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 |
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 |
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 |
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 |
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 ( ). 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.
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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.
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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.
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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.
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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).
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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.
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 |
DISCUSSION |
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.
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