pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis

Sajeda Meghji1, Matthew S. Morrison2, Brian Henderson1, and Timothy R. Arnett2

1 Oral and Maxillofacial Surgery, Eastman Dental Institute, London WC1X 8LD; and 2 Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the effects of HCO3- and CO2 acidosis on osteoclast-mediated Ca2+ release from 3-day cultures of neonatal mouse calvaria. Ca2+ release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H+ to culture medium (HCO3- acidosis). For example, addition of 4 meq/l H+ reduced pH from 7.12 to 7.03 and increased Ca2+ release 3.8-fold. The largest stimulatory effects (8- to 11-fold), observed with 15-16 meq/l added H+, were comparable to the maximal Ca2+ release elicited by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; 10 nM], parathyroid hormone (10 nM), or prostaglandin E2 (1 µM); the action of these osteolytic agents was attenuated strongly when ambient pH was increased from ~7.1 to ~7.3. CO2 acidosis was a less effective stimulator of Ca2+ release than HCO3- acidosis over a similar pH range. Ca2+ release stimulated by HCO3- acidosis was almost completely blocked by salmon calcitonin (20 ng/ml), implying osteoclast involvement. In whole mount preparations of control half-calvaria, ~400 inactive osteoclast-like multinucleate cells were present; in calvaria exposed to HCO3- acidosis and to the other osteolytic agents studied, extensive osteoclastic resorption, with perforation of bones, was visible. HCO3- acidosis, however, reduced numbers of osteoclast-like cells by ~50%, whereas 1,25(OH)2D3 treatment caused increases of ~75%. The results suggest that HCO3- acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones, rather than by inducing formation of new osteoclasts, and provide further support for the critical role of acid-base balance in controlling osteoclast function.

calcium release; carbon dioxide; bicarbonate ion; acid-base balance; osteolysis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SKELETON CONTAINS a massive reserve of base that is available as a "fail-safe" mechanism to buffer protons if the kidney and lungs are unable to maintain acid-base balance within narrow physiological limits (8, 15). The deleterious effects of systemic acidosis on the skeleton have been recognized at least since the early part of this century (5, 18). More recent in vivo studies have suggested that the bone loss associated with acidosis is not due to passive physicochemical processes but involves enhanced osteoclastic resorption (9, 22).

Experiments with disaggregated rat osteoclasts cultured on cortical bone or dentine wafers provided the first direct evidence for the stimulatory action of extracellular protons on cell-mediated bone resorption. Resorption pit formation by rat osteoclasts in culture media buffered nonphysiologically, with the use of HEPES only, was activated by progressive acidification from pH 7.4 to 6.8 (3). Cultured osteoclasts are also stimulated to resorb in physiologically buffered media when pH is reduced either by decreasing HCO3- concentration or by increasing PCO2 (2). Recent work has shown that rat osteoclasts are particularly sensitive to extracellular pH in the range 7.2-7.0, such that shifts of <0.1 unit are sufficient to cause changes of severalfold in pit formation (6).

Experiments with cultured mouse calvaria showed that calcium efflux from bones was stimulated much more strongly when extracellular pH was reduced by decreasing HCO3- concentration (metabolic acidosis) than by increasing PCO2 (respiratory acidosis) (11, 12). The aims of the present study were 1) to investigate the effects of small extracellular pH changes resulting from HCO3- and CO2 acidosis on osteoclastic resorption in cultured calvaria; 2) to compare the effects of acidosis on calvarial resorption with those of "classical" osteolytic agents and to determine whether the action of these agents was dependent on ambient acidification; and 3) to investigate whether acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones or by inducing formation of new osteoclasts.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] was kindly provided by Dr K. W. Colston (St. George's Hospital Medical School, London, UK). BWA 70C, a 5-lipoxygenase inhibitor of the iron ligand class, containing the hydroxamic acid chelating group, was a gift of the Wellcome Foundation (Beckenham, Kent, UK). MK 886, a selective inhibitor of the 5-lipoxygenase-activating protein (FLAP), was donated by Merck Frosst (Kirkland, QC, Canada). Other reagents, unless specified, were purchased from Sigma (Poole, Dorset, UK).

Mouse calvarial bone resorption assay. The method, which measures bone resorption as Ca2+ release from neonatal mouse calvaria, was similar to that described in detail by Meghji et al. (24). Briefly, 5-day-old MF1 mice were killed by cervical dislocation. The frontoparietal bones were removed and trimmed of any adhering connective tissue and interparietal bone, with care being taken not to damage the periosteum. Dissected calvaria were pooled, washed free of blood and adherent brain tissue in Hanks' balanced salt solution, and then divided along the sagittal suture. Half-calvaria were cultured individually on 1-cm2 stainless steel grids (Minimesh, FDP quality, Expanded Metal, West Hartlepool, UK) in 6-well plates with 1.5 ml of BGJ medium, 5% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (ICN Biomedicals, Basingstoke, Hants, UK) at the air-liquid interface in a humidified CO2 incubator. After an initial 24-h preincubation period, the medium was removed and replaced with control or test media. Prostaglandin E2 (PGE2), 1,25(OH)2D3, indomethacin, BWA 70C, and MK 886 were dissolved in ethanol vehicle for use; the final concentration of ethanol in cultures did not exceed 1:500. Each experimental group consisted of five individual cultures. Included in each experiment were groups of half-calvaria that had been killed by three cycles of freeze-thawing with liquid nitrogen. The cultures were then incubated for 72 h without further medium changes and without the incubator door being opened, so as to ensure constant CO2 levels and minimize pH fluctuations. Culture medium acidification was achieved either by adding small amounts of concentrated HCl to culture medium as described by Murrills et al. (31), resulting in decreased HCO3- concentration (metabolic acidosis) or by increasing incubator CO2 tension (respiratory acidosis).

After 72 h, experiments were terminated by withdrawal of culture medium and washing of bones once with PBS, followed by fixation in 95% ethanol-5% glacial acetic acid for 10 min. Incubator PCO2 was determined by immediate measurment of a culture medium sample by use of a blood gas analyzer (Radiometer ABL 330, Copenhagen, Denmark). The mean final pH of each treatment group was determined by removal and pooling of a 100-µl sample from each replicate; the pooled samples were then reequilibrated with CO2 in the incubator before measurement with the blood gas analyzer. Slight differences in CO2 tension between groups were normalized to the initially measured value using pH-PCO2 calibration curves constructed for BGJ medium, as previously described (31). Bicarbonate concentrations were calculated by the Radiometer ABL 330 blood gas analyzer by use of the Henderson-Hasselbalch equation.

Calcium concentrations in culture medium at the end of experiments were measured colorimetrically by autoanalyzer (Chem Lab Instruments, Essex, UK) with the following procedure. Samples were acidified with excess 1 M HCl and subjected to continuous flow dialysis against the metal complexing agent cresolphthalein complexone (CPC) to separate Ca2+ from proteins; 8-hydroxyquinoline (2.5 g/l) was added to samples to eliminate Mg2+ interference. Dialyzed Ca2+ bound to CPC was then determined after reaction with 2-amino-2-methylpropano-l-ol; the absorbance of the resultant purple-colored solution was measured at 570 nm. The basal calcium concentration of the BGJ medium after addition of 5% heat-inactivated fetal calf serum was 2.00 mM. All measurements were performed blind on coded samples.

Whole mount histology. After fixation/decalcification with 95% ethanol-5% glacial acetic acid, calvaria were stained for tartrate-resistant acid phosphatase (TRAP) (20, 23) by means of a Sigma kit 387-A and were mounted in melted glycerol jelly. The numbers of TRAP-positive multinucleated osteoclasts (two or more nuclei) were assessed blind on coded samples by means of transmitted light microscopy.

Statistics. Statistical comparisons, where appropriate, were made by one-way analysis of variance, with the use of Bonferroni's correction for multiple comparisons. Representative data are presented as means ± SE for 5 replicates. Results are shown for representative experiments that were each repeated at least three times.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of HCO3- acidosis on calvarial resorption. Basal levels of Ca2+ release in nonacidified control bones (pH 7.20-7.25) were very low after 3 days of culture. Acidification of culture medium by addition of small amounts of H+ as HCl resulted in a steep increase in Ca2+ release from live bones. For example, decreasing the pH of the culture medium from 7.21 (control) to 7.17 by addition of 3 meq/l H+ reduced HCO3- concentration from 13.2 to 12.2 mmol/l, resulting in a 2.8-fold increase in Ca2+ release, and decreasing the pH to 6.94 by addition of 15 meq/l H+ reduced HCO3- concentration to 7.1 mmol/l, causing an 8.3-fold stimulation of Ca2+ release. In contrast, in bones killed by freeze-thawing, there was a marked influx of Ca2+, evidenced by a decrease in culture medium Ca2+ concentration; the magnitude of this influx was slightly reduced with progressive acidification (Fig. 1).


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Fig. 1.   Stimulatory effect of small decreases in medium pH, achieved by adding H+ (i.e., HCO3- acidosis) as HCl, on Ca2+ release from live mouse half-calvaria cultured for 3 days (open bars). Addition of 0, 3, 6, 9, 12, and 15 meq/l H+ to culture medium resulted in calculated HCO3- concentrations of 13.2, 12.2, 10.9, 10.4, 8.5, and 7.1 mmol/l, respectively after 3 days of culture. In dead bones killed by freeze-thawing (hatched bars), a net Ca2+ influx occurred that was slightly reduced as pH decreased (triangle ). PCO2 was 36.3 mmHg. Values are means ± SE (n = 5). Significantly different from nonacidified control: *P < 0.05; **P < 0.01; ***P < 0.001.

Effect of CO2 acidosis. When culture medium was acidified from 7.12 to 6.90 by increasing incubator PCO2 from 54.5 to 87.2 mmHg, only a small, nonsignificant increase in Ca2+ release from calvaria was observed after 3 days of culture. The stimulation of Ca2+ release resulting from CO2 acidosis was markedly lower than that elicited by HCO3- acidosis at comparable pH values. However, further acidification to pH 6.81 by increasing PCO2 to 108.5 mmHg resulted in a 5.3-fold stimulation of Ca2+ release compared with control. This stimulation occurred in the face of an increase in HCO3- concentration from 15.8 to 22.5 mM (Fig. 2).


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Fig. 2.   Stimulation of Ca2+ release from mouse half-calvaria by HCO3- acidosis (produced by addition of 4, 8, 12, or 16 meq/l H+ to culture medium) was greater than the stimulation resulting from CO2 acidosis (produced by raising culture medium PCO2 to 87.2 or 108.5 mmHg) at comparable pH values. In dead bones (hatched bar), severe CO2 acidosis was associated with a small Ca2+ influx; effects of HCO3- acidosis on dead bones are shown in Fig. 1. Increasing PCO2 from 54.5 to 87.2 and 108.5 mmHg resulted in calculated HCO3- concentrations of 15.8, 21.6, and 22.5 mmol/l, respectively, whereas addition of 4, 8, 12, and 16 meq/l H+ to culture medium at constant PCO2 (54.5 mmHg) yielded calculated HCO3- concentrations of 13.5, 11.3, 9.7, and 8.4 mmol/l, respectively. Ca2+ release values are means ± SE (n = 5). Significantly different from control: **P < 0.01; ***P < 0.001.

Effects of inhibitors. HCO3- acidosis-stimulated Ca2+ release (resulting from addition of 15 meq/l H+) was completely blocked by salmon calcitonin (20 ng/ml; Fig. 3). Thus Ca2+ release stimulated by HCO3- acidosis was due to osteoclastic resorption. Ca2+ release resulting from severe CO2 acidosis was also blocked by salmon calcitonin (20 ng/ml; data not shown). HCO3- acidosis-stimulated Ca2+ release was additionally completely blocked by the cyclooxygenase inhibitors indomethacin (Fig. 4) and ibuprofen (data not shown) and was inhibited by the 5-lipoxygenase inhibitors, BWA 70C (Fig. 5A) and MK 886 (Fig. 5B), suggesting that the effect may be mediated by both prostaglandins and leukotrienes.


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Fig. 3.   Complete blockage of HCO3- acidosis-stimulated Ca2+ release from 3-day cultures of mouse half-calvaria by salmon calcitonin (sCT; 20 ng/ml). PCO2 was 36.3 mmHg. Values are means ± SE (n = 5). Significantly different from control: ***P < 0.001.



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Fig. 4.   Complete blockage of HCO3- acidosis-stimulated Ca2+ release from 3-day cultures of mouse half-calvaria by the cyclooxygenase inhibitor indomethacin. Hatched bar shows Ca2+ release in nonacidified control cultures. PCO2 was 64.0 mmHg. Values are means ± SE (n = 5). Significantly different from cultures treated with 12 meq/l H+ alone: *P < 0.05; **P < 0.01.



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Fig. 5.   Inhibition of HCO3- acidosis-stimulated Ca2+ release from 3-day cultures of mouse half-calvaria by the 5-lipoxygenase inhibitors BWA 70C (A) and MK 886 (B). Hatched bars show Ca2+ release in nonacidified control cultures. PCO2 was 64.0 mmHg. Values are means ± SE (n = 5). Significantly different from cultures treated with 12 meq/l H+ alone: *P < 0.05.

Effects of "classical" resorption stimulators. The maximal effects of the osteolytic agents 1,25(OH)2D3 (10 nM), bovine parathyroid hormone-(1-34) (PTH, 20 ng/ml) and PGE2 (1 µM) on Ca2+ release from 3-day calvarial cultures were similar and were equivalent in magnitude to the effect of HCO3- acidosis at pH 6.94 (15 meq/l added H+; Fig. 6). In most experiments, PTH, 1,25(OH)2D3, and PGE2 treatments themselves resulted in slight acidification of culture medium.


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Fig. 6.   Equivalence of the stimulatory action of HCO3- acidosis (15 meq/l H+; pH 6.94) on Ca2+ release from mouse half-calvaria to the maximal effects of the classical osteolytic agents prostaglandin E2 (PGE2), bovine parathyroid hormone-(1-34) (PTH), or 1,25-dihydroxyvitamin D3 (1,25 D3). PTH and 1,25(OH)2D3 treatment resulted consistently in slight acidification of culture medium. PCO2 was 54.5 mmHg. Values are means ± SE (n = 5). Significantly different from pH 7.21 control: ***P < 0.001.

In an additional series of experiments, we examined the dependence of the osteolytic action of PGE2, 1,25(OH)2D3, and PTH on ambient acidification in 3-day cultures of mouse half-calvaria. Addition of 15 meq/l OH- as NaOH increased final medium pH from ~7.1 in control cultures to ~7.3, resulting in marked attenuation of Ca2+ release in all treatment groups (Fig. 7). Addition of equivalent amounts of Na+ as NaCl to culture medium was without effect on bone resorption. In the case of PGE2-stimulated bones, increasing pH from 7.12 to 7.33 reduced resorption by 80%.


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Fig. 7.   Dependence of the osteolytic action of PGE2, 1,25(OH)2D3, and PTH on ambient acidification in 3-day cultures of mouse half-calvaria. Addition of 15 meq/l OH- as NaOH increased final medium pH from ~7.1 in control cultures to ~7.3, resulting in marked attenuation of Ca2+ release in all treatment groups. PCO2 was 57.2 mmHg. Values are means ± SE (n = 5). Significantly different from control group (C): *P < 0.05; ***P < 0.001; significantly different from respective, nonalkalinized treatment group: #P < 0.05 (PTH), ##P < 0.01 (PGE2).

Histology (TRAP staining) and cell counting. In whole mount preparations of half-calvaria cultured in control medium for 3 days and then stained to demonstrate tartrate-resistant acid phosphatase, ~200-400 inactive TRAP-positive osteoclast-like multinucleate cells were typically present; resorption cavities visualized by the TRAP staining were small and relatively inactive (Figs. 8A and 9). The appearance of freshly isolated bones (i.e., not cultured) was similar. In calvaria exposed to HCO3- acidosis (>= 12 meq/l H+), extensive osteoclastic resorption cavities, with characteristic scalloped edges, were evident (Fig. 8B); resorption was sometimes sufficiently aggressive to cause complete perforation of bones. In bones exposed to severe CO2 acidosis (108.5 mmHg) at pH 6.82, osteoclasts in scalloped resorption bays were also clearly evident (Fig. 8C). Extensive resorption cavities were also observed in bones treated with 10 nM 1,25(OH)2D3 (Fig. 8D), 20 ng/ml PTH-(1-34), or 1 µM PGE2. However, cell counts revealed that HCO3- acidosis reduced the numbers of osteoclast-like cells by ~50% compared with controls, whereas 1,25(OH)2D3 treatment caused increases of ~75% (Fig. 9).


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Fig. 8.   Whole mount mouse half-calvaria stained to demonstrate tartrate-resistant acid phosphatase (TRAP) after 72-h culture, viewed by transmitted light microscopy. A: nonacidified control, pH 7.208, showing small resorption cavities (arrowheads); scale bar = 500 µm. B: bone treated with 12 meq/l H+, pH 7.01 (HCO3- acidosis); scale bar = 500 µm. C: higher power detail of bone exposed to severe CO2 acidosis (108.5 mmHg) at pH 6.82; osteoclasts in scalloped resorption bays (arrowheads) are clearly evident; scale bar = 100 µm. D: bone treated with 10 nM 1,25(OH)2D3, pH 7.09; scale bar = 500 µm. Large areas of osteolysis, demonstrated by red-black TRAP staining of scalloped resorption fronts and osteoclasts (arrowheads), are visible in acidosis and 1,25(OH)2D3-treated bones.



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Fig. 9.   Stimulation of TRAP-positive osteoclast (OC)-like cell numbers in whole mount mouse half-calvaria by 10 nM 1,25(OH)2D3 (1,25 D3), but not 1 µM PGE2 or HCO3- acidosis resulting from addition of 15 meq/l H+. Significantly different from control (C): *P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study correlates Ca2+ release data with histological evidence to demonstrate that osteoclastic resorption in cultured mouse calvarial bones is extremely sensitive to activation by HCO3- acidosis, an effect which can cause bone destruction equivalent to the maximal osteolysis produced by agents such as 1,25(OH)2D3, PTH, or PGE2. Furthermore, our results show that the action of these classical osteolytic agents is attenuated markedly by slight alkalinization. We also found that severe CO2 acidosis resulted in increased osteoclastic resorption.

Three separate lines of evidence indicate that net Ca2+ release into the culture medium from mouse calvarial bones stimulated by HCO3- acidosis is almost entirely the result of osteoclast activity. First, salmon calcitonin (20 ng/ml) completely blocked the Ca2+ release from live bones stimulated by HCO3- acidosis. Second, in bones killed by freeze-thawing, HCO3- acidosis exerted only a minimal effect, i.e., a slight reduction of the net Ca2+ influx that was always observed. Although such dead bones do not provide a perfect control because freeze-thawing may render the cellular lining of calvarial surfaces more "leaky," our data suggest that the influence of HCO3- acidosis on physicochemical Ca2+ exchange was small, even at large acid loads. These results are in essential agreement with the earlier findings of Goldhaber and Rabadjija (17). Third, in calvaria exposed to HCO3- acidosis (>= 12 meq/l H+), extensive osteoclastic resorption cavities were present; such cavities were absent in calcitonin-treated bones.

Our findings show that bone resorption in cultured mouse calvaria may be more sensitive to small pH changes than was previously appreciated. Ca2+ release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H+ (i.e., HCO3- acidosis) to culture medium. Figure 2 shows that addition of 4 meq/l H+ reduced pH from 7.12 to 7.03 but increased Ca2+ release 3.8-fold; an 11-fold increase was observed with 16 meq/l added H+. The steep responses to HCO3- acidosis evident in Figs. 1 and 2 resemble closely the acid-activation curve for resorption pit formation by cultured rat osteoclasts in which resorption is essentially "switched off" above pH 7.2 and stimulated maximally at pH ~6.9 (6, 31). Osteoclasts derived from chick long bones (4, 28), human osteoclastoma tissue (19), or long-term mouse marrow cultures (29) show remarkably similar responses to extracellular pH changes. Taken together, these observations suggest that the local pH in the microenvironment of osteoclasts in cultured mouse parietal bones is close to that of the tissue culture medium and thus considerably lower than that of blood pH (~7.40).

We also demonstrated that CO2 acidosis is a less effective stimulator of Ca2+ release from calvarial bones than HCO3- acidosis over a similar pH range, a result that is in good agreement with data from the 45Ca2+ flux experiments of Bushinsky and colleagues (11, 12). One partial explanation for the smaller stimulatory effect of CO2 acidosis may be that hypercapnia promotes deposition of Ca2+, as carbonates, on bone surfaces (11). Nevertheless, our own results show that resorption is activated quite strongly by severe CO2 acidosis at low pH (PCO2 = 108.5 mmHg; pH = 6.81). It may be that, when PCO2 is increased, the "set point" at which resorption begins to be acid activated is shifted to lower pH values. It is also noteworthy that the striking stimulation of bone resorption caused by severe CO2 acidosis occurred despite an increase in HCO3- concentration (22.5 mM compared with control value of 15.8 mM). This result suggests that, in the main, it is the increased H+ concentration that is ultimately responsible for acidosis-stimulated osteclastic bone resorption. Experiments with cultured osteoclasts have not shown clear differences between the effects of CO2 and HCO3- acidosis in stimulating pit formation (Ref. 2; M. Morrison and T. R. Arnett, unpublished data). The reasons for this discrepancy are unknown. In vivo, HCO3- acidosis is associated with bone loss (reviewed in Ref. 5). In thyroparathyroidectomized rats, acute HCO3- acidosis induced by acid feeding results in a striking hypercalcemia that is prevented by calcitonin or colchicine, implying osteoclast involvement (22). The effects of CO2 acidosis in vivo are less well investigated; however, a recent study (32) reported that hormone replacement therapy causes a respiratory alkalosis in normal postmenopausal women and that changes in blood pH were inversely correlated with those in urinary excretion of hydroxyproline, an index of bone resorption.

The behavior of the calvarial system in response to acid stimulation also differs from that of cultured osteoclasts in another key respect. We found that resorption stimulated by HCO3- acidosis could be blocked by the cyclooxygenase inhibitors indomethacin and ibuprofen, in line with earlier findings (17, 33) and suggesting a requirement for endogenous prostaglandin synthesis. In contrast, resorption pit formation by cultured osteoclasts is stimulated by cyclooxygenase inhibitors (26, 27) and is inhibited by prostaglandins (4, 13). Our observation that HCO3- acidosis-activated resorption was additionally attenuated by two mechanistically distinct inhibitors of the 5-lipoxygenase pathway suggests that leukotrienes, which stimulate bone resorption in a number of systems (16, 25), are also involved in mediating the effect.

In this study, we correlated a biochemical index of calvarial bone resorption (Ca2+ release) with quantitative histological analysis of the same bones. Surprisingly, perhaps, this approach has not been used before. The simple whole mount histological technique we used was first described by Marshall and colleagues (20, 23), who reported that ~200 multinucleate, TRAP-positive osteoclasts were visible in single parietal bones of 4-day-old mice before being placed in culture; our own data for control bones agree well with these values. In calvaria exposed to HCO3- acidosis, extensive osteoclastic resorption cavities were evident, but TRAP-positive osteoclast numbers were reduced consistently. Whether this change in osteoclast numbers reflects reductions in osteoclast survival, formation, or both, is uncertain. Cell culture experiments have shown that osteoclast formation in 10-day mouse marrow cultures is inhibited at pH 6.9-7.0 (29); however, survival of mature osteoclasts in this pH range appears unimpaired (3, 6, 31). Thus the impressive resorption cavities seen in calvaria exposed to acidosis may reflect mainly the activation of preexisting quiescent osteoclasts rather than the formation of new osteoclasts. However, the possibility cannot be excluded that acidosis rapidly stimulates the formation of new osteoclasts, which are then immediately activated, alongside increased apoptosis of preexisting quiescent osteoclasts.

In contrast, the osteolytic effects of 1,25(OH)2D3 appeared to be related, at least in part, to increases in TRAP-positive osteoclast numbers, reflecting the stimulatory action of this hormone on osteoclast formation in long-term marrow cultures. Ca2+ release in 1,25(OH)2D3-treated cultures was similar to that in cultures exposed to HCO3- acidosis, but resorption on a per cell basis was ~3.5-fold lower, based on the cell counts shown in Fig. 9. Interestingly, cultures treated with 1,25(OH)2D3, PTH, or PGE2 generally exhibited slight acidification compared with controls. Similar effects of PTH have been described previously (10, 14); this is probably due to a stimulation of H+ efflux from osteoblastic target cells (7, 35). We have recently found that 1,25(OH)2D3 also stimulates extracellular acidification by cultured primary osteoblast-like cells derived from rodent calvaria (34). Other bone-resorbing agents reported to stimulate H+ efflux from osteoblast-like cells include insulin-like growth factor I (35), interleukin-1 (1, 34) and ATP (21). Obviously, such acidification could account for some of the osteolytic action of these agents (5, 30).

In conclusion, our findings indicate that the modulation of osteoclast activity by small pH changes is a key determinant of bone resorption in mouse calvarial cultures. Similar responses to extracellular acidification have been observed in all bone resorption systems examined to date. The great sensitivity of osteoclasts to extracellular protons may have evolved in vertebrates as a last line of defense against systemic acidosis.


    ACKNOWLEDGEMENTS

We are grateful to the Arthritis Research Campaign for support. M. S. Morrison was the recipient of a Medical Research Council PhD studentship.


    FOOTNOTES

Address for reprint requests and other correspondence: T. R. Arnett, Dept. of Anatomy and Developmental Biology, University College London, Gower St., London WC1E 6BT, UK (E-mail: t.arnett{at}ucl.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 May 2000; accepted in final form 6 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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