1 Department of Pharmacology, Faculty of Medicine, and 2 Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5G 1G6
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ABSTRACT |
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Osteoclasts are
multinucleated cells that resorb bone by extrusion of protons and
proteolytic enzymes. They display marked heterogeneity in cell size,
shape, and resorptive activity. Because high resorptive activity in
vivo is associated with an increase in the average size of osteoclasts
in areas of greater resorption and because of the importance of proton
extrusion in resorption, we investigated whether the activity of the
bafilomycin A1-sensitive vacuolar-type
H+-ATPase (V-ATPase) and amiloride-sensitive
Na+/H+ exchanger differed between large and
small osteoclasts. Osteoclasts were obtained from newborn rabbit bones,
cultured on glass coverslips, and loaded with the pH-sensitive
indicator
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).
Intracellular pH (pHi) was recorded in single osteoclasts by monitoring fluorescence. Large (10 nuclei) and small (
5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pHi, their pHi was decreased by bafilomycin
A1 addition or removal of extracellular Na+,
and the realkalinization upon readdition of Na+ was
bafilomycin A1 sensitive. After acid loading, a
subpopulation of large osteoclasts (40%) recovered by V-ATPase
activity alone, whereas all small osteoclasts recovered by
Na+/H+ exchanger activity. Interestingly, in
60% of the large osteoclasts, pHi recovery was mediated by
both the Na+/H+ exchanger and V-ATPase
activity. Our results show a striking difference between
pHi regulatory mechanisms of large and small osteoclasts
that we hypothesize may be associated with differences in the potential
resorptive activity of these cells.
vacuolar-type hydrogen-adenosinetriphosphatase; sodium/hydrogen exchanger; nuclear number; intracellular pH
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INTRODUCTION |
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OSTEOCLASTS ARE
MULTINUCLEATED cells responsible for bone resorption.
Osteoclastic bone resorption is a complex process starting with
migration and attachment of osteoclasts to the bone surface. This is
followed by the development of a sealing zone, the formation of a
ruffled border, and the subsequent extrusion of protons, Cl, and proteolytic enzymes in the extracellular
resorption zone between the ruffled border and the bone surface
(52). Proton extrusion is accomplished by a vacuolar-type
H+-ATPase (V-ATPase) localized on the ruffled border
(8, 53). Maintenance of intracellular pH (pHi)
is believed to be controlled mainly by Na+/H+
exchangers and Cl
/HCO3
exchangers on
the basolateral membrane (21, 35, 49).
Osteoclasts from humans, rats, and rabbits can under normal circumstances contain anywhere from 2 to 30 nuclei, with an average of 3 to 10 nuclei. However, in diseases characterized by increased bone resorption such as Paget's disease, end-stage renal disease, periodontal disease, and rheumatoid arthritis, osteoclast size is generally increased (1, 28, 32, 45). In Paget's disease, osteoclasts have been reported to reach a diameter of 100 µm and to contain up to 100 nuclei (45).
Osteoclasts become multinucleated as a result of cell fusion, either with other multinuclear osteoclasts or with mononuclear osteoclast precursors (17, 25), and certain enzymes and receptors are up- or downregulated upon formation of multinuclear osteoclasts from their mononuclear precursors (14, 56). However, it is not known whether increased multinuclearity in osteoclasts is associated with changes in gene expression, as has been reported for other multinucleated cells (15, 16, 34, 38, 54). Piper et al. (39) studied the relationship between osteoclast size (as determined by the number of nuclei) and the resorptive ability of these cells and found a positive correlation between the size of an osteoclast and the volume of the resorption pit made. They also found that the volume resorbed per nucleus tended to decrease with increasing nuclear number. However, their observations included only osteoclasts associated with a resorption pit and did not take into account the possibility that osteoclasts can form several pits within a given period of time (27). Thus they may have underestimated the activity of their osteoclasts. We recently found that, in cultures with significantly greater numbers of large osteoclasts, both pit size and pit number were increased, indicating that large osteoclasts were not only resorbing a larger surface area per pit but also formed more pits than smaller osteoclasts (30).
One striking characteristic of osteoclasts is the marked heterogeneity
observed in their behavior and responsiveness to inhibitory and
stimulatory agents. Kanehisa (26) investigated
calcitonin-induced inhibition of osteoclast function and found that,
although the majority of cultured rabbit osteoclasts exposed to
calcitonin stopped migrating and started contracting, others showed no
discernible changes in cytoplasmic motility or general morphology. In
addition, although osteoclasts at 9 of 23 separate resorption sites
regained resorptive activity due to spontaneous escape from
calcitonin-induced contraction, the remaining 14 did not. Another
example was reported by Owens and Chambers (37), who found
that although macrophage colony stimulating factor increased the
percentage of migrating rat osteoclasts from 10 to 60%, the remaining
40% of osteoclasts did not respond to this cytokine. Equally
striking were the observations of Hall et al. (22), who
found that although 75% of rat osteoclasts required continued mRNA and
protein synthesis to resorb bone when cultured in vitro, some cells
still actively resorbed in the presence of the inhibitors actinomycin D
and cycloheximide. In addition, Yu and Ferrier (58)
reported that only 27% of osteoclasts in 1-day cultures responded to
interleukin-1 with an increase in intracellular Ca2+
concentration, whereas 84% responded in 3-day cultures. Heterogeneity has also been observed with regard to pHi regulation; under
conditions of chronic extracellular acidosis and induced acid load,
40% of osteoclasts recovered by V-ATPase activity, whereas the
remaining 60% did not (36, 40). Although some
investigators have associated variable responses with osteoclast size
or shape (2, 40), the reasons for the variable responses
have not been elucidated.
Proton extrusion and pHi regulation have a pivotal role in
the resorptive process. Interestingly, heterogeneity of osteoclasts with regard to activity of the proton pumps has also been noted (36, 40). Therefore, we sought to determine in this study whether small (2-5 nuclei) and large (10 nuclei) osteoclasts differed with regard to V-ATPase and Na+/H+
exchanger activities. We found that basal pHi in large and
small osteoclasts differed significantly, as did the responses to
Na+ removal and to the addition of the V-ATPase inhibitor
bafilomycin A1. Upon acid loading, small osteoclasts
recovered by an Na+-dependent and amiloride-sensitive
mechanism, whereas large osteoclasts consisted of the following two
groups: an Na+-dependent group that was maximally inhibited
by the addition of both amiloride and bafilomycin A1 and an
Na+-independent group that was sensitive only to
bafilomycin A1. These results clearly show that large and
small osteoclasts differ with regard to the activity of proton
extrusion mechanisms.
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EXPERIMENTAL PROCEDURES |
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Materials.
Medium 199, -minimum essential medium (
-MEM), FCS, and
antibiotics (penicillin G, gentamicin, fungizone) were obtained from GIBCO-BRL (Burlington, ON). HEPES-buffered HCO3
-free
RPMI, bafilomycin A1, and HEPES were purchased from Sigma. 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
and nigericin were from Molecular Probes (Eugene, OR). Amiloride was obtained from ICN Biochemicals (Montreal, PQ). All other chemicals were
purchased from Sigma or Fisher.
Osteoclast isolation and identification.
Osteoclasts were isolated from rabbit long bones as described
previously (3, 27). Briefly, the femurs, tibiae, humeri, and radii of 1-day-old New Zealand White rabbits were dissected out.
After removal of adherent soft tissues, the shafts were placed in a
sterile petri dish containing medium 199 and cut longitudinally, and
the interior surfaces were curetted to release the bone cells. The
medium containing cells and bone fragments was agitated by pipetting to
release additional cells attached to the bone fragments. These bone
marrow preparations containing osteoclasts were centrifuged at 200 g for 10 min, and the cells obtained from the bones of one
rabbit were resuspended in 8 ml of -MEM (pH 7.4) with 10% FCS and
antibiotics (100 µg/ml penicillin G, 0.5 µg/ml gentamicin, and 0.3 µg/ml fungizone). The osteoclast-containing cell suspension was
plated on glass coverslips in six-well tissue culture dishes (100 µl/coverslip) and allowed to attach overnight in
-MEM containing 10% FCS and antibiotics in humidified air (37°C and 5%
CO2). The cells were then cultured for 3-8 h in 25 mM
HEPES-buffered, HCO3
-free RPMI with 10% serum at pH
7.4 and 37°C in an air incubator before measurement of
pHi. In some cases, the cells were cultured for 24 h
under these conditions, but the results were pooled as no differences
were found between short- and long-term culture in HEPES-buffered
HCO3
-free medium.
pHi measurements. The pHi was determined using the microfluorometric technique described previously for osteoclasts (35). Briefly, coverslips with cells were placed in a Leiden coverslip dish and were maintained at 37°C for the remainder of the experiment. The cells were loaded with the H+-sensitive fluorescent dye BCECF by incubation with 1 µM of the parent acetoxymethyl ester for 10 min and then were washed with RPMI and incubated in Na+-containing medium (in mM: 140 NaCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 20 HEPES, pH 7.4). For incubation in Na+-free conditions, Na+ was replaced with either K+ or N-methyl-D-glucamine (NMG+). Test solutions were applied to the cells by pipetting 3 ml of the desired medium (with or without inhibitors) into the chamber. Previously used culture medium was removed by aspiration. In some experiments, treatments were applied sequentially to the same cells (see Figs. 2 and 3). Because the effects of amiloride are reversible, it was added first and then was washed out with Na+-containing medium alone, after which bafilomycin A1 was added. In the experiments shown in Figs. 5 and 6, treatments with transport blockers were all independent observations on different cells; however, a few cells in Fig. 6 (19 out of a total of 131) received sequential treatment as in the experiments shown in Figs. 2 and 3. No statistical difference was seen between the results of sequential and independent addition of inhibitors in the experiments shown in Fig. 6, and thus the results were pooled. The rate of pHi recovery shown in Figs. 5 and 6 is the initial maximal rate of change.
Fluorescence intensity in single cells was measured using a Nikon TMD-Diaphot microscope attached to an M-series dual-wavelength illumination system from Photon Technologies. Illumination was on for 2 s and off for 8 s (to minimize photobleaching), and the photometric data were recorded at a rate of 5 points/s. The excitation light was directed to the cells via a 510-nm dichroic mirror. Fluorescence emission collected by a Nikon Fluor ×40 oil-immersion objective traversed a 542 ± 64-nm band-pass filter.Acid loading of osteoclasts. For acid loading, we used the NH4+ "prepulse" method (47). The adherent cells were first exposed to 40 mM NH4Cl for 5-8 min, during which time there was a rapid rise in pHi as NH3 entered the cell and combined with H+ to form NH4+, followed by a slow decline in pHi due to influx of NH4+, which dissociated to form NH3 and H+. Osteoclasts were then transferred to NH4+-free medium, resulting in a rapid loss of NH3, subsequent dissociation of internal NH4+ into NH3 and H+, and a decrease in pHi to a level much lower than the starting pHi. Recovery of osteoclast pHi was then monitored in the cells in the presence of different media and inhibitors of pHi regulatory mechanisms.
pHi calibration.
Calibration of the fluorescence ratio vs. pH of each individual
osteoclast was performed using the K+/H+
ionophore nigericin. Upon completion of the pH experiments, the cells
were equilibrated in K+ medium (140 mM) of varying pH in
the presence of 5 µM nigericin, and calibration curves were
constructed by plotting the extracellular pH [pHo; which
is assumed to be identical to pHi (50)]
against the corresponding fluorescence ratio. Additionally, some cells were calibrated by the null-point method, as originally described by
Eisner et al. (13), to verify the values found with the
nigericin technique. The principle of this method is to find the
mixture of permeant acid (A) and base (B) that, upon entering the cell, produces no change in pHi. The pHi can then be
calculated from the composition of this mixture, according to the
following equation: pHi = pHo 0.5log([A]/[B]), where brackets denote concentration. Thus, with
pHo of 7.4, we prepared Na+-containing
solutions of pH 6.8-7.8 using butyric acid as the weak acid and
trimethylamine as the weak base.
Statistics. Data were analyzed statistically by one-way ANOVA and with an unpaired, double-sided Student's t-test. All results are expressed as means ± SE for n number of cells.
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RESULTS |
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Size distribution of osteoclasts. The osteoclast-containing cell cultures obtained from newborn rabbit long bones were comprised of a variety of stromal cell types and osteoclasts. Osteoclasts were defined as cells having two or more nuclei under phase-contrast microscopy and staining positively for TRAP after fixation. We have reported previously that cells so identified bind calcitonin and resorb bone when plated on bone slices (43, 51). The size and nuclear number of the osteoclasts ranged from cells with a diameter of 30 µm and 2 nuclei to cells with a diameter of 90 µm and 23 nuclei. We studied the following two groups of osteoclasts: small osteoclasts that contained 2-5 nuclei (60% of the total number of osteoclasts) and large osteoclasts that contained 10 or more nuclei (10% of the osteoclasts); we investigated the pHi changes in these cells. This choice was arbitrary and was made to decrease the likelihood of overlap for parameters that might increase or decrease in value gradually.
Determination of pHi in osteoclasts by two independent
methods of calibration.
BCECF was uniformly distributed in the cytoplasmic compartment. Small
osteoclasts maintained a steady resting pHi of 7.34 ± 0.02 (n = 94) when cultured on glass coverslips for
2-6 h in HCO3-free HEPES-buffered RPMI with 10%
FCS, as determined by the nigericin calibration technique. In contrast,
large osteoclasts had a significantly higher pHi of
7.70 ± 0.02 (n = 156) under the same conditions (P < 0.001). This difference in steady-state
pHi between large and small osteoclasts was confirmed when
another calibration technique (the null-point method) was used
(n = 12, Fig.
1A). pHi tracings for a single small osteoclast and a single large osteoclast, calibrated by both the null-point method and then the nigericin method
(y-axis), are shown in Fig. 1, B and
C. The pHi of the representative small osteoclast in Fig. 1B decreased when a null solution of pH
7.05 was added but increased after addition of a null solution of pH 7.45, indicating that this osteoclast had a resting pHi
between these values, i.e., ~7.2. A similar procedure applied to the
large osteoclast shown in Fig. 1C indicates a resting
pHi of ~7.7. Because of the excellent agreement between
the two calibration methods and the greater ease of performing
nigericin calibration, all future calibrations were performed with the
nigericin technique.
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Effect of amiloride and bafilomycin A1 on osteoclast
resting pHi.
Upon addition of amiloride (an inhibitor of the
Na+/H+ exchanger) to Na+-containing
medium, the pHi of both small and large osteoclasts did not
change significantly (Fig.
2A). Upon the removal of
amiloride by washing and the addition of bafilomycin A1
(the specific and widely used inhibitor of the V-ATPase) in
Na+-containing medium to these same cells, the
pHi of the large osteoclasts decreased significantly from
7.59 ± 0.04 to 7.37 ± 0.05 at a rapid initial rate of
0.16 ± 0.01 pH units/min, whereas the pHi of small
osteoclasts did not change. When these same cells were treated with
both amiloride and bafilomycin A1, there was a significant drop in pHi of small osteoclasts from 7.34 ± 0.03 to
7.19 ± 0.05, whereas that of large osteoclasts remained at about
the same level as seen with bafilomycin A1 alone.
Representative traces of a small (Fig. 2B) and a large (Fig.
2C) osteoclast treated with amiloride and then with
bafilomycin A1 are shown.
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Cl channels and V-ATPase activity in small and large
osteoclasts.
Because V-ATPase-mediated proton transport is often charge coupled to
passive Cl
permeability, we wished to ascertain whether
the observed differences in V-ATPase activity between small and large
osteoclasts were due to differences in Cl
channels in
these cells. To evaluate this, the Cl
channel inhibitor
DIDS was added to Na+-containing medium in addition to
bafilomycin A1. Once again, the pHi of small
osteoclasts did not change significantly compared with control
Na+-containing medium (Fig.
3A). Under the same
conditions, the pHi of large osteoclasts decreased
significantly from 7.55 ± 0.05 to 7.35 ± 0.06. Interestingly, however, DIDS alone raised the pHi of both
large and small osteoclasts by 0.2 pH units. Representative traces of a
small and a large osteoclast are shown in Fig. 3, B and
C, respectively.
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The importance of extracellular Na+
in maintaining high pHi in large osteoclasts.
When Na+-containing medium was replaced by K+-
or NMG+-containing medium, the pHi of small
osteoclasts was not affected (Fig. 4,
A and B). Similar treatment of large osteoclasts
resulted in a decrease in pHi by 0.40 pH units. This drop
in pHi of large osteoclasts occurred rapidly (within
20 s) and was maintained until Na+ was added back to
the external medium (Fig. 4C).
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Effect of amiloride and bafilomycin A1 on osteoclast
pHi recovery.
To determine which pH regulatory mechanisms were involved in the pH
recovery of large osteoclasts after readdition of Na+,
the effects of various inhibitors were evaluated independently in
different cells (Fig.
5A). Under
Na+-free conditions, recovery was negligible, averaging
0.01 pH units/min. The addition of Na+ alone resulted in
immediate and rapid recovery at a rate of 0.20 pH units/min. Addition
of amiloride resulted in a 25% inhibition in the initial maximal rate
of Na+-induced pHi recovery. Addition of
bafilomycin A1 resulted in a 70% reduction in the rate of
pHi recovery of large osteoclasts in the presence of
Na+. Complete inhibition of pHi recovery was
seen with addition of both amiloride and bafilomycin A1,
suggesting that both the Na+/H+ exchanger and
the V-ATPase contribute to pHi recovery in the presence of
Na+. Representative traces of three different large
osteoclasts and their recovery in the presence of amiloride,
bafilomycin A1, or amiloride and bafilomycin A1
are shown in Fig. 5, B-D, respectively.
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Effect of amiloride and bafilomycin A1 on osteoclast
pHi recovery after acid load.
Because the results shown above strongly suggested that both the
Na+/H+ exchanger and V-ATPase activity operated
to different degrees in large and small osteoclasts at physiological
pHi, we decided to investigate whether under more acidic
conditions similar differences would be detectable. We used the
ammonium prepulse technique to decrease pHi. Small
osteoclasts did not recover in the absence of Na+ but did
recover in the presence of Na+ (Fig.
6A).
Recovery of pHi in small osteoclasts in the presence of
Na+ was completely inhibited by amiloride, indicating that
the recovery was mediated by amiloride-sensitive
Na+/H+ exchange.
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DISCUSSION |
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The production and extrusion of protons and the maintenance of
pHi are essential processes in the function and activity of osteoclasts. It is widely accepted that V-ATPases found in the ruffled
border of osteoclasts pump protons into the extracellular resorption zone, thus acidifying the bone surface and dissolving the
mineral component of bone (8, 46, 53). Proton extrusion into the extracellular resorption zone is accompanied by passive Cl flux through a Cl
channel. The driving
force behind Cl
movement is the potential difference that
arises from electrogenic proton transport across the ruffled membrane
during bone resorption (7, 41, 44). With regard to
maintenance of an appropriate pHi, Teti et al.
(49) demonstrated that, during bone resorption, one of the
mechanisms of alkaline export was an Na+-independent
Cl
/HCO3
exchanger. Other reports
indicate the presence and function of an amiloride-sensitive
Na+/H+ exchanger (20, 21, 31, 35)
and a proton conductance (35). However, in many cases,
differences in pHi regulatory responses between osteoclasts
of the same preparation have been reported, in particular with regard
to Na+/H+ exchange and V-ATPase activities for
which the investigators have had no explanation or attributed them to
differences in substrate, stage of resorption cycle, or cell shape
(20, 31, 35, 40, 59, 60).
The first striking finding in the present investigation was the
existence of a significant difference (0.4 pH units) in steady-state pHi of small and large osteoclasts maintained in
HEPES-buffered Na+-containing medium at pH 7.4. The value
obtained for small osteoclasts was in good agreement with that observed
in a number of other cell types and with other reports on osteoclasts
cultured on glass in nominally HCO3-free,
HEPES-buffered medium at pH 7.4 (35, 36, 40, 49, 60).
However, large osteoclasts had an unusually high pHi of 7.70. Because Boyarsky and colleagues (10) recently
reported that the nigericin technique to calibrate BCECF in vascular
smooth muscle cells could lead to errors of up to 0.2 pH units in
estimating steady-state pHi, we verified our results using
the null-point method. This method has been reported to be more
reliable than the nigericin method in some instances because it is
independent of K+ concentration, thus eliminating the error
involved in estimating intracellular K+ (4, 10, 11,
13, 57). The results of the two methods of calibration were in
excellent agreement, indicating that the difference in pHi
between large and small osteoclasts is not an artifact of the recording
system. Interestingly, Ravesloot et al. (40) in their
experiments with rat osteoclasts also found that a small percentage of
the cells tested had high initial pHi values, but they did
not attempt to associate this with a specific population of
osteoclasts. Other reports on osteoclast pHi regulation do
not mention the size of osteoclasts studied, although Nordstrom et al.
(35) did specifically state that the majority of their studies were performed on compact osteoclasts containing three to five
nuclei. The great abundance of small osteoclasts compared with large
ones (60 vs. 10%) in rabbit, rat, and chick osteoclast preparations
cultured on glass coverslips (our results and Refs. 3 and 39) and the
greater ease of performing single-cell experiments with cells of a
smaller diameter would suggest that other investigators may also have
concentrated mainly on smaller osteoclasts.
The cause of the high basal pHi in large osteoclasts was
further investigated. First, we excluded the possibility that the high
pHi in large osteoclasts was a result of maintaining the cells in HEPES-buffered medium. Buckler et al. (11) had
shown that in type I carotid body cells of neonatal rats the remarkably alkaline pHi (7.8) was due to the transfer of these cells
from CO2-containing to HEPES-buffered
(CO2-free) medium. They observed that, when measuring
pHi within 1 h of transfer, the pHi was
7.8, but if the cells were cultured in HEPES-buffered medium for
24 h, the pHi was 7.3. However, this was not the case
for osteoclasts in our study; large osteoclasts cultured for 24 h
in HEPES-containing medium had the same alkaline pHi as
those cultured for 2 h, whereas small osteoclasts had the same low
pHi (7.3) at the two time points (results not shown). It is
also relevant in this regard that Teti et al. (49) found
that avian osteoclasts attached to bone and transferred to
HCO3-free HEPES-containing medium became slightly
acidified, not alkaline, within 2 min of transfer and then recovered to
resting pHi within 20 min.
We next discovered that, although Na+ was required for large osteoclasts to maintain their alkaline pHi, the Na+/H+ exchanger was not the primary mechanism involved in this process, as addition of amiloride did not lower the resting pHi of large cells. However, amiloride added after Na+ removal slightly inhibited the recovery of these cells (25% inhibition) to their starting pHi upon readdition of Na+ to the extracellular medium, suggesting a small role for the Na+/H+ exchanger. Interestingly, the specific V-ATPase inhibitor bafilomycin A1 inhibited the Na+-dependent alkalinization by 70%, whereas the combination of bafilomycin A1 and amiloride completely inhibited the recovery. The fact that V-ATPase activity was Na+ dependent in these cells was surprising because V-ATPase activity is generally not linked to the presence of Na+ in the extracellular medium. However, Mernissi et al. (33) did report Na+-dependent ATPase activity in the rat nephron that was clearly not a Na+-K+-ATPase, K+-ATPase, or Ca2+-ATPase, but they did not look at pH regulation or bafilomycin sensitivity to determine whether it was a V-ATPase. Taken together, our findings suggest that both small and large osteoclasts cultured on glass contain active proton pumps but that their role in pHi regulation is much more important in large osteoclasts than in small osteoclasts. This was further supported by our observation that the addition of bafilomycin A1 resulted in a significant decrease in basal pHi in large osteoclasts but not in small ones.
One possible explanation for the difference in V-ATPase activities
between large and small osteoclasts could be the variation in number or
activities of Cl channels in these cells. Because
V-ATPase activity is often charge coupled to passive Cl
permeability, a lack of anion conductance due to insufficient Cl
channels may inhibit further V-ATPase activity due to
the electrogenic nature of this pump (9, 44). Thus lower
V-ATPase activity in small osteoclasts could be due to fewer
Cl
channels in these cells compared with large
osteoclasts and not directly as a result of fewer or less active
V-ATPases. Various reports have indeed demonstrated the presence of
Cl
channels in osteoclasts (7, 9, 29, 41,
44). Interestingly, Sims et al. (44) and Kelly et
al. (29) reported that only 30-50% of their
osteoclasts had a basal Cl
current; however, they
used very high concentrations of the Cl
channel
inhibitors DIDS and SITS (0.5-1 mM). Such high concentrations may
have inhibited other ion currents as well, including V-ATPases (9). To study Cl
channel activity in our
cells, we added 10-100 µM DIDS [shown by Blair et al.
(9) to reversibly inhibit Cl
channels in
osteoclasts without affecting other proton transport mechanisms] in
addition to 200 nM bafilomycin A1. The pHi of
small osteoclasts did not change significantly, whereas that of large osteoclasts did decrease, thus indicating that the differences in small
and large osteoclast responses to bafilomycin A1 were not
caused by differences in Cl
channel activity.
Surprisingly, DIDS addition alone resulted in a rise in pHi
in both large and small osteoclasts, likely due to inhibition of
Cl
/HCO3
exchange activity in these
cells. That this transporter continues to be active in our cells is
possible, because significant accumulation of HCO3
by
carbonic anhydrase activity may continue in osteoclasts maintained in
nominally HCO3
-free medium as a result of
CO2 production in association with glycolysis and ATP
generation in the mitochondria (6). The fact that both
large and small osteoclasts exhibited a similar increase is further
evidence that anion translocation is not different in these cells, only
V-ATPase activity.
To further study the characteristics of the pHi regulatory mechanisms in large and small osteoclasts, we next examined the recovery of these cells from an acid load in the presence of various inhibitors. In agreement with previously reported data for osteoclasts (35, 60), the recovery of small osteoclasts from very low pHi was Na+ dependent, amiloride sensitive, and bafilomycin A1 resistant. All of these data suggest a classical Na+/H+ exchanger as the primary mechanism of realkalinization in these cells. The fact that this antiporter was very active in recovery from an acid load in small osteoclasts but was not very active at physiological pH in these cells is consistent with other reports that Na+/H+ exchangers only become active below a set-point pH of ~6.8 (5, 19, 23, 55).
The recovery from an acid load by large osteoclasts, however, was more complex. Surprisingly, 40% of the large osteoclasts recovered at a very fast rate from an acid load in the absence of Na+ (Na+ independent), whereas 60% of the large osteoclasts recovered from an acid load in an Na+-dependent manner (Na+ dependent). The Na+-independent cells did not respond to amiloride but were sensitive to inhibition by bafilomycin A1, indicating that V-ATPase was exclusively responsible for the realkalinization. Ravesloot et al. (40) also described a subpopulation of cells with active proton pumps in rat osteoclasts cultured on glass. Similar to our results, they found that these cells recovered from an acid load in the absence of Na+ and also had a higher mean initial pHi. They attempted to correlate the presence of high initial pHi and recovery from an acid load in the absence of Na+ with cell shape (round and well spread vs. polygonal with cytoplasmic extensions) but concluded that osteoclast shape was not sufficient to explain the differences. They did not report on the number of nuclei seen in their cells. We also examined the shape of our rabbit osteoclasts and found that the great majority of our cells were round and did not display irregularly shaped cytoplasmic extensions as described by Ravesloot et al. (40). This may be due to the fact that rat osteoclasts take on a stellar appearance when transferred to serum-free medium (18), whereas similarly treated rabbit osteoclasts do not (Lees and Heersche, unpublished observations). Ingber et al. (24) and Schwartz et al. (42) reported that spreading of bovine capillary endothelial cells and normal fibroblasts leads to rapid activation of the Na+-H+ antiporter and elevation of pHi. They also found that fully spread cells had a higher pHi than round cells and that local clustering of integrins led to activation of the Na+-H+ antiporter and hence the rise in pHi. The evidence available for osteoclasts suggests that this explanation for the difference in pHi of large and small osteoclasts is not likely, because pHi did not seem to correlate with the degree of spreading but rather with nuclear number.
In the Na+-dependent category of large osteoclasts, pHi recovery was only partially inhibited by amiloride and bafilomycin A1, but when amiloride and bafilomycin A1 were added together, pHi recovery was completely inhibited. The presence and activity of both a V-ATPase and Na+/H+ exchanger in the same cell has also been reported in other cell types, i.e., alveolar macrophages, rabbit outer medullary collecting duct cells, rat papillary collecting duct cells, and guinea pig pancreatic duct cells (5, 12, 23, 48). The findings of De Ondarza and Hootman (12) in guinea pig pancreatic duct cells were similar to ours in that neither amiloride nor bafilomycin A1 alone significantly inhibited pHi recovery from an acid load, whereas both inhibitors added together completely abolished this recovery.
Osteoclasts cultured on bone slices cycle between an actively resorbing phase and a migratory phase (27). Lehenkari et al. (31) reported that active, bone-resorbing osteoclasts regulate their pHi differently from osteoclasts cultured on glass or nonresorbing cells cultured on bone. The active osteoclasts on bone had greater V-ATPase activity than Na+/H+ exchange activity and had a slightly higher pHi than inactive cells. It is tempting to hypothesize that in our experiments the category of large Na+-independent cells that rely on V-ATPase activity exclusively for regulating their pHi represent the actively resorbing population. If large resorbing osteoclasts remain activated when transferred to our culture system, then this could account for our finding of two categories of large osteoclasts. This could imply that small osteoclasts and the category of large Na+-dependent osteoclasts are in an inactive or nonresorbing state. We are currently performing experiments using a system in which we can distinguish between actively resorbing and nonresorbing osteoclasts to test this hypothesis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Lamara Shrode for technical assistance and Dr. Sergio Grinstein for valuable comments and critical review of the manuscript.
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FOOTNOTES |
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This work was supported by the Arthritis Society of Canada.
Portions of this work were presented in part as an abstract at the 19th Annual Meeting of the American Society for Bone and Mineral Research, Cincinnati, OH, 1997.
Address for reprint requests and other correspondence: J. N. M. Heersche, Faculty of Dentistry, Rm. 403, Univ. of Toronto, 124 Edward St., Toronto, Ontario, Canada M5G 1G6 (E-mail: johan.heersche{at}utoronto.ca).
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. §1734 solely to indicate this fact.
Received 24 November 1999; accepted in final form 27 March 2000.
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REFERENCES |
---|
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---|
1.
Aota, S,
Nakamura T,
Suzuki K,
Tanaka Y,
Okazaki Y,
Segawa Y,
Miura M,
and
Kikuchi S.
Effects of indomethacin administration on bone turnover and bone mass in adjuvant-induced arthritis in rats.
Calcif Tissue Int
59:
385-391,
1996[ISI][Medline].
2.
Arkett, SA,
Dixon SJ,
and
Sims SM.
Substrate influences rat osteoclast morphology and expression of potassium conductances.
J Physiol (Lond)
458:
633-653,
1992[Abstract].
3.
Asotra, S,
Gupta AK,
Sodek J,
Aubin JE,
and
Heersche JNM
Carbonic anhydrase II mRNA expression in individual osteoclasts under "resorbing" and "nonresorbing" conditions.
J Bone Miner Res
9:
1115-1122,
1994[ISI][Medline].
4.
Babcock, DF.
Examination of the intracellular ionic environment and of ionophore action by null point measurements employing the fluorescein chromophore.
J Biol Chem
258:
6380-6389,
1983
5.
Bidani, A,
Brown SES,
and
Heming TA.
pHi regulation in alveolar macrophages: relative roles of Na+-H+ antiport and H+-ATPase.
Am J Physiol Lung Cell Mol Physiol
266:
L681-L688,
1994
6.
Blair, HC.
How the osteoclast degrades bone.
Bioessays
20:
837-846,
1998[ISI][Medline].
7.
Blair, HC,
and
Schlesinger PH.
Purification of a stilbene sensitive chloride channel and reconstitution of chloride conductivity into phospholipid vesicles.
Biochem Biophys Res Commun
171:
920-925,
1990[ISI][Medline].
8.
Blair, HC,
Teitelbaum SL,
Ghiselli R,
and
Gluck S.
Osteoclastic bone resorption by a polarized vacuolar proton pump.
Science
245:
855-857,
1989[ISI][Medline].
9.
Blair, HC,
Teitelbaum SL,
Tan H-L,
Koziol CM,
and
Schlesinger PH.
Passive chloride permeability charge coupled to H+-ATPase of avian osteoclast ruffled membrane.
Am J Physiol Cell Physiol
260:
C1315-C1324,
1991
10.
Boyarsky, G,
Hanssen C,
and
Clyne L.
Inadequacy of high K+/nigericin for calibrating BCECF. I. Estimating steady-state intracellular pH.
Am J Physiol Cell Physiol
271:
C1131-C1145,
1996
11.
Buckler, KJ,
Vaughan-Jones RD,
Peers C,
and
Nye PCG
Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat.
J Physiol (Lond)
436:
107-129,
1991[Abstract].
12.
De Ondarza, J,
and
Hootman SR.
Confocal microscopic analysis of intracellular pH regulation in isolated guinea pig pancreatic ducts.
Am J Physiol Gastrointest Liver Physiol
272:
G124-G134,
1997
13.
Eisner, DA,
Kenning NA,
O'Neill SC,
Pocock G,
Richards CD,
and
Valdeolmillos M.
A novel method for absolute calibration of intracellular pH indicators.
Pflügers Arch
413:
553-558,
1989[ISI][Medline].
14.
Elleder, M.
Enzyme patterns in human endocytotic multinucleate giant cellsa histochemical study.
Acta Histochem
79:
1-10,
1986[ISI][Medline].
15.
Enelow, RI,
Sullivan GW,
Carper HT,
and
Mandell GL.
Cytokine-induced human multinucleated giant cells have enhanced candidacidal activity and oxidative capacity compared with macrophages.
J Infect Dis
166:
664-668,
1992[ISI][Medline].
16.
Fais, S,
Burgio VL,
Silvestri M,
Capobianchi MR,
Pacchiarotti A,
and
Pallone F.
Multinucleated giant cells generation induced by interferon-gamma. Changes in the expression and distribution of the intercellular adhesion molecule-1 during macrophage fusion and multinucleated giant cell formation.
Lab Invest
71:
737-744,
1994[ISI][Medline].
17.
Fischman, DA,
and
Hay ED.
Origin of osteoclasts from mononuclear leucocytes in regenerating newt limbs.
Anat Rec
143:
329-334,
1962[ISI].
18.
Fuller, K,
Owens JM,
Jagger CJ,
Wilson A,
Moss R,
and
Chambers TJ.
Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.
J Exp Med
178:
1733-1744,
1993[Abstract].
19.
Grinstein, S,
and
Furuya W.
Characterization of the amiloride-sensitive Na+-H+ antiport of human neutrophils.
Am J Physiol Cell Physiol
250:
C283-C291,
1986
20.
Gupta, A,
Edwards JC,
and
Hruska KA.
Cellular distribution and regulation of NHE-1 isoform of the Na-H exchanger in the avian osteoclast.
Bone
18:
87-95,
1996[ISI][Medline].
21.
Hall, TJ,
and
Chambers TJ.
Na+/H+ antiporter is the primary proton transport system used by osteoclasts during bone resorption.
J Cell Physiol
142:
420-424,
1990[ISI][Medline].
22.
Hall, TJ,
Schaeublin M,
and
Chambers TJ.
The majority of osteoclasts require mRNA and protein synthesis for bone resorption in vitro.
Biochem Biophys Res Commun
195:
1245-1253,
1993[ISI][Medline].
23.
Hays, SR,
and
Alpern RJ.
Apical and basolateral membrane H+ extrusion mechanisms in inner stripe of rabbit outer medullary collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
259:
F628-F635,
1990
24.
Ingber, DE,
Prusty D,
Frangioni JV,
Cragoe EJJ,
Lechene C,
and
Schwartz MA.
Control of intracellular pH and growth by fibronectin in capillary endothelial cells.
J Cell Biol
110:
1803-1811,
1990[Abstract].
25.
Jaworski, ZFG,
Duck B,
and
Sekaly G.
Kinetics of osteoclasts and their nuclei in evolving secondary Haversian systems.
J Anat
133:
397-405,
1981[ISI][Medline].
26.
Kanehisa, J.
Time course of "escape" from calcitonin-induced inhibition of motility and resorption of disaggregated osteoclasts.
Bone
10:
125-129,
1989[ISI][Medline].
27.
Kanehisa, J,
and
Heersche JNM
Osteoclastic bone resorption: in vitro analysis of the rate of resorption and migration of individual osteoclasts.
Bone
9:
73-79,
1988[ISI][Medline].
28.
Kaye, M,
Zucker SW,
Leclerc YG,
Prichard S,
Hodsman AB,
and
Barré P-E.
Osteoclast enlargement in endstage renal disease.
Kidney Int
27:
574-581,
1985[ISI][Medline].
29.
Kelly, MEM,
Dixon SJ,
and
Sims SM.
Outwardly rectifying chloride current in rabbit osteoclasts is activated by hyposmotic stimulation.
J Physiol (Lond)
475:
377-389,
1994[Abstract].
30.
Lees, RL,
and
Heersche JNM
Macrophage colony stimulating factor increases osteoclastic bone resorption in long-term osteoclast-containing rabbit bone marrow cultures by increasing osteoclast size.
J Bone Miner Res
14:
937-945,
1999[ISI][Medline].
31.
Lehenkari, PP,
Laitala-Leinonen T,
Linna T-J,
and
Vaananen HK.
The regulation of pHi in osteoclasts is dependent on the culture substrate and on the stage of the resorption cycle.
Biochem Biophys Res Commun
235:
838-844,
1997[ISI][Medline].
32.
Makris, GP,
and
Saffar JL.
Quantitative relationship between osteoclasts, osteoclast nuclei and the extent of the resorbing surface in hamster periodontal disease.
Arch Oral Biol
27:
965-969,
1982[ISI][Medline].
33.
Mernissi, GE,
Barlet-Bas C,
Khadouri C,
Marsy S,
Cheval L,
and
Doucet A.
Characterization and localization of ouabain-insensitive Na-dependent ATPase activities along the rat nephron.
Biochim Biophys Acta
1064:
205-211,
1991[ISI][Medline].
34.
Nishii, H,
Ashitaka Y,
Maruo M,
and
Mochizuki M.
Studies on the effect of thyroid hormone and epidermal growth factor on the cultured human cytotrophoblast.
Endocrinol Jpn
38:
279-286,
1991[Medline].
35.
Nordstrom, T,
Rotstein OD,
Romanek R,
Asotra S,
Heersche JNM,
Manolson MF,
Brisseau GF,
and
Grinstein S.
Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance.
J Biol Chem
270:
2203-2212,
1995
36.
Nordstrom, T,
Shrode LD,
Rotstein OD,
Romanek R,
Goto T,
Heersche JNM,
Manolson MF,
Brisseau GF,
and
Grinstein S.
Chronic extracellular acidosis induces plasmalemmal vacuolar type H+ ATPase activity in osteoclasts.
J Biol Chem
272:
6354-6360,
1997
37.
Owens, J,
and
Chambers TJ.
Macrophage colony-stimulating factor (M-CSF) induces migration in osteoclasts in vitro.
Biochem Biophys Res Commun
195:
1401-1407,
1993[ISI][Medline].
38.
Papadimitriou, JM,
and
Van Bruggen I.
Evidence that multinucleate giant cells are examples of mononuclear phagocytic differentiation.
J Pathol
148:
149-157,
1986[ISI][Medline].
39.
Piper, K,
Boyde A,
and
Jones SJ.
The relationship between the number of nuclei of an osteoclast and its resorptive capability in vitro.
Anat Embryol (Berl)
186:
291-299,
1992[ISI][Medline].
40.
Ravesloot, JH,
Eisen T,
Baron R,
and
Boron WF.
Role of Na-H exchangers and vacuolar H+ pumps in intracellular pH regulation in neonatal rat osteoclasts.
J Gen Physiol
105:
177-208,
1995[Abstract].
41.
Schlesinger, PH,
Blair HC,
Teitelbaum SL,
and
Edwards JC.
Characterization of the osteoclast ruffled border chloride channel and its role in bone resorption.
J Biol Chem
272:
18636-18643,
1997
42.
Schwartz, MA,
Lechene C,
and
Ingber DE.
Insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integrin , independent of cell shape.
Proc Natl Acad Sci USA
88:
7849-7853,
1991[Abstract].
43.
Shibutani, T,
and
Heersche JNM
Effect of medium pH on osteoclast activity and osteoclast formation in cultures of dispersed rabbit osteoclasts.
J Bone Miner Res
8:
331-336,
1993[ISI][Medline].
44.
Sims, SM,
Kelly ME,
and
Dixon SJ.
K+ and Cl currents in freshly isolated rat osteoclasts.
Pflügers Arch
419:
358-370,
1991[ISI][Medline].
45.
Singer, FR,
and
Roodman GD.
Paget's disease of bone.
In: Principles of Bone Biology, edited by Bilezikian JP,
Raisz LG,
and Rodan GA.. San Diego, CA: Academic, 1996, p. 969-977.
46.
Sundquist, K,
Lakkakorpi P,
Wallmark b.,
and
Vaananen K.
Inhibition of osteoclast proton transport by bafilomycin A1 abolishes bone resorption.
Biochem Biophys Res Commun
168:
309-313,
1990[ISI][Medline].
47.
Swallow, CJ,
Grinstein S,
and
Rotstein OD.
Regulation of cytoplasmic pH in resident and activated peritoneal macrophages.
Biochim Biophys Acta
1022:
203-210,
1990[ISI][Medline].
48.
Takeda, K,
Oohara T,
Tabel K,
and
Asano Y.
Dual regulatory mechanisms of proton transport in rat papillary collecting duct cells in culture.
Jpn J Physiol
39:
397-410,
1989[ISI][Medline].
49.
Teti, A,
Blair HC,
Teitelbaum SL,
Kahn AJ,
Koziol C,
Konsek J,
Zambonin-Zallone A,
and
Schlesinger PH.
Cytoplasmic pH regulation and chloride/bicarbonate exchange in avian osteoclasts.
J Clin Invest
83:
227-233,
1989[ISI][Medline].
50.
Thomas, JA,
Buchsbaum RN,
Zimniak A,
and
Racker E.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
18:
2210-2218,
1979[ISI][Medline].
51.
Turksen, K,
Kanehisa J,
Opas M,
Heersche JNM,
and
Aubin JE.
Adhesion patterns and cytoskeleton of rabbit osteoclasts on bone slices and glass.
J Bone Miner Res
3:
389-400,
1988[ISI][Medline].
52.
Vaananen, K.
Osteoclast function: biology and mechanisms.
In: Principles of Bone Biology, edited by Bilezikian JP,
Raisz LG,
and Rodan GA.. San Diego, CA: Academic, 1996, p. 103-113.
53.
Vaananen, HK,
Karhukorpi EK,
Sundquist K,
Wallmark B,
Roininen I,
Hentunen T,
Tuukkanen J,
and
Lakkakorpi P.
Evidence for the presence of a proton pump of the vacuolar H+-ATPase type in the ruffled borders of osteoclasts.
J Cell Biol
111:
1305-1311,
1990[Abstract].
54.
Vignery, A,
Wang F,
Qian HY,
Benz EJ, Jr,
and
Gilmore-Hebert M.
Detection of the Na+-K+-ATPase 3-isoform in multinucleated macrophages.
Am J Physiol Renal Fluid Electrolyte Physiol
260:
F704-F709,
1991
55.
Wakabayashi, S,
Fafournoux P,
Sardet C,
and
Pouyssegur J.
The Na+/H+ antiporter cytoplasmic domain mediated growth factor signals and controls "H+-sensing".
Proc Natl Acad Sci USA
89:
2424-2428,
1992[Abstract].
56.
Woods, C,
Domenget C,
Solari F,
Gandrillon O,
Lazarides E,
and
Jurdic P.
Antagonistic role of vitamin D3 and retinoic acid on the differentiation of chicken hematopoietic macrophages into osteoclast precursor cells.
Endocrinology
136:
85-95,
1995[Abstract].
57.
Yamashiro, DJ,
and
Maxfield FR.
Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.
J Cell Biol
105:
2713-2721,
1987[Abstract].
58.
Yu, H,
and
Ferrier J.
Interleukin-1 alpha induces a sustained increase in cytosolic free calcium in cultured rabbit osteoclasts.
Biochem Biophys Res Commun
191:
343-350,
1993[ISI][Medline].
59.
Yu, H,
and
Ferrier J.
Osteoclast ATP receptor activation leads to a transient decrease in intracellular pH.
J Cell Sci
108:
3051-3058,
1995
60.
Zimolo, Z,
Weslowski G,
and
Rodan GA.
Acid extrusion is induced by osteoclast attachment to bone. Inhibition by alendronate and calcitonin.
J Clin Invest
96:
2277-2283,
1995[ISI][Medline].