(Received for publication, August 21, 1995; and in revised form, February 15, 1996)
From the
We used highly purified avian osteoclasts and isolated membranes
from osteoclasts to study effects of tamoxifen, 4-hydroxytamoxifen,
calmodulin antagonists, estrogen, diethylstilbestrol, and the
anti-estrogen ICI 182780 on cellular degradation of H-labeled bone in vitro and on membrane HCl
transport. Bone resorption was reversibly inhibited by tamoxifen,
4-hydroxytamoxifen, and trifluoperazine with IC
values of
1 µM. Diethylstilbestrol and 17-
-estradiol had
no effects on bone resorption at receptor-saturating concentrations,
while ICI 182780 inhibited bone resorption at concentrations greater
than 1 µM. At these concentrations ICI 182780, like
tamoxifen, inhibits calmodulin-stimulated cyclic nucleotide
phosphodiesterase activity. Membrane HCl transport, assessed by
ATP-dependent acridine orange uptake, was unaffected by
17-
-estradiol and diethylstilbestrol at concentrations up to 10
µM, while ICI 182780 inhibited HCl transport at
concentrations greater than 1 µM. In contrast HCl
transport was inhibited by tamoxifen, 4-hydroxytamoxifen, and the
calmodulin antagonists, trifluoperazine and calmidazolium, with
IC
values of 0.25-1.5 µM. These results
suggested the presence of a membrane-associated non-steroid receptor
for tamoxifen in osteoclasts. Tamoxifen binding studies demonstrated
saturable binding in the osteoclast particulate fraction, but not in
the nuclear or cytosolic fractions. Membranes enriched in ruffled
border by differential centrifugation following nitrogen cavitation
showed binding consistent with one site, K
1 µM. Our findings indicate that tamoxifen
inhibits osteoclastic HCl transport by binding membrane-associated
target(s), probably similar or related to calmodulin antagonist
targets. Further, effects of estrogens or highly specific
anti-estrogens on bone turnover do not support the hypothesis of a
direct effect on osteoclasts by these compounds in this species.
Transport of Ca into and out of bone is
critical for maintenance of serum calcium activity. This requires
continuous bone turnover at variable rates, which is mediated by the
osteoclast. However, skeletal mineral is also structurally vital, so
osteoclastic activity is regulated by multiple factors, often acting in
opposing directions. Several hormonal signals are involved in this
regulation, including peptides and low molecular weight
factors(1) . Steroids including estrogens have major effects on
bone turnover(2) , but the receptors and intermediary signaling
involved are not established. This study was performed to determine the
mechanism of steroid-related effects on central biochemical elements of
osteoclastic activity.
A limiting biochemical step and the central
regulated element of bone turnover is secretion of HCl to dissolve the
bone mineral. This is driven by a vacuolar-like
H-ATPase that is highly expressed in a unique
osteoclastic organelle, the ruffled membrane(3) . Multiple
intermediary cell signals influence the activity of acid secretion, but
one of critical interest is intracellular calcium activity and the
ubiquitous calcium-binding protein, calmodulin. The unique
acid-dependent dissolution of calcium salts produces high local
extracellular calcium activity(4) , which is reflected in an
elaborate osteoclastic calcium regulatory mechanism including a
calmodulin-dependent calcium ATPase(5) , and factors
influencing osteoclastic intracellular calcium activity such as matrix
attachment(6) . The vacuolar-like H
-ATPase
driving acid secretion in osteoclasts is also calmodulin-dependent, and
osteoclasts concentrate calmodulin at the ruffled membrane(7) .
The anti-estrogenic compound, tamoxifen, reduces bone turnover(8) , suggesting that tamoxifen may be a particularly useful tool to dissect osteoclast control pathways. Tamoxifen, a known calmodulin antagonist (9) , is a triphenylethylene derivative with low toxicity and strong antitumor activity, particularly in breast cancer, properties ascribed to its anti-estrogenic activity(10) . Tamoxifen may thus regulate osteoclastic activity by either calmodulin or steroid receptor interactions. In contrast to expectations that tamoxifen would cause bone loss because of anti-estrogenic properties(11) , it preserves bone mass (8, 12) and has estrogen-like effects on human bone metabolism (13) . Tamoxifen has mixed estrogenic and anti-estrogenic effects, which can be tissue-specific. The ethoxyaminoalkyl side chain of tamoxifen is known to be essential for both the anti-estrogenic and calmodulin antagonistic effects(14) . The estrogen receptor is a calmodulin-binding protein(15) , and derivatives of estrogen substituted with the ethoxyaminoalkyl side chain of tamoxifen prevent the binding of calmodulin to the estrogen receptor(16) .
Consequently, we studied the effects of tamoxifen and its active metabolite, 4-hydroxytamoxifen, on osteoclastic HCl transport and cellular activity, and compared these effects to those of calmodulin antagonists, estrogens, and a specific anti-estrogen. We report that tamoxifen and 4-hydroxytamoxifen inhibit membrane acid transport and osteoclastic bone resorption with dose responses similar to the calmodulin antagonists, while neither estrogens nor a highly specific steroidal anti-estrogen showed measurable effects at relevant concentrations. In keeping with these findings, tamoxifen binds to osteoclast cell membrane fractions with a dissociation constant consistent with concentrations observed to have functional effects on whole cell activity and cell membrane ATP-dependent HCl transport. These data support a model for control of osteoclastic acid secretion by tamoxifen and related compounds that is not directly related to estrogen receptors, and further suggest that compounds such as tamoxifen act on the acid-secreting membrane by a mechanism similar to other calmodulin antagonists.
Figure 1: Characterization of osteoclast preparations. Osteoclasts were isolated by bone affinity binding (see ``Experimental Procedures'') and characterized by 121F monoclonal antibody and tartrate-resistant acid phosphatase activity (20, 21) . Scale markers indicate 15 µm. A, a field of cells is shown by transmitted light; bone fragments used in the isolation are seen as refractile angular acellular material (arrows). B, fluorescein-labeled 121F antibody tags all of the cells in the same field as in panel A. Some cells show a rim of bright stain suggesting membrane-associated reactivity (arrow). C, tartrate-resistant acid phosphatase was demonstrated using naphthol 6-bromo-2-phospho-3-naphthoyl-2-methoxyanilide phosphate as substrate, in 4 mM tartrate at pH 5.6, and fast garnet 2-methyl-4-[(2-methylphenyl)-azo]benzene diazonium hydrochloride to show the product as red color. The affinity isolated cells are all positive, while bone fragments used in the procedure do not react (arrows). D, control cells similar to those in panel B, but reacted with non-immune serum at the same concentration.
17--Estradiol and
diethylstilbestrol did not inhibit resorption of metabolically labeled
bone by affinity-purified avian osteoclasts at concentrations
meaningful with respect to receptor-mediated effects (Fig. 2).
Estradiol had no measurable effect on osteoclastic bone resorption (Fig. 2A). A trend toward increased activity, on the
order of 10%, was seen with diethylstilbestrol at concentrations
greater than 10
M (Fig. 2B), but was not statistically different.
Affinity constants of estrogen receptors for these ligands are on the
order of 10
M, so that concentrations of
10
M, and certainly 10
M, would be saturating even if the 10% serum proteins in
the assay medium reduced the effective free steroid activity by an
order of magnitude. Similarly, the specific anti-estrogen ICI 182780
had no effect on activity of affinity-purified avian osteoclasts at
physiologically meaningful concentrations (Fig. 2C).
ICI 182780 inhibited bone resorption only at concentrations greater
than 10
M, with 50% inhibition at
approximately 10
M. The inhibition was
reversible on removal of the compound, and so is not related to cell
death. Estrogen, diethylstilbestrol and anti-estrogen effects on
degradation of [
H]proline-labeled bone were also
tested in partially purified osteoclast preparations, made by serum
sedimentation of cells extracted from medullary bone of
calcium-deprived laying hens but without bone affinity purification.
These results were qualitatively similar to the assays performed with
affinity-purified osteoclasts.
Figure 2:
Effect of estrogenic compounds and the
anti-estrogen ICI 182780 on osteoclastic bone resorption. Avian
osteoclasts purified by bone binding (Fig. 1) were incubated for
4 days with 200 µg of 25-50-µm
[H]-labeled bone fragments in charcoal-stripped,
phenol red-free medium (see ``Experimental Procedures'')
without addition (controls) and with increasing concentrations of
17-
-estradiol (A), diethylstilbestrol (B), and
ICI 182780 (C) (horizontal axis).
H
released to the media, representing degraded bone (vertical
axis), was determined by scintillation counting. All results are
standardized as percent of control activity to eliminate interassay
differences in cellular activity. At 10
,
10
, and 5
10
M, n = 4; other points are means of multiple quadruplicate
tests: n = 12 (10
,
10
, 10
M); n = 16 (control, 10
, 10
M). Mean ± S.E. Average control resorption was
30% of total substrate.
Tamoxifen, 4-hydroxytamoxifen, and
the calmodulin antagonist trifluoperazine inhibited bone resorption by
purified osteoclasts at physiologically relevant concentrations (Fig. 3), in contrast to the estrogen-related compounds which
were ineffective at receptor-saturating concentrations (Fig. 2).
Tamoxifen, 4-hydroxytamoxifen and trifluoperazine inhibited bone
resorption in a concentration-dependent manner with maximal inhibitions
near 7 µM. Effects were saturating and reversible: Half
maximal inhibition was 1 µM for all compounds.
Removal of the substances resulted in return of osteoclastic bone
resorption to control levels during an additional 3-day incubation
(data not shown), indicating that these concentrations did not kill the
osteoclasts. While the effective concentrations of these compounds are
10
- to 10
-fold greater than those relevant to
steroid receptors, these IC
values are typical for
calmodulin-antagonist effects and are similar to peak serum levels
obtained for tamoxifen in the treatment of breast cancer, 0.5-1
µM. As with estrogen and anti-estrogen experiments,
similar results were obtained when partially purified osteoclasts were
used.
Figure 3:
Inhibition of avian osteoclast bone
resorption by tamoxifen and trifluoperazine. Purified avian osteoclasts
were incubated 4 days with 200 µg of 20-40-µm
[H]-labeled bone fragments in micromolar
concentrations of tamoxifen (A), 4-hydroxytamoxifen (B), or trifluoperazine (C). Note that the effects of
the calmodulin antagonist trifluoperazine are similar to those of
tamoxifen and its metabolite in this concentration range. Inhibition of
bone resorption was calculated relative to bone degradation by
osteoclasts without drug addition (vertical axis) from
H released into the media, less no-cell controls. Data for
tamoxifen and trifluoperazine are from three quadruplicate experiments (n = 12); data for 4-hydroxytamoxifen are from one
quadruplicate experiment (n = 4). Mean ±
S.E.
Because ICI 182780 inhibited osteoclasts at concentrations
above 1 µM where the calmodulin antagonists were also
effective, we tested whether this anti-estrogen is also a calmodulin
antagonist. This would not be inconsistent with the specificity of ICI
182780 anti-estrogenic effects (27) because in that capacity
its K is 10
- to 10
-fold
lower. Further, a variety of compounds with hydrophobic planar groups
and flexible polar side chains, a description that fits ICI 182780, are
calmodulin antagonists. To test calmodulin inhibition without
introducing systematic bias, we used an unrelated in vitro calmodulin-stimulated phosphodiesterase assay. Both tamoxifen and
ICI 182780 inhibited this calmodulin-dependent system similarly, with
half-maximal effects at
2-4 µM (Fig. 4).
Figure 4:
Effects
of tamoxifen and ICI 182780 on calmodulin-stimulated cyclic nucleotide
phosphodiesterase activity. The indicated concentrations of tamoxifen (closed symbols) and ICI 182780 (open symbols) were
added to the cyclic nucleotide phosphodiesterase assay (see
``Experimental Procedures''). In this assay all values are
compared to a control assay run in the presence of carrier alone
(100%). Note that both compounds are inhibitors in this assay, with
IC
2-4 µM. Data are means of two
to five determinations; error bars average
10% and are omitted for
clarity.
Figure 5:
Effects of various concentrations of
tamoxifen and trifluoperazine in combination. A, avian
osteoclast-enriched fractions from serum sedimentation (not further
purified by bone affinity binding; see ``Experimental
Procedures'') were incubated with labeled bone for 3 days in
carrier (column 1), 0.7 µM tamoxifen (column
2), 0.7 µM trifluoperazine (column 3), or
0.7 µM tamoxifen plus 0.7 µM trifluoperazine (column 4). Bone degraded was calculated from H
released into the media as percent control (vertical axis).
Data are compiled from three separate experiments each performed in
quadruplicate, mean ± S.E. Statistical difference compared to
carrier: *, p = 0.03;**, p = 0.06. Bone
resorption in the presence of both substances (column 4) is
less than that with tamoxifen alone (p < 0.05) or
trifluoperazine alone (p < 0.02). B,
affinity-purified avian osteoclasts were incubated with labeled bone
for 3 days in carrier alone (column 1), 3 or 7 µM tamoxifen (columns 2 and 3), 3 or 7 µM trifluoperazine (columns 4 and 5), or 7
µM tamoxifen plus 3 µM trifluoperazine (column 6), and bone degraded was determined as in A.
The data are representative of two experiments performed in
quadruplicate, means ± S.E. There is no significant difference
between column 6 and columns 3 and 5.
Vesicle acidification was not affected by 1 µM 17--estradiol (Fig. 6A), and neither
diethylstilbestrol nor ICI 182780 had measurable effects at meaningful
concentrations relative to steroid receptors (10
M). Tamoxifen and the calmodulin antagonist
trifluoperazine completely inhibited vesicle acidification at
concentrations consistent with their effects on cellular activity (Fig. 6, B and C, respectively). Concentration
dependence of inhibition of ATP-dependent membrane acid transport by
tamoxifen, trifluoperazine, and another calmodulin antagonist,
calmidazolium, are summarized in Fig. 7. Half-maximal inhibitory
concentrations were 0.25, 1.5, and 1.0 µM for tamoxifen,
trifluoperazine, and calmidazolium, respectively. The half-maximal
inhibitory concentrations of tamoxifen and trifluoperazine on vesicle
acidification were similar to their IC
values on bone
resorption (Fig. 3). Membrane vesicle acidification was
inhibited by 4-hydroxytamoxifen, the major metabolite of tamoxifen,
similarly to tamoxifen. ICI 182780 inhibited vesicle acidification at
10
M, in keeping with effects observed in
the bone resorption experiments and with its inhibition of
calmodulin-dependent phosphodiesterase activity at this concentration.
Figure 6:
Effect of 17--estradiol, tamoxifen,
and trifluoperazine on osteoclast membrane vesicle acidification.
ATP-dependent acid uptake was measured in avian osteoclast membranes
isolated by nitrogen cavitation and differential centrifugation. Acid
transport was monitored by measuring the decrease in fluorescence at
540 nm with excitation at 468 nm of acridine orange due to uptake into
vesicles (vertical axis) as a function of time (horizontal
axis). Assays were performed with vehicle alone (closed
symbols) or with test compounds (open symbols) 1
µM 17-
-estradiol (A), 2 µM tamoxifen (B), and 10 µM trifluoperazine (C). Test compounds were added to the assay mixture 5 min
prior to addition of 2 mM MgCl
(open
arrow, left) to initiate the reaction. Specificity for
acid transport was confirmed by fluorescence recovery on washout of the
fluorescent weak base with 1 mM NH
Cl (closed
arrow, right).
Figure 7:
Dose-dependent inhibition of ruffled
membrane vesicle acidification by tamoxifen, calmidazolium, and
trifluoperazine. Avian osteoclast vesicle acidification was measured as
a function of inhibitor concentration by quenching
Mg-ATP-dependent acridine orange fluorescence as in Fig. 6. Recovery on addition of 1 mM NH
Cl
at steady state was used to calculate inhibition of acid transport as a
percentage of matched vehicle-only controls (vertical axis).
Tamoxifen (closed circles), calmidazolium (open
circles), and trifluoperazine (open squares) were added
at indicated concentrations (horizontal axis) to membrane
vesicles 5 min prior to initiation of acid transport by addition of 2
mM MgCl
. Control acidification was
2
fluorescence units; the data are typical of three experiments using
different membrane vesicle preparations, which gave similar results.
100% inhibition indicates no fluorescence change on NH
Cl
addition.
Figure 8:
Tamoxifen binding to membrane, cytosolic,
and nuclear osteoclast fractions. Aliquots of fractions, produced as
described under ``Experimental Procedures,'' were incubated
with 0.63 nM [H]tamoxifen and 100 nM to 10 µM unlabeled tamoxifen. Samples were
precipitated, washed in polyethylene glycol, and counted to determine
bound tamoxifen. A, tamoxifen binding in membrane (closed
circles), cytosolic (closed squares), and nuclear
fractions (open circles). Data are representative of three
separate experiments; triplicate determinations are shown as mean
± S.E.
Figure 9:
Tamoxifen binding to osteoclast membranes
containing a high proportion of HCl transporting membrane. Aliquots of
membrane fractions produced by nitrogen cavitation (see
``Experimental Procedures'') were incubated with 0.63 nM [H]tamoxifen and 0-30 µM unlabeled tamoxifen. Samples were precipitated, washed in
polyethylene glycol, and counted to determine bound and free tamoxifen.
Binding at 30 µM unlabeled tamoxifen was subtracted as
nonspecific binding. A, tamoxifen binding in ruffled
membrane-enriched preparations. Means of duplicate determinations are
shown; these data are typical of results from three separate
experiments. B, Scatchard analysis of binding data for ruffled
membrane-enriched preparations. The apparent K
is 1 µM, with 5
10
sites/cell. This result suggests that tamoxifen at micromolar
concentrations binds an abundant membrane-associated protein in
osteoclasts.
Estrogens are bone sparing agents, but their mechanism of action is not clear. Which bone cells have estrogen receptors is controversial. Further, whether estrogenic effects are mechanistically related to the effects of bone-sparing compounds such as tamoxifen is enigmatic. It has been proposed that tamoxifen inhibits osteoclast activity by antagonizing estradiol binding(8, 26) . However, other mechanisms might lead to similar findings. For example, the calmodulin antagonist trifluoperazine inhibits breast cancer cell growth similarly to tamoxifen, but independently of the estrogen receptor(27) , and tamoxifen is both an anti-estrogen and a calmodulin antagonist. We studied effects of estrogen, tamoxifen, and related compounds on osteoclastic bone resorption and a key regulated process in bone resorption, membrane HCl transport, to resolve these points.
Bone affinity-purified avian osteoclasts were used to limit observations to direct osteoclast interactions (Fig. 1). This technique uses a small quantity of fragmented bone with a large surface area to concentrate osteoclasts, which attach to bone; other cells attach nonspecifically according to the substrate where they settle, which is 99% cell culture plastic (using 10-cm tissue culture plates and 2 mg of 20-40-µm bone). Thus, the ratio of osteoclasts to contaminants in the bone-attached fraction is, under ideal conditions, improved by 2 orders of magnitude, and non-osteoclastic effects, such as signals that may be generated from steroid receptors in other bone cells, are eliminated. This simplifies interpretation of results, and permits more detailed biochemical dissection of the bone resorption process.
We find that the estrogens 17--estradiol and
diethylstilbestrol, as well as the highly specific steroidal
anti-estrogen ICI 182780, have no effect on osteoclastic activity at
receptor-saturating concentrations (Fig. 2). At extremely high
concentrations, over 10
M, effects were
variable; cell activity in the presence of diethylstilbestrol was
slightly increased, although the difference was not statistically
different. On the other hand, ICI 182780 inhibited bone resorption at
concentrations over 10
M. ICI 182780 binds
the estrogen receptor with an apparent K
of
10
M(25) , and physiological
estrogen concentrations are 10
to 10
M. In postmenopausal women, plasma 17-
-estradiol is
1.5
10
M, while premenopausal
levels are
7.4
10
M(28) , and receptor affinities are in this range. Thus,
these effects of ICI 182780 are observed at 1000-fold or greater than
their receptor K
values, and do not represent
estrogen receptor-mediated effects. Estrogen and diethylstilbestrol did
not effect bone resorption at all concentrations tested
(10
to 10
M).
There are numerous reports of effects of estrogen on bone mass not directly attributable to osteoclastic estrogen-receptor binding(2, 8, 29, 30, 31, 32) . Our findings are not inconsistent with these results, which point to effects on osteoblasts(29) , membrane-associated estrogen binding different from the classical estrogen receptor and of uncertain significance(30) , or to effects on cell number or activity related to cell differentiation(8, 31, 32) . None of these processes are modeled in our system, which specifically examined purified osteoclasts and osteoclast membrane HCl transport.
On the other hand, it has been reported that 17--estradiol
directly inhibits avian osteoclastic bone resorption, almost
completely, with half-maximal effects at
10
M(24) . Our results are not consistent with this
study. Because of this controversial report, our cell preparations were
carefully characterized (Fig. 1) and results were repeated with
multiple cell preparations. In addition to estradiol, the general
estrogen agonist diethylstilbestrol and the highly specific
anti-estrogen ICI 182780 were tested. None of these compounds affected
bone resorption at or below 10
M. Assays
measuring pit formation by avian and rat osteoclasts were run in an
attempt to demonstrate an estradiol effect at submicromolar levels;
differences from controls were not seen. (
)We conclude that
the results reported by Oursler et al.(24) are not
reproduced under the conditions used here, which included essentially
homogeneous preparations of osteoclasts, phenol-red free medium and
charcoal-stripped sera to avoid possible steroid-like effects of the
phenol red and effects of serum steroids. The report of estradiol
effects in avian osteoclast cultures (24) may thus reflect
effects of non-osteoclastic cells, in vitro differentiation,
or medium components not present at measurable levels in our system.
In contrast, we found that the triphenylethylene compound tamoxifen
had clear inhibitory effects on osteoclastic bone resorbing activity (Fig. 3A). 4-Hydroxytamoxifen (the principal tamoxifen
metabolite) and the calmodulin antagonist trifluoperazine had similar
effects (Fig. 3, B and C, respectively). In
addition, the effects of tamoxifen and trifluoperazine were additive at
submaximal inhibitory concentrations (Fig. 5A).
Additivity was not observed when one compound was present at maximal
inhibitory concentration (Fig. 5B). These results
suggest that the inhibitory effect of tamoxifen are related to a
calmodulin-dependent signaling mechanism. Tamoxifen and
trifluoperazine inhibit calmodulin-dependent cyclic
nucleotide phosphodiesterase activity with IC
values of
1-3 µM, supporting this hypothesis. On the other
hand, tamoxifen binds both estrogen (10) and anti-estrogen (33) receptors, and these properties are believed to be the
basis of its beneficial effects in malignancies such as breast cancer.
However, the lack of observed estrogenic or anti-estrogenic effects on
osteoclastic activity at receptor-saturating concentrations indicate
that neither of these mechanisms are responsible for the observed
osteoclastic effects. The calmodulin antagonist activity of tamoxifen (9, 34) depends on its ethoxyaminoalkyl side chain,
which is also essential for its anti-estrogenic effects(16) .
Earlier work on osteoclastic activity points to an important role for
calmodulin interactions in acid secreting activity, including a high
concentration of calmodulin at the acid secreting ruffled membrane and
calmodulin antagonist inhibition of osteoclast membrane acid
transport(7) .
We investigated this hypothesis further by comparing the effects of tamoxifen and the calmodulin antagonists trifluoperazine and calmidazolium on ATP-dependent membrane acid transport (Fig. 6Fig. 7). Tamoxifen, trifluoperazine, and the highly specific calmodulin antagonist calmidazolium were all potent inhibitors of HCl transport. Estrogen and diethylstilbestrol had no effect. The anti-estrogen ICI 182780 inhibited vesicle acidification at concentrations over 1 µM, as did the calmodulin antagonists. However, it is also a calmodulin antagonist at these concentrations (Fig. 4). ICI 182780, tamoxifen, and other calmodulin antagonists all act consistently at low micromolar concentrations by inhibiting bone resorption, osteoclast membrane vesicle acidification, and phosphodiesterase activity. This argues for a related inhibitory mechanism for these compounds, a disruption of calmodulin-dependent signaling. However, calmodulin-dependent control mechanisms are very complex and involve a large number of specific calmodulin-protein interactions with different properties. For example, the half-maximal inhibitory concentration of tamoxifen differed by severalfold in the membrane transport assay (Fig. 6) and in the phosphodiesterase assay (Fig. 4); this is likely due to differences in the particular calmodulin-protein interactions present in the different assay procedures.
A potential problem with membrane
transport experiments such as those shown in Fig. 6is that the
antagonists tested are themselves weak bases, which could affect
acridine orange distribution. Accumulation of weak bases in acid
compartments depends mainly on concentration, membrane permeability of
the free base, and pK; high molecular weight
alkylamines such as tamoxifen typically have pK
11 and their membrane-permeable uncharged forms are present
at concentrations too low, at pH 7.4 (the assay buffer pH), to compete
effectively with acridine orange (pK
9.4).
Further, effective concentrations of the antagonists were 0.25-1
µM (Fig. 7), 8-30% of the acridine
concentrations, suggesting that such artifacts would be much smaller
than the observed effects even if pK
were near
that of acridine orange. However, to rule out such effects, control
assays were run with 1 µM NH
Cl included
(pK
9.3, competes with 3.3 µM acridine at 300 µM; see ``Experimental
Procedures''). This reduced acridine orange quenching less than
5%, indicating that the effects of the compounds tested cannot be due
to nonspecific effects of their amine groups on the assay.
The
results of the membrane transport assays suggested that a tamoxifen
binding site is present in osteoclast membranes. Tamoxifen binding was
saturable in crude membrane fractions (Fig. 8). In contrast,
tamoxifen binding in the nuclear and cytosolic fractions was
non-saturable (Fig. 8), indicating nonspecific binding. Because
ATP-dependent vesicle acid uptake was directly inhibited, a simple
binding to the acid-secreting membrane was hypothesized. Ruffled
border-rich cell membranes produced by nitrogen cavitation and
differential centrifugation demonstrated saturable high affinity
membrane binding with a single apparent K of 1
µM (Fig. 9). This result is obtained under
conditions that may vary substantially from those in living cells in
terms of calcium activity, buffer composition, and other variables.
Despite these limitations, binding of tamoxifen at low micromolar
concentration to this osteoclast fraction enriched in acid-transporting
membrane likely reflects a key molecular interaction of the inhibitor
with an osteoclastic protein. There were 5
10
binding sites/cell, consistent with an abundant
membrane-associated protein, but not a steroid receptor. It is likely
that this represents interaction with membrane-bound calmodulin or
calmodulin-binding proteins.
Our observations indicate that
tamoxifen directly inhibits osteoclast membrane acid transport by a
mechanism independent of cytosolic steroid receptors. Further, our
results show that critical elements in osteoclastic acid secretion are
similarly affected by tamoxifen, calmodulin antagonists, and, at high
concentrations, ICI 182780. Whether the effects of calmodulin on
osteoclast acid secretion derive from a direct effect of calmodulin on
the H-ATPase, its charge-coupled Cl
conductance, or are mediated secondarily by calmodulin-binding
proteins is unknown. Further, identification of specific protein
interactions of tamoxifen will be required to determine whether the
similarity of pharmacological effects of tamoxifen and calmodulin
antagonists reflect interactions with the same or related proteins.