Activation of P2Y but not P2X4 nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts

A. Frederik Weidema, S. Jeffrey Dixon, and Stephen M. Sims

Department of Physiology and Division of Oral Biology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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<UP><SUB>o</SUB><SUP>2+</SUP></UP>), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca2+-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of [Ca2+]i. After store depletion in the absence of Ca<UP><SUB>o</SUB><SUP>2+</SUP></UP>, addition of Ca<UP><SUB>o</SUB><SUP>2+</SUP></UP> led to a rise of [Ca2+]i consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La3+. In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X4 nucleotide receptors, but with no rise of [Ca2+]i. We conclude that nucleotide-induced elevation of [Ca2+]i in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.

adenosine 5'-triphosphate; bone; cytosolic calcium; purinoceptor; phospholipase C; store-operated calcium influx


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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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INTRODUCTION
METHODS
RESULTS
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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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (26 mM), HEPES (25 mM) and supplemented with antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B) and heat-inactivated fetal bovine serum (15% vol/vol). Rabbit osteoclasts were isolated and purified with pronase, as previously described (24). Rat osteoclasts were studied within 14 h of isolation, whereas rabbit osteoclasts were used for up to 5 days, during which time the Ca2+ responses described here did not change noticeably. Osteoclasts were identified as cells having multiple nuclei (>= 3), 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)]beta , 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 beta  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).

In experiments involving simultaneous patch clamp and fluorescence, cells were loaded with fura 2 before perforated-patch configuration was established. In cases where the whole cell configuration was used, 30 µM fura 2 salt was also included in the electrode solution and EGTA was reduced to 0.01 mM with no added Ca2+ to minimize Ca2+ buffering. Current and Ca2+ traces were recorded simultaneously to ensure proper temporal alignment.

Test solutions were applied to cells by local superfusion from micropipettes (5-10 µm diameter) positioned 30-50 µm from the cell (Picospritzer II; General Valve, Fairfield, NJ). This was sufficient to replace the solution around cells by >95% with a delay of 50-150 ms, as determined by the delay in the shift of the reversal potential of the inwardly rectifying K+ current on application of 135 mM K+ solution. Application of control solutions did not cause appreciable changes in membrane currents or [Ca2+]i.

Agonists tested included ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), 2-methylthioadenosine 5'-triphosphate (2meSATP), AMP, adenosine 5'-O-(2-thiodiphosphate) (ADPbeta S), and UTP. 2meSATP was from Research Biochemicals International (Natick, MA). Unless otherwise indicated, chemicals were from Sigma Chemical or BDH (Toronto, Ontario, Canada). Values are presented as means ± SD.


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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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Fig. 1.   Multiple subtypes of nucleotide receptors in mammalian osteoclasts. Nucleotides were applied to cells for the times indicated by the bars below the Ca2+ traces in this and subsequent figures. A: ATP (20 µM) applied to an isolated rat osteoclast caused rapid rise of cytosolic free Ca2+ concentration ([Ca2+]i) with transient and sustained components. After 5 min for recovery, the P2 receptor antagonist Cibacron blue (100 µM) was applied by bath superfusion, blocking the subsequent response to ATP (middle). After washout and recovery, the osteoclast again responded to ATP (right trace). B: the poorly hydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S; 10 µM) induced an increase in [Ca2+]i with transient and sustained phases. After 3 min of recovery, UTP (10 µM, an agonist at P2Y2 and P2Y4 receptors), elicited similar elevation of [Ca2+]i in the same cell. C: nucleotides also elicited oscillatory changes of [Ca2+]i in some osteoclasts. 2-Methylthioadenosine 5'-triphosphate (2meSATP; 1 µM) elicited a rise of [Ca2+]i followed by oscillations. After 3 min of recovery, a similar response was elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbeta S; 1 µM). D and E: evidence for multiple nucleotide receptors in individual cells. Prolonged application of UTP (100 µM) induced a transient elevation of [Ca2+]i that desensitized after ~50 s. Subsequent stimulation with ATP (100 µM) in the continued presence of UTP elicited a rise of [Ca2+]i. When the agonists were applied in the reverse order to another osteoclast, application of ATP elicited the typical rise of [Ca2+]i, but subsequently UTP elicited little response (E). Responses in A-C were recorded from rat osteoclasts, and in D and E from rabbit osteoclasts. All responses were observed in osteoclasts from both species. Cells were allowed to recover for 3-5 min between applications of nucleotides, as indicated on the time scales below the Ca2+ traces.

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 ATPgamma S (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 ADPbeta 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, ATPgamma S, 2meSATP, ADP, ADPbeta 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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Fig. 2.   Nucleotide-induced rise of [Ca2+]i involves release of Ca2+ from intracellular stores. 2meSATP (10 µM, applied for the periods indicated by the bars below the traces) induced a large, transient Ca2+ elevation followed by a plateau. The cell was periodically stimulated, allowing at least 5 min recovery period between challenges. After removal of extracellular Ca2+ (6 min bath perfusion with solution containing 0 mM Ca2+ and 0.5 mM EGTA), 2meSATP continued to elicit elevations of [Ca2+]i. The peak amplitude was progressively reduced, but not abolished, in Ca2+-free solution. Readdition of Ca2+ to the bath restored the nucleotide-induced rise of Ca2+ (at right). Traces are all from a single rat osteoclast and are representative of results in 5 osteoclasts.

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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Fig. 3.   Blockade of Ca2+-ATPase leads to store depletion. A: Ca2+ transients were repeatedly induced by 2meSATP (10 µM) in a Ca2+-containing buffer and persisted after removal of extracellular Ca2+ (0 Ca2+ with 0.5 mM EGTA). The trace illustrated was the second stimulation after 8 min in Ca2+-free solution. Subsequent addition of cyclopiazonic acid (CPA; 2 µM) to inhibit the endoplasmic reticulum Ca2+-ATPase resulted in a small increase in basal [Ca2+]i, and the Ca2+ peak first elicited by 2meSATP was comparable to control response. Subsequent application of 2meSATP failed to elicit any response, indicating depletion of Ca2+ stores. Full recovery of the cell is shown at right after washout of CPA and readdition of Ca2+, where 2meSATP again elicited a large rise of [Ca2+]i. B: expansion of the traces (i and ii) from A reveal that CPA prolonged the recovery of [Ca2+]i to resting levels. C: rate of decline of [Ca2+]i was determined for the same responses shown in A and B, as described in METHODS. CPA reduced the rate of decline at all levels of [Ca2+]i, indicating a role for the endoplasmic reticulum Ca2+-ATPase in Ca2+ homeostasis in osteoclasts. Traces shown are for a rat osteoclast and are representative of results from 5 osteoclasts.

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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Fig. 4.   Store-operated Ca2+ influx in osteoclasts. A: stores were depleted by repeated stimulation with 2meSATP (10 µM) in Ca2+-free solution with CPA (2 µM), with ~10 min recovery between stimulations (traces illustrated are from a rabbit osteoclast). The third application of 2meSATP induced only a small rise of Ca2+, indicating store depletion. On readdition of Ca2+, a large and transient increase in [Ca2+]i was evident (without application of nucleotide). As illustrated at right, the 2meSATP-induced response fully recovered after washout of CPA for 18 min. B: control studies in another rabbit osteoclast revealed that Ca2+ influx was minimal without store depletion. Bathing cells in Ca2+-free solution, but without CPA in this case, caused a gradual decrease in Ca2+ transient (4th stimulation with ATPgamma S in 0 mM Ca2+ illustrated). Note that readdition of bath Ca2+ in this case caused essentially no rise of [Ca2+]i and that recovery of the ATPgamma S-induced Ca2+ transient occurs (at right). Similar results were obtained in 4 osteoclasts.

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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Fig. 5.   Properties of Ca2+ influx induced by store depletion. A: [Ca2+]i was monitored in rat osteoclast while Ca2+ levels in the bath were varied, as indicated above the recording. Under control conditions, removing extracellular Ca2+ did not alter basal [Ca2+]i. Local application of the irreversible Ca2+-ATPase inhibitor thapsigargin (Tg; 2 µM, indicated by bar) caused a slow increase of [Ca2+]i, which was reversed by removal of extracellular Ca2+, indicative of Ca2+ influx (4 of 4 osteoclasts tested). B: in another rat osteoclast treated with thapsigargin (2 µM) to increase [Ca2+]i, addition of the channel blocker La3+ (100 µM) caused a prompt decline of [Ca2+]i (7 of 7 cells tested). C: a fura 2-loaded rat osteoclast was clamped at various membrane potentials in perforated-patch configuration, while [Ca2+]i was simultaneously monitored following treatment with thapsigargin. The peak [Ca2+]i is plotted as a function of membrane potential for this representative cell, showing that the rise of Ca2+ was large at negative potentials and much reduced at positive potentials. Similar voltage dependence was observed in 6 other cells.

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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Fig. 6.   Involvement of phospholipase C (PLC) in nucleotide-induced Ca2+ transients. Ca2+ transients were repeatedly elicited by ATP (10 µM). A: U-73122 (10 µM), an inhibitor of PLC applied by bath perfusion to a rat osteoclast, completely inhibited the response to ATP (observed in 68 of 86 cells tested). B: as a control, the inactive analog U-73343 (10 µM) was applied in a similar protocol, and ATP continued to elicit Ca2+ transients (2 responses shown here for a rabbit osteoclast), indicating no nonspecific disruption of nucleotide signaling (representative of responses recorded in 49 of 58 cells tested).



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Fig. 7.   No rise of [Ca2+]i accompanies activation of P2X4 cation current. [Ca2+]i and membrane currents were studied with combined patch-clamp and fluorescence recording. Control response is illustrated at left, showing that ATP (20 µM) induced a large elevation of [Ca2+]i as well as inward current, measured at -60 mV (recorded using voltage ramp commands from -100 to +50 mV in 340 ms, every 2 s). To eliminate the contribution of stores, the same cell was treated with the PLC inhibitor U-73122 (10 µM, bath application) for 5 min before stimulation. After such treatment, no rise of [Ca2+]i was elicited by ATP, even though there was little change in the P2X current (8 of 8 cells tested). This indicates that Ca2+ entry through P2X channels was negligible. Currents in fura 2-loaded rat osteoclast were recorded using the nystatin-perforated-patch configuration.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 ADPbeta S, 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 ADPbeta S, 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 PLCbeta . Inhibition of PLCbeta 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, ATPgamma S was found to stimulate resorption by human osteoclast-like cells cultured on resorbable substrate (6). In contrast to the effects of ATPgamma 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.


    ACKNOWLEDGEMENTS

We thank Yang Jiao for assistance with the fluorescence recording and Lin N. Naemsch for helpful comments on the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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