Department of Physiology and Division of Oral Biology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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Extracellular nucleotides cause elevation of
cytosolic free Ca2+ concentration
([Ca2+]i) in osteoclasts, although the
sources of Ca2+ are uncertain. Activation of P2Y receptors
causes Ca2+ release from stores, whereas P2X receptors are
ligand-gated channels that mediate Ca2+ influx in some cell
types. To examine the sources of Ca2+, we studied
osteoclasts from rat and rabbit using fura 2 fluorescence and patch
clamp. Nucleotide-induced rise of [Ca2+]i
persisted on removal of extracellular Ca2+
(Ca
adenosine 5'-triphosphate; bone; cytosolic calcium; purinoceptor; phospholipase C; store-operated calcium influx
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INTRODUCTION |
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EXTRACELLULAR NUCLEOTIDES are important signaling molecules that mediate a number of functions. For example, ATP acts as a rapid neurotransmitter in the brain and in smooth muscle and as a paracrine mediator in responses of tissues to injury and inflammation (10-12, 14, 25, 26, 28). Nucleotides act via two types of receptors: P2X receptors, which are ligand-gated ion channels that allow Ca2+ influx in some systems, and P2Y receptors, which are heptahelical receptors that act via G proteins to raise cytosolic free Ca2+ concentration ([Ca2+]i) (25, 28). Cloning strategies have identified seven P2X and five P2Y receptor subtypes in mammals that are currently accepted as valid nucleotide receptors (28).
Osteoclasts are multinucleated cells responsible for the resorption of bone and other mineralized tissues. Osteoclasts are regulated by cell-cell and cell-matrix interactions, as well as by local and systemic mediators (19, 31). Endocrine and paracrine factors that regulate osteoclast formation and activity include peptides such calcitonin and RANK ligand, ions such as Ca2+ and H+, and nucleotides such as ATP (9, 31, 37). Nucleotides regulate osteoclast activity in several ways. In vitro, low concentrations of ATP, but not adenosine, stimulate formation of mouse osteoclasts and the resorptive activity of rat osteoclasts (23). In the absence of extracellular Mg2+ and Ca2+, high concentrations of ATP (2 mM) cause the formation of pores in murine osteoclasts and macrophages, but not in osteogenic or chondrogenic cells (22). Earlier studies revealed that nucleotides caused elevation of [Ca2+]i in rabbit osteoclasts (35, 36), with responses elicited by ATP and ADP, but not by UTP. However, the reported lack of response to UTP in rabbit osteoclasts is puzzling, in light of more recent reports indicating that the P2Y2 receptor (at which UTP is a potent agonist) is expressed in human osteoclast-like cells (5, 6) and rat osteoclasts (17). Furthermore, UTP has been found to elevate [Ca2+]i in rat osteoclasts (34), suggesting that responses may be species dependent. There is also evidence for the expression of P2X receptors in rat and rabbit osteoclasts (17, 24), with electrophysiological characteristics indicative of the P2X4 subtype (24, 33). However, the contribution of P2X receptors in mediating influx of Ca2+ in osteoclasts has not been determined.
The purpose of this study was to examine the effects of nucleotides on activation of Ca2+ influx and/or Ca2+ mobilization from stores and to compare responses in rat and rabbit osteoclasts. Using Ca2+-sensitive fluorescent dyes and patch-clamp recording, we found that mammalian osteoclasts express multiple subtypes of P2Y receptors. Nucleotide-induced rise of [Ca2+]i occurs primarily due to P2Y-mediated release of Ca2+ from intracellular stores. Furthermore, store-operated Ca2+ entry could be elicited by depletion of intracellular stores. However, we found no evidence for influx of Ca2+ through P2X4 ATP-gated channels.
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METHODS |
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Osteoclast isolation.
All procedures involving animals were approved by the Council on Animal
Care at the University of Western Ontario and complied with the
guidelines of the Canadian Council on Animal Care. Osteoclasts were isolated from femora and tibiae of rat or rabbit pups (up to 1 wk
old) that were killed by decapitation. Cells were plated onto plain or
type I collagen-coated glass coverslips as described previously
(2, 33) and maintained in medium 199 (GIBCO Laboratories, Burlington, Ontario, Canada) with HCO3), and
the identity of rounded osteoclasts was confirmed by counting nuclei on
disruption of the cell with a pipette at the end of recording.
Fluorescence recording of Ca2+.
[Ca2+]i of single osteoclasts was monitored
using fura 2. Cells were loaded by incubation with 1.5 µM fura 2-AM
(Molecular Probes) for 30-60 min at room temperature and then were
washed and incubated at 37°C for 30-60 min to allow for ester
hydrolysis. Coverslips containing fura 2-loaded cells were placed in a
chamber (0.75 ml) mounted on a Nikon Diaphot inverted microscope and
were continuously superfused with Na+ solution consisting
of (in mM) 130 NaCl, 5 KCl, 10 glucose, 1 MgCl2, 1 CaCl2, and 20 HEPES, pH 7.4 (adjusted with NaOH),
280-290 mosM. Whole cell [Ca2+]i was
monitored by alternate epifluorescence illumination of individual fura
2-loaded osteoclasts as described previously (33, 34),
with 340/380 nm excitation from a RatioMaster illumination system
(Photon Technology International, London, Canada) using a Nikon Fluor
×40 objective. The emission signal was filtered with a 510-nm
band-pass filter, detected with a photomultiplier, and sampled at
5-20 ratios/s. The ratio R was the fluorescence intensity at 340 nm divided by that at 380 nm. [Ca2+]i was
calculated from the relationship [Ca2+]i = Kd[(R Rmin)/(Rmax
R)]
, where
Kd (dissociation constant for the fura
2-Ca2+ complex) was 224 nM, Rmin and
Rmax were the values of R at low and saturating
Ca2+ concentrations, respectively, and
was the ratio of
fluorescence intensity at 380 nm measured at low and saturating
Ca2+ concentrations (16). Calibration
constants were determined using fura 2 salt in high-K+
buffer, and all recordings were corrected for background. The rate of
change of [Ca2+]i was determined as the slope
of lines fit to successive regions of smoothed Ca2+ traces
and was plotted as a function of [Ca2+]i
averaged for the same time periods.
Electrophysiology.
For recording macroscopic currents, we used nystatin perforated-patch
or conventional whole cell configuration. K+ electrode
solution contained (in mM) 140 KCl, 20 HEPES, 1 MgCl2, 0.4 CaCl2, and 1 EGTA (~100 nM free Ca2+), pH 7.2 (adjusted with KOH), 280-290 mosM. To block K+
currents in some experiments, we used electrode solution in which CsCl
was substituted for KCl (140 mM CsCl). Cells were superfused (1-2
ml/min) with Na+ solution as given above. Currents were
recorded with Axopatch 1D or 200A amplifiers (Axon Instruments, Foster
City, CA), filtered (3dB at 1 kHz) and digitized at 2-5 kHz
using pCLAMP 6.0 (Axon Instruments). Currents were also stored on
videotape using a pulse code modulator. Current-voltage relationships
were obtained using voltage ramp protocols, where voltage was shifted
from
100 to +100 mV over 340 ms. Experiments were performed at room
temperature (21-25°C).
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RESULTS |
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We examined responses to nucleotides in >500 rat and rabbit
osteoclasts, with similar findings in cells from both species. Osteoclasts responded to ATP with a rapid rise of
[Ca2+]i consisting of both a transient and
sustained phase (Fig.
1A). Such responses
were elicited by ATP in 147 of 154 rat osteoclasts, and 74 of 118 rabbit osteoclasts tested. Mean values in 50 representative osteoclasts
were basal [Ca2+]i of 134 ± 52 nM, peak
response to ATP of 806 ± 444 nM, and a plateau level of 270 ± 129 nM. Our goal was to understand the nature of the receptors and
signaling pathways mediating these responses, so we first employed the
P2 receptor antagonist Cibacron blue, an anthraquinone-sulfonic acid
derivative and isomer of reactive blue 2. Pretreatment of cells with
Cibacron blue (40-100 µM) abolished the rise of
[Ca2+]i in response to ATP (Fig. 1A,
middle, 7 of 7 osteoclasts). The failure of cells to respond under
these conditions was not due to desensitization, as full recovery was
observed following washout of the blocker and subsequent stimulation
with ATP (Fig. 1A, right). Another widely used
nucleotide receptor blocker, suramin, is autofluorescent at the
wavelengths used for fura 2, precluding its use in these studies.
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Osteoclasts have been shown to exhibit both "spread" and "rounded" morphologies, with a greater proportion of rat osteoclasts exhibiting a rounded morphology when plated on collagen, as opposed to glass substrate (2). Rat osteoclasts of the rounded morphology as well as rabbit osteoclasts showed the most reproducible responses and were therefore used for many of the studies detailed below. In contrast, Ca2+ transients in spread rat osteoclasts often diminished with three to five challenges, making it difficult to carry out prolonged recordings from individual cells.
Ectonucleotidases can rapidly degrade extracellular ATP, so we next
tested whether breakdown products of ATP could contribute to the rise
of Ca2+, using two approaches. First, we applied poorly
hydrolyzable analogs, such as ATPS (1-100 µM), which also
induced elevations of [Ca2+]i comparable to
those elicited by ATP (Fig. 1B) (29 of 37 rat and 48 of 62 rabbit osteoclasts showed positive responses). Another analog,
2meSATP (0.1-10 µM) elicited responses in 77 of 105 rat osteoclasts tested (Fig. 1C). It is notable that ATP and
nucleotide analogs elicited oscillatory changes of
[Ca2+]i in some osteoclasts (Fig.
1C), a feature observed most commonly in response to
2meSATP. The second approach to determine the class of receptors
underlying the responses was to examine the effects of ADP, AMP, and
adenosine. ADP (0.1-50 µM) elicited a rise of [Ca2+]i in 12 of 18 rat osteoclasts, as did
ADP
S (0.1-10 µM, 15 of 19 rat osteoclasts). In contrast,
neither AMP (10-50 µM) nor adenosine (10-50 µM) elicited
Ca2+ responses in any osteoclast tested (10 rat osteoclasts
tested for each ligand). In each case, we confirmed the responsiveness of osteoclasts by subsequently recording
[Ca2+]i elevations on application of ATP or 2meSATP.
It has been reported that rabbit osteoclasts do not respond to UTP with a rise of [Ca2+]i (35). However, P2Y2 nucleotide receptors (at which both UTP and ATP are potent agonists) are expressed in human osteoclastoma giant cells (5, 6) and in rat osteoclasts (17). Moreover, rat osteoclasts respond to UTP with a rise of [Ca2+]i (34). To determine whether responsiveness to UTP might be species dependent, we examined the effects of UTP on rat and rabbit osteoclasts and found that cells from both species did respond to UTP (10-100 µM; Fig. 1B). Such responses to UTP were observed in 21 of 42 rabbit osteoclasts and 10 of 15 rat osteoclasts tested.
We next examined whether multiple receptor subtypes might exist on individual osteoclasts, relying on desensitization of the receptors with continued application of agonist. Prolonged application of UTP induced a transient elevation of [Ca2+]i that desensitized after ~50 s, whereupon subsequent stimulation with ATP (in the continued presence of UTP) still elicited a rise of [Ca2+]i (Fig. 1D; representative of results observed in 4 of 4 rabbit and 3 of 3 rat osteoclasts). When the agonists were applied in the reverse order, prolonged application of ATP elicited the typical rise of [Ca2+]i, but subsequent responses to UTP were abolished (Fig. 1E; representative of observations in 3 of 3 rabbit and 3 of 3 rat osteoclasts). Control responses on the same days revealed that four of six rabbit and four of four rat osteoclasts responded to UTP alone, so these results are consistent with the presence of multiple nucleotide receptor subtypes in individual osteoclasts.
To summarize, elevation of [Ca2+]i is
elicited in mammalian osteoclasts by several nucleotides, including
ATP, ATPS, 2meSATP, ADP, ADP
S, and UTP, but not AMP or adenosine.
Due to variability between cells and within cells, as well as the
method used to apply agents focally to cells, it was not possible to
obtain full concentration-dependence curves. Based on these findings,
we conclude that nucleotide-induced Ca2+ elevation is
mediated by P2 (nucleotide) but not by P1 (adenosine) receptors.
Moreover, these results indicate that multiple nucleotide receptor
subtypes coexist in individual osteoclasts.
Nucleotide-induced elevation of [Ca2+]i could
be due to Ca2+-release from stores, via P2Y nucleotide
receptors and/or Ca2+ influx via the P2X4
nucleotide receptor identified previously in osteoclasts
(24). To investigate the source of Ca2+,
nucleotides were applied before and during superfusion with Ca2+-free solution containing 0.5 mM EGTA. Nucleotides
continued to elicit large Ca2+ transients in
Ca2+-free conditions (5 of 5 osteoclasts tested), even
after repeated stimulation (Fig. 2). Note
that a slight reduction in basal [Ca2+]i was
observed in Ca2+-free solutions, confirming the effective
superfusion of the cells. Subsequent applications of nucleotide
resulted in a progressive decrease in the peak amplitude of the
response, which is indicative of store depletion and not
desensitization, since responses were fully restored after readdition
of bath Ca2+ (Fig. 2, right). To examine the
possibility that changes in membrane potential also contribute to the
Ca2+ transients, we depolarized cells with solution
containing 135 mM extracellular K+, thereby clamping the
membrane potential close to 0 mV. ATP continued to elicit
Ca2+ transients under this condition (7 osteoclasts,
data not shown), indicating that changes of membrane potential are not
essential for the Ca2+ transients studied here. Based on
these results, Ca2+ mobilization from intracellular stores
appeared to be the dominant, but perhaps not exclusive, source of
Ca2+.
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We next used several strategies to deplete the Ca2+ stores. We first examined effects of caffeine as an activator of the endoplasmic reticulum (ER) Ca2+ release channel, the ryanodine receptor, which is also proposed to reside in the plasma membrane of osteoclasts and act as a Ca2+ influx pathway (1, 37). In our studies, caffeine (5 mM) failed to alter [Ca2+]i in rat or rabbit osteoclasts, and it did not have any effect on subsequent stimulation with ATP (10 of 10 rabbit osteoclasts and 4 of 4 rat osteoclasts tested). As a control, we confirmed that caffeine did cause elevation of [Ca2+]i and contraction of isolated smooth muscle cells, as described previously (29), indicating that the caffeine used for these studies was effective.
We next studied the effects of inhibiting the ER
Ca2+-ATPase on nucleotide responses. Application of the
reversible inhibitor cyclopiazonic acid (CPA; 1-2 µM) to cells
in Ca2+-free solution resulted in a small but consistent
increase in basal [Ca2+]i (Fig.
3A). The initial stimulation
with nucleotides in the presence of CPA elicited a Ca2+
transient of similar amplitude, but subsequent responses were essentially abolished (Fig. 3A, right, representative of
responses in 5 osteoclasts). The disruption of signaling did not
reflect nonspecific rundown, since nucleotide responses recovered fully after washout of CPA (10-15 min) and readdition of
Ca2+ to the bath (Fig. 3A, far right). Thus
depletion of the Ca2+ store by CPA abolished responses to
nucleotides, confirming the dominant role of Ca2+ stores in
mediating the responses.
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We note that although acute exposure of cells to CPA caused little change in the peak of the initial response to nucleotides, the time course for recovery of [Ca2+]i was consistently prolonged (Fig. 3B). Such an effect indicates that reuptake of Ca2+ by the ER contributed to restoration of basal [Ca2+]i. To examine this in further detail, we determined the rate of decline of [Ca2+]i for control responses and following treatment with CPA. When plotted as a function of [Ca2+]i, the maximum rate of decline of [Ca2+]i was reduced following treatment with CPA to 36% of control levels (187 ± 50 nM/s compared with 68 ± 35 nM/s following treatment with CPA, n = 7; Fig. 3C). These findings provide direct evidence that the ER Ca2+ stores not only provide a source of Ca2+ in osteoclasts, but also contribute to the restoration of basal Ca2+ levels following a transient increase.
Based on the evidence for involvement of Ca2+ stores, we
considered the possibility that store emptying activates an influx pathway (i.e., capacitative Ca2+ influx). After stores were
depleted with a combination of CPA, Ca2+-free solution and
repeated nucleotide stimulation, reintroduction of extracellular
Ca2+ caused a substantial rise of
[Ca2+]i that peaked then declined toward
baseline, consistent with store-operated influx (Fig.
4A, n = 4).
Functional evidence for refilling of the stores was obtained in the
same cells, where ATP again elicited a rise of Ca2+
comparable with control responses (Fig. 4A). Osteoclasts are thought to express a Ca2+-sensing receptor
(37), so we also considered the possibility that removal
and readdition of Ca2+ to osteoclasts, independent of store
depletion, influenced [Ca2+]i. Cells were
subjected to the same protocol of removing then reintroducing bath
Ca2+, except CPA was not present to deplete stores,
whereupon reapplication of Ca2+ to cells did not elicit
sizable changes of [Ca2+]i (Fig.
4B).
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To further examine properties of the store-operated influx, we treated
cells with the irreversible Ca2+-ATPase inhibitor
thapsigargin (1-5 µM), which resulted in substantial rise of
[Ca2+]i (Fig.
5A, 5 of 5 cells tested). Near
the peak of the response, superfusion with Ca2+-free
solution caused a sharp decline of [Ca2+]i,
indicating that influx contributed at least in part to the sustained
elevation of [Ca2+]i (Fig. 5A,
right, seen in 4 of 4 cells tested). As a control, we first
determined that perfusion of each osteoclast with Ca2+-free
solution before thapsigargin caused negligible change in [Ca2+]i (Fig. 5A, left). Further
evidence that store depletion by thapsigargin activated a
Ca2+-influx pathway was obtained by testing the effects of
an influx blocker. Addition of La3+ (100 µM) to cells at
the peak of the response to thapsigargin resulted in a prompt decline
of [Ca2+]i (Fig. 5B, 7 of 7 cells
tested). To study the voltage dependence of this influx pathway, we
used combined patch-clamp and fluorescence methods. Under these
conditions where membrane potential could be controlled, the peak of
the thapsigargin-activated rise of [Ca2+]i
was larger when cells were held at more negative potentials to increase
the force driving Ca2+ entry (Fig. 5C, data from
1 cell, representative of 7 cells). This was apparent as a nonlinear
voltage dependence, consistent with that seen for store-operated
currents in other systems (27).
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P2X receptors are nucleotide-gated ion channels that are cation
selective and, in some cases, exhibit sizable Ca2+
permeability (28). We considered the possibility that
release of Ca2+ from stores could have masked influx
through P2X4 channels. However, the absence of selective
agonists or antagonists made it difficult to discern the contribution
of P2X receptors in isolation. Moreover, the presence of store-operated
Ca2+ influx meant that stores could not simply be depleted
to investigate other components. Accordingly, we tested the effects of
an inhibitor of phospholipase C (PLC), as a way of selectively blocking
P2Y signaling. Treatment of osteoclasts with U-73122 (10 µM) was
effective in reducing the peak of the nucleotide-induced
Ca2+ transient to <10% of initial response in 68 of 86 osteoclasts tested (Fig. 6A).
We applied the inactive analog U-73343 in a similar way as a control,
and it did not reduce nucleotide-induced responses (Fig. 6B,
49 of 58 cells continued to exhibit elevation of
[Ca2+]i). Having established that inhibition
of PLC was effective, we used combined patch-clamp and fluorescence
recordings to examine the relationship between P2X4 current
and [Ca2+]i. A representative experiment is
shown in Fig. 7, where ATP induced a rise
of [Ca2+]i (shown above) accompanied by
P2X4 current, shown below as the current recorded at 60
mV during voltage ramp commands (Fig. 7, left). Treatment of
the same cell with U-73122 (5 min, 10 µM) inhibited the rise of
Ca2+, with little change in the amplitude or time course of
the P2X current (Fig. 7, right, n = 8).
Thus, based on these experiments, we could not identify functional
evidence for Ca2+ influx associated with P2X4
current.
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DISCUSSION |
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Previous studies have suggested that functional P2X and P2Y
receptors are expressed in osteoclasts (24, 33-36).
The results presented here are consistent with earlier reports but have
now revealed evidence for the coexistence of multiple P2Y subtypes in
individual osteoclasts. Elevation of [Ca2+]i
by UTP is consistent with the presence of P2Y2 and/or
P2Y4 receptors, at which UTP is a potent agonist. Responses
to ATP in the continued presence of UTP indicate the presence of
additional subtypes of P2Y receptors in the same cell. Further evidence
is the effectiveness of 2meSATP and ADPS, which are agonists at P2Y
receptors other than P2Y2 and P2Y4
(28). In contrast to previous reports (6,
35), we observed responses to UTP in both rat and rabbit
osteoclasts, possibly indicating the presence of functional
P2Y2 or P2Y4 receptors. In this regard,
P2Y2 receptors are expressed in human osteoclastoma
(5, 6), and in situ hybridization studies reveal
expression of P2Y2 but not P2Y4 receptors in
rat osteoclasts (17). Based on these findings, the effects of UTP that we observed are most likely mediated by P2Y2 receptors.
The coexistence of P2X and P2Y receptor subtypes in osteoclasts raises
the question of the source of Ca2+ contributing to the rise
of [Ca2+]i. A number of cloned and
heterologously expressed P2X channel subtypes allow Ca2+
entry (25). For example, the ratio of the permeabilities
to Ca2+ and Na+ ratios for P2X1 and
P2X4 are relatively high [4.1 and 4.2, respectively (13, 30)]. Although the Ca2+ permeability of
the endogenous P2X1 channel is high in smooth muscle
(3), the Ca2+ permeability of many other
native P2X channels is not known. Evidence presented in this report
suggests that most, if not all, of the Ca2+ response
elicited by nucleotides in osteoclasts arises by P2Y-mediated release
from intracellular stores, rather than influx through P2X4
channels. First, Ca2+ responses were inhibited by Cibacron
blue, which does not block P2X4 in heterologous expression
systems (28) or in osteoclasts (24).
Second, UTP and ADPS, which are weak or inactive agonists at all P2X
receptors (28), caused substantial elevation of
[Ca2+]i. Third, initial
nucleotide-induced elevations of [Ca2+]i
were virtually identical in the presence or absence of extracellular Ca2+. Fourth, most P2Y receptors are coupled through G
proteins to PLC
. Inhibition of PLC
by the inhibitor U-73122
abolished the elevation of [Ca2+]i elicited
by ATP, without greatly reducing the P2X4 current. Taken
together, these findings support a dominant role for P2Y-mediated release of Ca2+ from intracellular stores in mediating
Ca2+ responses in osteoclasts.
Repeated application of nucleotides in Ca2+-free medium led to the gradual diminution of responses, consistent with depletion of intracellular Ca2+ stores. This process was hastened by the ER Ca2+-ATPase inhibitor, CPA, indicating that nucleotides cause release of Ca2+ from intracellular stores. Notably, CPA also caused significant reduction in the rate of recovery of [Ca2+]i following nucleotide stimulation, indicating a role for the ER in restoration of basal Ca2+ levels in osteoclasts. Inhibition of nucleotide-induced elevation of [Ca2+]i by U-73122 is consistent with release from stores being mediated by IP3. The lack of effects of caffeine on [Ca2+]i in the present study does not support the presence of functional ryanodine receptor Ca2+ release channels in osteoclasts, in contrast to earlier reports (1, 37).
Depletion of stores by CPA or thapsigargin led to prolonged elevation
of [Ca2+]i. This effect was dependent on the
presence of extracellular Ca2+ and was inhibited by
La3+, properties suggesting the activation of a
store-operated, or capacitative Ca2+ entry pathway. When
membrane potential of osteoclasts was controlled using voltage clamp,
the rise of [Ca2+]i due to influx exhibited a
voltage dependence similar to that shown previously for store-operated
Ca2+ influx in a number of cell types, such as lymphocytes
(21). For example, the rise of Ca2+ was
greater at more negative potentials, and there was a suggestion of
inward rectification, consistent with the properties of
Ca2+ release-activated Ca2+ current, referred
to as CRAC current (27). However, without blockade of
overlapping K+ and Cl currents (18,
33), it was not possible to resolve the store-operated Ca2+ influx current, so its characteristics in osteoclasts
remain to be determined.
Recently, nucleotides have been shown to stimulate osteoclastic bone
resorption. Low concentrations of ATP, but not adenosine, enhance
formation of mouse osteoclasts and the resorptive activity of rat
osteoclasts in vitro (23). Moreover, ATPS was found to
stimulate resorption by human osteoclast-like cells cultured on
resorbable substrate (6). In contrast to the effects of ATP
S, UTP (which activates P2Y2 and P2Y4 but
not P2X4 receptors) had no effect on osteoclastic
resorption. Thus the functional effects of nucleotides on
osteoclasts appear to be mediated by a receptor other than
P2Y2 or P2Y4, possibly P2X4 or
another subtype of P2Y receptor. Nucleotides could stimulate resorption
by increasing the activity or number of osteoclasts. ATP is a
secretagogue in several cell types, and activation of osteoclastic
resorption involves formation of the ruffled border by an exocytotic
process, which could be induced by ATP. It is also possible that
nucleotide receptors play a role in osteoclast formation by mediating
fusion of osteoclast precursors, in a manner similar to that described for fusion of macrophages in culture (8).
Osteoclasts may be exposed to nucleotides from a number of sources. These include nucleotides released locally in response to cell damage or mechanical stimulation. During inflammation and hemostasis, nucleotides are secreted by activated leukocytes and platelets (4). Further, ATP may be released as a neurotransmitter from neurons innervating bone. Nucleotides are released into the extracellular fluid by a number of cell types in response to mechanical stimulation. Endothelial cells release ATP in response to fluid flow (7). Similarly, epithelial cells release ATP and astrocytoma cells release UTP in response to fluid flow or deformation of the substratum (15, 20, 32). Although the mechanism underlying mechanically induced release of nucleotides is unclear, it may involve exocytosis, efflux through a membrane channel or transporter, or reversible "microtrauma" to the plasma membrane.
The downstream events activated by P2 receptors that ultimately lead to increased resorptive activity are not yet understood. It is known that nucleotide binding leads to elevation of [Ca2+]i, cation influx, and depolarization, signals that may influence a number of critical processes such as cell matrix interactions, formation of the ruffled border, and gene expression. Nucleotides, released at sites of trauma or inflammation and physiologically in response to mechanical stimulation, may act through P2 receptors to enhance the activity of osteoclasts and the resorption of bone and other mineralized tissues.
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ACKNOWLEDGEMENTS |
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We thank Yang Jiao for assistance with the fluorescence recording and Lin N. Naemsch for helpful comments on the manuscript.
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FOOTNOTES |
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This work was supported by The Arthritis Society and the Canadian Arthritis Network. S. M. Sims was supported by a Scientist Award from the Medical Research Council of Canada.
Address for reprint requests and other correspondence: S. M. Sims, Dept. of Physiology, The Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (E-mail: stephen.sims{at}med.uwo.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. Section 1734 solely to indicate this fact.
Received 30 May 2000; accepted in final form 18 January 2001.
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