in response to oxaloacetate, pyruvate, phosphoenolpyruvate (PEP), and O2*
Experimental condition/compound addedMolar ratio of additional lactate production: additional glucose uptakeMedian ratioMean ± SD of ratioP vs. theoretical mean of 2.00
  • *

    Bovine articular cartilage was incubated for 4.5 hours at 37°C under anoxic conditions in medium containing 0.3 gm glucose/liter, with 20 mM oxaloacetate, pyruvate, or PEP added to the medium as sodium salts. Alternatively, cartilage was incubated under aerobic conditions with no addition to the medium. Glucose uptake and lactate production were determined concurrently. The amounts of additional glucose taken up, and of additional lactate produced, were calculated by reference to unsupplemented anoxic controls within the same experiment. Table includes results from 8 experiments altogether.

  • Individual values are from separate experiments.

  • PEP was added in place of pyruvate.

  • §

    Unsupplemented medium in contact with air; each comparison was made with respect to anoxic unsupplemented medium in the same experiment.

Anoxic/oxaloacetate1.88, 1.98, 2.02, 2.272.002.04 ± 0.17>0.8
Anoxic/pyruvate, PEP1.97, 1.99, 2.06, 2.222.022.06 ± 0.11>0.6
Aerobic/O2 (air)§1.65, 1.78, 1.86, 1.91, 1.96, 1.97, 2.13, 2.151.941.93 ± 0.17>0.6

Iodoacetamide and 2-deoxyglucose are powerful inhibitors of glycolysis in aerobic cartilage (4, 5, 17), acting on G3PDH (32) and glucose-6-phosphate isomerase (33), respectively. Iodoacetamide (10 mM) or 2-deoxyglucose (50 mM) prevented oxaloacetate or pyruvate from increasing lactate production (Figure 6) or 35S-sulfate incorporation (results not shown) under anoxia. Increasing the gas-phase CO2 concentration to 5% did not enhance the effect of 20 mM pyruvate on anoxic lactate production or 35S-sulfate incorporation (data not shown).

Certain metabolites closely related to oxaloacetate or pyruvate, or connected with earlier stages of the glycolytic pathway, were tested but exhibited markedly smaller effects than those seen with oxaloacetate, pyruvate, or PEP at the same concentrations. Thus, the glycolytic intermediate 3-phospho-D-glycerate, the Krebs cycle acids L-malate, fumarate, and α-ketoglutarate, and the reducible substrate acetaldehyde (each at 20 mM) caused significant stimulation of lactate production and 35S-sulfate incorporation under anoxic conditions (P < 0.01 with each compound). Treatments without significant effect (P > 0.05) on either lactate production or 35S-sulfate incorporation included succinate, acetate, L-alanine, L-aspartate, L-glutamine (each at 20 mM), phosphocreatine (18 mM), and L-lactate (20 mM, measuring anoxic 35S-sulfate incorporation only).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Oxygen has a powerful influence on cartilage metabolism even though chondrocyte energy is derived mainly from glycolysis (4, 5, 8, 9). Under anoxia, although chondrocytes remained viable for many days (34), rates of both sulfate incorporation and lactate production were low; addition of O2 increased both rates substantially, in a concentration-dependent manner (5, 21). The results of the present study show that this effect was not specific to O2: other oxidants could also stimulate metabolism significantly. When the dyes MEB and DCIP, the inorganic complex FECY, or the keto-acids oxaloacetate and pyruvate were added to anoxic cartilage, lactate production and 35S-sulfate incorporation were greatly increased (Figures 3–6). At the highest availability of MEB, DCIP, or FECY tested (0.1–0.5 mM; Figure 3), these substances increased anoxic glycolysis (lactate production) as effectively as did similar concentrations of O2 (∼0.2 mM dissolved O2 in air-saturated medium at 37°C [35]). The keto-acids oxaloacetate or pyruvate (or PEP) stimulated anoxic glycolysis to a somewhat greater extent (Figure 5). It should be noted, however, that these compounds were supplied at a far higher concentration (20 mM), and the dose-dependence of their action was not investigated.

When O2 or other oxidants were supplied to anoxic cartilage, 35S-sulfate incorporation was stimulated proportionally more strongly than was lactate production (Figures 4 and 5). Figure 7 shows that the same numeric relationship between lactate production and 35S-sulfate incorporation persisted over a wide range of rates, irrespective of which class of oxidant was tested (reducible dyes and FECY in Figure 7A, keto-acids in Figure 7B, O2 in Figures 7A and B).

thumbnail image

Figure 7. Relationship between lactate production and sulfate incorporation in anoxic cartilage supplied with artificial oxidants or O2 (air-saturated medium). The results in A are from Figure 4 and include anoxic, MEB, DCIP, FECY, and aerobic treatments. The results in B are from Figure 5 and include anoxic, OAA, PYR, PEP, and aerobic treatments. Because the 2 variables (lactate production, sulfate incorporation) had broadly comparable experimental error, neither could be regarded as the “independent” variable. Therefore, for each graph, regressions of y on x and of x on y were calculated, and the mean slope of the 2 lines was taken. The mean slopes thus obtained were 1.75 in A and 1.61 in B. The correlation coefficients (r) between lactate production and sulfate incorporation were +0.89 in A and +0.90 in B. See Figures 1 and 5 for definitions.

Download figure to PowerPoint

Iodoacetamide and 2-deoxyglucose have their principal sites of action at different points in the glycolytic pathway (32, 33). The presence of either inhibitor largely prevented oxaloacetate or pyruvate from stimulating glycolysis under anoxia (Figure 6). From this we conclude that a fully functional glycolytic pathway is needed for rapid anoxic lactate formation in the presence of artificial oxidants, as it is for rapid lactate formation in response to O2 itself (5).

Within the limits of our measurements, neither O2 nor the keto-acids affected the stoichiometry of the glycolytic pathway between glucose and lactate, despite a marked increase in flux. The additional anaerobic glycolytic flux that oxaloacetate, pyruvate, or PEP promoted showed a near-theoretical 2:1 molar ratio of lactate produced:glucose consumed, the same molar ratio as was seen when glycolysis was stimulated with O2 (Table 1). These findings demonstrate that irrespective of the rate of glycolysis or how it is stimulated, a close balance is maintained between the production of NADH in the reaction catalyzed by G3PDH and its consumption in the reaction catalyzed by lactate dehydrogenase. Glucose thus remains the principal carbon source for the glycolytic pathway when it is stimulated by other oxidants in place of O2. The fact that pyruvate or oxaloacetate increased lactate formation even when the cartilage was in glucose-free medium (see Results) showed that these keto-acids did not act by overcoming a restriction on the entry of glucose into the cells. The same reasoning applies in the case of O2 as well (5). Articular chondrocytes in vivo are known to contain glycogen (12), which can evidently support continued lactate formation—albeit at a somewhat lower rate—in the absence of external glucose.

Our results also confirm that stimulation of metabolism occurs as the result of reduction of the dyes MEB and DCIP, the inorganic complex FECY, and the keto-acids oxaloacetate and pyruvate; in undergoing reduction, all function as oxidants (electron acceptors) with respect to cartilage. That MEB, DCIP, and FECY were reduced by live cartilage was evident from very obvious color changes (Figure 1). Oxaloacetate could function as an oxidant for the cartilage if it was reduced to L-malate in the reaction catalyzed by L-malate dehydrogenase (EC 1.1.1.37). Malate was present in the used incubation medium (Figure 2). The cytosolic form of malate dehydrogenase, which would be most accessible to externally supplied oxaloacetate, has been detected at high activity in rat articular cartilage (6) and in rabbit articular chondrocytes grown in monolayer culture (36). There is a precedent for compounds such as oxaloacetate serving as oxidants in vivo: the blood parasite Leishmania uses certain 4-carbon acids (oxaloacetate, malate, fumarate) as electron acceptors, reducing them to succinate by partial reversal of the Krebs cycle as a means of reoxidizing NADH (37). The powerful response elicited by oxaloacetate, the smaller but positive effects of L-malate and fumarate, and the inactivity of succinate (see Results) would be consistent with the notion of a similar process operating here.

Like oxaloacetate, externally supplied pyruvate (or PEP) increased glycolysis in anoxic cartilage (Figures 5 and 6). However, little if any of this exogenous pyruvate or PEP can have been reduced to lactate, for if the latter reaction had occurred to a significant extent, the lactate:glucose molar ratio would have risen above the observed value of 2:1 (Table 1). The fate of externally supplied pyruvate in anoxic articular cartilage remains unclear.

A likely mode of action common to both O2 and the other oxidants would be the removal of a reductant from the cells. Whereas MEB or DCIP might function intracellularly, FECY does not penetrate the plasma membrane (38, 39), so it must act at the cell surface, perhaps via a transmembrane oxidoreductase. Under conditions of sustained abundant supply of MEB, DCIP, or FECY, the quantities of these compounds reduced were of the same order as the quantity of additional lactate formed, relative to that in anoxia-only controls (Figure 3). In terms of reductant, decolorization of MEB, DCIP, or FECY at the maximum rates observed in anoxic cartilage (see Results) would consume ∼40–70 microequivalents of electrons/gm dry weight cartilage in 4.5 hours. This flux greatly exceeds the flow of reductant to O2 in aerobic cartilage, which is typically ∼9–18 microequivalents of electrons/gm dry weight cartilage in 4.5 hours (calculated from Table 1 in ref. 1). In short-term studies at least, lipid breakdown needs to be considered as a possible source of the abundant reductant going to MEB, DCIP, and FECY, and also perhaps to externally supplied keto-acids. Lipid droplets are commonly observed in electron microscopy studies of articular chondrocytes (12), and “neutral fats” have been demonstrated with a specific staining procedure (7). Beta-hydroxyacyl dehydrogenase (β-hydroxybutyrate dehydrogenase), a key enzyme in β-oxidation of fatty acids, has been detected cytochemically in articular cartilage (7, 8).

Modulation of the intracellular oxidation-reduction status by oxidants other than O2 has been demonstrated directly in other cell types, usually by measuring the ratio of intracellular pyruvate:lactate concentrations (which reflects the ratio of NADH:NAD+ concentrations in the cytosol). As the following examples show, a change toward a more oxidized intracellular milieu is often beneficial. Thus, in human erythrocytes, FECY decreased the cytosolic NADH:NAD+ ratio (i.e., a shift toward a more oxidized status) and promoted the pentose phosphate pathway and lactate production (40). Brief treatment with other mild oxidants (diamide, H2O2) also promoted erythrocyte glycolysis (41). FECY restored ATP synthesis in erythrocytes when this was partially inhibited by iodoacetate (38). In isolated hepatocytes, hypoxic injury (assessed by loss of ability to exclude trypan blue) was correlated with an increased cytosolic NADH:NAD+ ratio (42). Oxaloacetate, acetoacetate, MEB, or DCIP reduced this ratio and protected the hepatocytes against damage during hypoxia, whereas substrates that favored NADH production (including sorbitol, glycerol, and ethanol) made the injury more severe (42).

Over a longer term, tissue culture studies demonstrate many instances of oxidants improving the growth of cells, even in air-saturated media. Reducible keto-acids, especially glyoxylic, pyruvic, α-ketoglutaratic, and oxaloacetic acid, increased division and colony formation by a variety of cell types in aerobic, serum-free medium (43–45), while the corresponding reduced (hydroxy) forms of the acids were ineffective. Attachment and growth of human melanoma cells in a low-serum medium was enhanced by addition of FECY or pyruvate (46, 47). FECY, or the auto-oxidizable compounds naphthoquinone sulfonate or indigo tetrasulfonate, stimulated growth of HeLa cells in aerobic medium (48, 49). The stimulatory effects of exogenous oxidants on cartilage metabolism are thus consistent with those found in other systems.

For normal function, articular cartilage appears to require exogenous oxidants to stimulate glycolysis, a pathway which evidence from other studies (15–17) strongly suggests is the principal source of ATP for metabolic processes in this tissue, including (for example) the synthesis of extracellular matrix. Under physiologic conditions, O2 acts as this oxidant, but its role can be adequately assumed by other agents. How these oxidants influence glycolysis remains unclear.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Mutchmeats, Witney, UK for supplying abattoir material.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES