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J. Biol. Chem., Vol. 276, Issue 42, 39107-39114, October 19, 2001

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Activity-dependent Development of P2X₇ Current and Ca²⁺ Entry in Rabbit Osteoclasts^*

Lin N. Naemsch, S. Jeffrey Dixon, and Stephen M. Sims

From the Canadian Institutes of Health Research Group in Skeletal Development and Remodeling, Department of Physiology and Division of Oral Biology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada

Received for publication, June 25, 2001, and in revised form, August 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bone remodeling is regulated by local factors and modulated by mechanical stimuli. Mechanical stimulation can cause release of ATP, an agent that stimulates osteoclastic resorption at low concentrations and inhibits at high concentrations. We examined whether osteoclasts express P2X₇ receptors, which are activated by high concentrations of ATP and can behave as ion channels or cause the formation of membrane pores. Rabbit osteoclasts were studied using patch clamp techniques. Successive or prolonged applications of 2'- & 3'-O-(4-benzoylbenzoyl)-ATP (BzATP, a relatively potent P2X₇ agonist) or high concentrations of ATP caused the development of a slowly deactivating inward current. The underlying channel was permeable only to small cations, ruling out pore formation. Divalent cations reduced current magnitude, consistent with the presence of P2X₇ receptors, a finding confirmed in rat osteoclasts by immunocytochemistry. Successive applications of BzATP also elicited [Ca²⁺]_i elevations that required extracellular Ca²⁺. The BzATP-induced current and the rise of [Ca²⁺]_i were temporally associated, and both were inhibited by PPADS, a P2X₇ antagonist. This study demonstrates that high concentrations of ATP activate P2X₇ receptors and provides the first functional evidence for an extracellular ligand-gated Ca²⁺ influx pathway in osteoclasts. ATP released in response to mechanical stimuli may act through P2X₇ receptors to inhibit osteoclastic resorption.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nucleotides are released as neurotransmitters during inflammation and by a number of cell types in response to mechanical stimulation (1-3). Extracellular nucleotides bind to P2 receptors to mediate diverse biological responses (4). P2 receptors vary in their selectivity for nucleotides and are subdivided into two families, P2X and P2Y. The seven P2X subtypes form ligand-gated channels nonselective for cations (5). Six subtypes of P2Y receptors have been identified in mammals and are G protein-coupled receptors that in many cases are coupled through phospholipase C to the release of Ca²⁺ from intracellular stores (6). Functional P2X receptors are multimeric, composed of at least three subunits (7) that can combine to form homomeric and heteromeric channels (8). Some P2X receptors change ion selectivity within seconds because of the formation of membrane pores (9-11). Cells can express multiple P2 receptors that respond to various ligands, likely accounting for the diversity of biological responses to extracellular nucleotides.

The precise balance of bone resorption and formation during remodeling is regulated by local factors and modulated by mechanical stimuli; however, the underlying mechanisms are unclear. Nucleotides have multiple effects on the functions of bone cells, including osteoclasts, the cells responsible for bone resorption (12). In vitro, low concentrations of ADP, ATP, or their analogs stimulate the resorptive activity of rodent osteoclasts and human osteoclast-like cells (13, 14). In addition, low concentrations of ATP promote, whereas high concentrations inhibit, the formation of murine osteoclasts (14). Thus, it is possible that nucleotides, released locally in response to mechanical or other stimuli, contribute to the regulation of bone remodeling. However, it is not clear which receptor or receptors mediate the effects of nucleotides on osteoclast function. The P2Y₂ receptor, which is activated by both ATP and UTP, has been cloned from human osteoclastoma (15) and identified by in situ hybridization in rat osteoclasts (16). ADP activates the P2Y₁ receptor, the expression of which has been demonstrated in rat osteoclasts by in situ hybridization and immunocytochemistry (17). Rat and rabbit osteoclasts respond to several P2 agonists with an elevation of the cytosolic free calcium concentration ([Ca²⁺]_i)¹ (18-20). These responses involve activation of multiple subtypes of P2Y receptors leading to release of Ca²⁺ from intracellular stores, but the contribution of Ca²⁺ influx is unclear. Nucleotides induce an inward current caused by activation of P2X receptors (21), and the P2X₄ receptor has been identified functionally and at the molecular level in osteoclasts (22). Combined patch clamp and fluorescence studies revealed that the P2X₄ receptor in osteoclasts does not mediate Ca²⁺ entry (20). However, it is possible that other P2X subtypes are present which do permit Ca²⁺ influx.

It is of interest to identify and characterize Ca²⁺ entry pathways in osteoclasts. Electrophysiological studies do not provide evidence for voltage-gated Ca²⁺ channels (23). However, osteoclasts respond to mechanical stimulation with transient elevation of [Ca²⁺]_i which involves both influx and release from stores (19, 24), effects that may be mediated by P2 receptors. The objective of this study was to examine whether P2X receptor subtypes, in addition to P2X₄, are expressed functionally in osteoclasts and to investigate their role in Ca²⁺ influx.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 the long bones of neonatal New Zealand White rabbits or Wistar rats (modified from Ref. 25). Animals were killed by decapitation, and long bones were isolated, minced, and placed in a culture tube containing medium 199 (Life Technologies, Inc., Burlington, ON, Canada), Earle's salts, 25 mM sodium bicarbonate, 0.7 mM L-glutamine, 25 mM HEPES, fetal bovine serum (15% v/v), and antibiotics (100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B). After bone fragments had settled, the suspended cells were plated on 12-mm glass coverslips. For studies of rat osteoclasts, cells were maintained at 37 °C in 5% CO₂ for 30-60 min, after which the coverslips were rinsed with phosphate-buffered saline (PBS) to remove nonadherent cells and submerged in fresh culture medium. Rat osteoclasts were used on the day of isolation. For studies of rabbit osteoclasts, cells were maintained at 37 °C in 5% CO₂ for 2 h, after which coverslips with adherent osteoclasts were submerged in fresh culture medium. The day of isolation was denoted as experimental day 0, and rabbit osteoclasts were studied on days 2-7. On the day of the experiments, the majority of non-osteoclastic cells were removed from rabbit cell preparations using Pronase E (0.001% in PBS with 0.5 mM EDTA) with intermittent agitation at 22 °C (modified from Ref. 26). When the majority of non-osteoclastic cells had been removed (<5 min), the saline was replaced with fresh medium. Osteoclasts were identified by their characteristic morphology under phase-contrast microscopy, and only cells having three or more nuclei were studied. In selected preparations, cytochemical staining for tartrate-resistant acid phosphatase was used to confirm the identity of these cells as osteoclasts.

Electrophysiology-- For recording of macroscopic currents, a conventional whole cell configuration was used with the electrode solution containing (in mM): 136 CsCl, 20 HEPES, 1 MgCl₂, 10 tetraethylammonium chloride, 0.4 CaCl₂, 0.1 EGTA, adjusted with CsOH to pH 7.2, 280-290 mosmol/liter, to block K⁺ currents. When recording K⁺ currents, in some experiments, the electrode solution contained KCl instead of CsCl, and tetraethylammonium chloride was excluded. Greater than 80% series resistance compensation was used. Liquid junction potentials were less than 2 mV with these solutions, and reported potentials were not corrected. Cells were superfused continuously (1-2 ml/min) in Na⁺ solution containing (in mM): 135 NaCl, 5 KCl, 10 glucose, 1 CaCl₂, 20 NaHEPES, pH 7.4, 280-290 mosmol/liter. Where noted, 1 mM MgCl₂ was added to the superfusion solution. Ca²⁺-free solution was prepared by omitting CaCl₂ and adding 0.5 mM EGTA. Na⁺-free solution was prepared by replacing Na⁺ with N-methyl-D-glucamine (NMG⁺; molecular mass, 196 Da). Currents were recorded using an Axopatch-1D amplifier (Axon Instruments, Foster City, CA), filtered (3 db at 1 kHz), and digitized at 2-5 kHz using pClamp 6.0 (Axon Instruments). Experiments were performed at room temperature (22-25 °C).

Agonists and antagonists were applied by superfusion or locally to individual osteoclasts using pressure ejection from micropipettes (5-10 µm diameter) positioned 30-50 µm from the cell (Picospritzer II, General Valve Corporation, Fairfield, NJ). Test substances were dissolved in the corresponding bath solution. When test substances were applied locally, the concentrations reported were those in the pipette solution; the concentrations at the cell surface were necessarily lower. Application of control solutions did not cause appreciable changes in membrane currents or [Ca²⁺]_i. Unless otherwise indicated, chemicals were from Sigma (St. Louis, MO). Values are presented as the mean ± S.E.

Fluorescence Measurement of [Ca²⁺]_i-- [Ca²⁺]_i was measured using the fluorescence from single osteoclasts (27). Cells plated on coverslips were loaded by incubation with 1.5 µM fura-2 acetoxymethylester (Molecular Probes, Eugene, OR) for 30-60 min at room temperature, washed, placed in fresh medium 199, and incubated for 30-60 min at room temperature to allow for ester hydrolysis. Coverslips containing fura-2-loaded cells were placed in a chamber, superfused continuously with buffer solutions, and examined using a Nikon Diaphot inverted microscope. Cells were excited with alternating 340 and 380 nm light, and the emission light was detected using a photomultiplier, sampling at 10 ratios/s (Photon Technologies International, London, ON, Canada). Fluorescence intensities at 340 and 380 nm were corrected for background by subtraction of values obtained from a region with no cells, and the ratio was used to calculate [Ca²⁺]_i (28).

In some experiments, [Ca²⁺]_i and currents were monitored simultaneously using combined fluorescence and patch clamp recording. Cells were loaded with fura-2 prior to establishing whole cell configuration; fluorescence and current were recorded simultaneously to ensure proper temporal alignment. Currents were also recorded at a higher bandwidth on digital videotape and/or digitized using pCLAMP software.

Immunocytochemistry-- Immunocytochemistry was carried out on rat osteoclasts using an affinity-purified anti-P2X₇ polyclonal antibody raised in rabbit (Alomone Laboratories, Jerusalem, Israel). Cells were fixed at room temperature in 4% paraformaldehyde for 10 min, rinsed in PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide in methanol for 30 min at room temperature. Cells were rinsed in PBS, incubated with 1% normal goat serum in PBS for 1 h, and then primary antibody (1:100) was added, and cells were incubated overnight at 4 °C. Cells were washed and incubated with secondary antibody (biotinylated goat anti-rabbit IgG, 1:200) followed by avidin and biotinylated horseradish peroxidase complex, according to the manufacturer's instructions (Vectastain ABC Elite Kit, Vector Labs, Burlingame, CA). Cells were rinsed with PBS, incubated with 3',3'-diaminobenzidine (Sigma, 2 mg/ml H₂O) until color developed, and then counterstained with Harris' modified hematoxylin. In control experiments, primary antibody was either preincubated with excess antigen peptide or omitted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activity-dependent Inward Current-- To characterize the P2X channels in osteoclasts, we used patch clamp techniques to record whole cell currents. In the initial studies, we used a CsCl electrode solution to block the K⁺ currents previously identified in these cells. The initial application of 100 µM ATP evoked an inward current that activated rapidly and then declined during agonist application. This current was identified previously as a P2X₄ nonselective cation current (22). This inward current desensitized rapidly upon successive stimulation with ATP (Fig. 1A, representative of 9 of 9 cells). Current voltage (I-V) relationships were studied using voltage ramp commands, where subtraction of the control current revealed the nucleotide-induced current. The initial ATP-activated current was inwardly rectifying (Fig. 1B) and reversed direction at 7 ± 1 mV (n = 9). In contrast to 100 µM ATP, 100 µM BzATP, which is a more potent agonist than ATP at P2X₇ receptors, evoked the initial inward current followed by an inward current that increased in amplitude with successive stimulations (Fig. 1C). This result is representative of that seen in 18 of 21 cells studied, with the remaining 3 cells exhibiting an initial P2X₄ current but no later component. This later component of the BzATP-activated current was inwardly rectifying and reversed at 9 ± 1 mV (Fig. 1D). Such a current was also observed in response to 1 mM ATP (4 of 5 cells, with a reversal of 9 ± 4 mV, not shown). In 7 of the 18 responding cells, BzATP evoked inward currents without delay. Thus, nucleotides appear to activate two distinct P2X currents: an initial P2X₄ current that desensitizes followed by an activity-dependent current.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Development of inward current in response to successive nucleotide applications. Whole cell currents were recorded from rabbit osteoclasts at a holding potential of 30 mV with the electrode containing CsCl solution to block K+ currents. Voltage ramp commands (0.6 V/s) were used to obtain I-V relationships where subtraction of the control current revealed the nucleotide-induced current, here and in subsequent figures. Symbols in panels A and C represent current recorded at 100 mV. Bars above the current trace represent the length of agonist application. Panel A, 100 µM ATP was applied for the times indicated, with the initial application inducing an inward current and little response to successive applications. Panel B, I-V relationships for the same cell as in panel A. The initial stimulation (1) of osteoclasts with ATP induced an inwardly rectifying current that reversed near 0 mV. Little current was elicited upon the 5th, 10th, or 15th stimulation. Panel C, whole cell current recorded from a different osteoclast in response to 100 µM BzATP. After the initial response to BzATP, successive stimulations led to the progressive development of an inward current of increasing amplitude. Panel D, I-V relationships for the cell in panel C reveal that both early and developing BzATP-induced currents reversed close to 0 mV.

Kinetics of Nucleotide-induced Currents-- The kinetics of P2X currents vary depending on the receptor subtype. The inward P2X₄ current activated by 100 µM ATP reached a maximum then declined rapidly in the continued presence of agonist (Fig. 2A, left), as reported previously in osteoclasts (22). Subsequent applications of 100 µM ATP elicited a current that was of reduced amplitude (Fig. 2A, right) but still inactivated in the presence of agonist. In contrast, 100-300 µM BzATP induced an inward current that persisted during and after agonist application, for periods of up to 1 min, considerably longer than the time required for solution exchange in these studies (Fig. 2B). These properties were observed in all cells studied with BzATP and also with high concentrations of ATP (1 mM, Fig. 2C). P2X₂ receptors have been identified in rat osteoclasts using immunocytochemistry (16). However, the characteristics of the activity-dependent current argue against involvement of P2X₂, which deactivates promptly upon agonist washout (29). Taken together, the persistence of the activity-dependent current during agonist application and its delayed deactivation upon agonist removal suggest the presence of P2X₇ receptors in osteoclasts.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Kinetics of P2X currents evoked by ATP and BzATP. Whole cell configuration was used to record currents from rabbit osteoclasts, with symbols illustrating the current at 100 mV. Panel A, the initial stimulation of an osteoclast with 100 µM ATP elicited an inward current caused by activation of P2X4 channels which declined during the continued presence of ATP (left). Subsequent stimulation of the same cell with 100 µM ATP evoked an inward current of smaller amplitude which declined in the presence of ATP (right). Panel B, after an initial stimulation, the application of 300 µM BzATP to a different osteoclast evoked a sustained inward current that deactivated slowly after agonist removal (left). Subsequently, BzATP elicited an even larger sustained inward current (right). Panel C, a higher concentration of ATP (1 mM) also evoked an initial rapid inward current (left), whereas later stimulation of the same cell by ATP induced an inward current that slowly deactivated on washout (right). The pipette contained CsCl electrode solution, to block K+ current, and the bath contained standard Na+ solution.

Nonselective Cation Channel-- Ion substitution was used to examine the selectivity of this later component of the BzATP-induced current. Replacement of Na⁺ with the large cation NMG⁺ caused the current at 30 mV to reverse from inward to outward (Fig. 3A) because of a shift of the I-V relationship to more negative potentials (Fig. 3B). This result is representative of responses recorded in 7 cells, with a mean reversal in NMG⁺ solution of 50 ± 5 mV. Thus, the BzATP-activated conductance exhibits low permeability to NMG⁺. Slow deactivation upon agonist washout accounts for the undershoot of the current recorded at 30 mV in NMG⁺ solution, as extracellular levels of Na⁺ are restored before channel closure (Fig. 3A). It has been reported that prolonged stimulation of P2X₇ or heterologously expressed P2X₂ or P2X₄ receptors can result in a change of ion selectivity within seconds because of the formation of pores that are permeable to NMG⁺ (9-11). To assess the possibility of pore formation, we examined the effects of prolonged stimulation of cells superfused in NMG⁺ solution. The persistence of outward current at 30 mV, even after a 3-min stimulation with BzATP (Fig. 3C), demonstrates that the channel remains impermeable to NMG⁺. Taken together, these data indicate that BzATP activates an activity-dependent P2X channel that is permeable to small cations and does not behave as a pore.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The BzATP-activated channel is impermeable to large cations. Whole cell configuration was used to record currents from rabbit osteoclasts, with a holding potential of 30 mV. CsCl electrode solution was used to block K+ currents. Panel A, the application of 300 µM BzATP from micropipettes, in the presence of extracellular Na+ (in micropipette and bath), activated a sustained inward current shown at both 30 and 100 mV. In contrast, the application of 300 µM BzATP in NMG+ solution (in micropipette) activated an outward current at 30 mV. The cell had been stimulated multiple times prior to the traces shown. Panel B, I-V relationships of BzATP-activated currents for the same cell as in panel A at the times indicated by i and ii. Replacement of extracellular Na+ with NMG+ shifted the reversal of the BzATP-activated current to more negative potentials. Panel C, the effect of a prolonged application of BzATP was examined in another cell. The cell was superfused continuously with Na+-free solution, and BzATP in NMG+ solution was applied by micropipette for the time indicated by the bar above the current trace. Even prolonged stimulation (up to 3 min) with BzATP did not result in a time-dependent change in the selectivity of the current. The outward current persisted in Na+-free solution, indicating that NMG+ did not permeate the channels.

Sensitivity of the P2X Current to Divalent Cations and Antagonists-- Because divalent cations play an important role in determining the kinetics and amplitude of P2X currents, we examined their effects on the activity-dependent current. In these experiments, solutions did not contain Mg²⁺ unless indicated. BzATP elicited a sustained inward current in both the presence and absence of Ca²⁺ (Fig. 4A). However, 1 mM Ca²⁺ reversibly reduced the amplitude of the BzATP-induced current. In solutions that contained Ca²⁺, BzATP elicited a sustained inward current in both the presence and absence of Mg²⁺, although 1 mM Mg²⁺ reduced the BzATP-induced current (Fig. 4B). Similarly, 10 µM Zn²⁺ reversibly reduced the amplitude of the BzATP-induced current (Fig. 4C). Data are representative of responses in 9 cells for Ca²⁺ (amplitude of current reduced by 43 ± 8%), 3 cells for Mg²⁺ (amplitude of current reduced by 70 ± 2%), and 5 cells for Zn²⁺ (amplitude of current reduced by 27 ± 8%). Notably, the addition of these divalent cations did not cause any marked change in the reversal potential or time course of deactivation. Suppression of this current by divalent cations resembles that reported for P2X₇ current (9). Moreover, reduction of the current by Zn²⁺ argues against mediation of the activity-dependent current by P2X₄ receptors, as Zn²⁺ potentiates the P2X₄ current (8, 22).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Sensitivity of the activity-dependent current to divalent cations. Whole cell configuration was used to record currents from individual rabbit osteoclasts, with the current at 100 mV plotted as a function of time. Panel A, 150 µM BzATP in Ca2+- and Mg2+-free solution containing 0.5 mM EGTA activated a sustained inward current that was reversibly diminished by 1 mM extracellular Ca2+. The I-V relationships (right) of BzATP-activated currents indicate the diminished current amplitude in Ca2+ (ii) compared with Ca2+-free conditions (i and iii). Panel B, similarly, 150 µM BzATP in Mg2+-free solution containing 1 mM Ca2+ elicited an inward current that was reversibly diminished in the presence of extracellular 1 mM Mg2+. On the right, I-V relationships illustrate the diminished current in the presence of Mg2+ (ii) with recovery in Mg2+-free solution (iii). Panel C, 150 µM BzATP in Mg2+-free solution containing 1 mM Ca2+ activated a sustained inward current that was reversibly diminished in the presence of 10 µM Zn2+. The I-V relationships (right) of BzATP-activated currents indicate the diminished current amplitude in the presence of Zn2+ (ii). Cells in panels A-C had been stimulated multiple times prior to the traces shown. Ca2+, Mg2+, and Zn2+ did not alter the reversal potential or rate of current deactivation appreciably.

P2 receptor antagonists also inhibited the activity-dependent current in osteoclasts. PPADS is an inhibitor of several P2 receptors including P2X₇ (5, 6). PPADS did not block the initial P2X₄ current in osteoclasts (n = 7), whereas the activity-dependent current was progressively inhibited (Fig. 5, n = 8). Oxidized ATP, an irreversible antagonist at the P2X₇ receptor (30), reduced the activity-dependent current (6 of 8 cells, not shown). Thus, the electrophysiological and pharmacological characteristics of the activity-dependent current in osteoclasts are similar to those reported for P2X₇ currents in other systems.

View larger version (7K): [in this window] [in a new window]   Fig. 5.   PPADS inhibits the activity-dependent current induced by BzATP. Whole cell configuration was used to record current in rabbit osteoclasts, with data illustrating the current measured at 100 mV. A CsCl electrode solution was used to block the K+ current. Panel A, successive stimulations with 200 µM BzATP induced a sustained inward current. The eighth stimulation is shown on the left. The BzATP-induced inward current was reduced progressively with a bath application of 100 µM PPADS. Panel B, in another cell, pretreatment with 100 µM PPADS for 5 min did not block the rapidly inactivating P2X4 current induced by the initial application of 150 µM BzATP. Breaks in the current traces represent 5 min.

Immunocytochemical Identification of P2X₇ Receptors in Osteoclasts-- Immunocytochemistry was used to confirm the expression of P2X₇ receptors in osteoclasts. These experiments were carried out using rat osteoclasts because of the binding of the secondary antibody to endogenous immunoglobulins present in preparations of rabbit osteoclasts. Positive immunostaining for P2X₇ was observed in most multinucleated cells (Fig. 6A; 143 of 146 osteoclasts from three separate experiments). In contrast, positive staining was observed in only 30% of the mononucleated cells, most likely monocytes and osteoclast precursors. Staining was not apparent in spindle-shaped stromal cells also found in these preparations. As negative controls, primary antibody was preincubated with antigen peptide or omitted. In these preparations, virtually all cytochemical staining was abolished (Fig. 6B).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Immunocytochemical staining reveals P2X7 immunoreactivity in osteoclasts. Immunocytochemical staining was carried out on cells isolated from the long bones of neonatal rats. Nuclei were counterstained with Harris' modified hematoxylin (blue). Osteoclasts (indicated by arrows) were identified as having  3 nuclei. Panel A, in this bright-field photomicrograph, positive immunostaining (brown) for P2X7 was observed in a multinucleated osteoclast. The cell shown is representative of 143 of 146 osteoclasts from three separate experiments. Panel B, as a negative control, primary antibody was incubated with excess antigen peptide for P2X7, which abolished immunostaining.

Functional Effects of Nucleotides on [Ca²⁺]_i in Osteoclasts-- To determine whether endogenous P2X₇ receptors elevate [Ca²⁺]_i, we measured changes of [Ca²⁺]_i in isolated rabbit osteoclasts in response to repetitive agonist stimulation. 100 µM ADPS (a P2Y-selective agonist) or 100 µM ATP induced transient elevations of [Ca²⁺]_i that desensitized with successive applications (Fig. 7, A and B, observed in 5 of 5 and 6 of 6 cells tested, respectively). Similarly, 100 µM UTP (a P2Y₂ agonist) elicited only an initial response in 8 of 10 cells (2 cells did not respond). 100-300 µM BzATP or a high concentration of ATP (1 mM) induced an initial transient elevation of [Ca²⁺]_i, but notably continued applications led to the progressive development of a sustained [Ca²⁺]_i elevation (Fig. 7, C and D). This activity-dependent rise of [Ca²⁺]_i was observed in 44 of 52 osteoclasts stimulated in this way but was not observed in response to ADPS, UTP, or lower concentrations of ATP, distinguishing it from P2Y-mediated responses. Thus, successive stimulation with BzATP elicits elevation of [Ca²⁺]_i with a pattern resembling the activity-dependent P2X₇ current described above.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Activity-dependent elevation of [Ca2+]i in rabbit osteoclasts. Cells were loaded with fura-2 to monitor [Ca2+]i in individual osteoclasts. Nucleotides were applied for 5 s every 30 s using pressure ejection from micropipettes, as indicated by bars below the traces. 100 µM ADPS (panel A) or 100 µM ATP (panel B) elicited a transient rise of [Ca2+]i caused by the activation of P2Y receptors. The P2Y-mediated response desensitized with successive applications, and [Ca2+]i returned to basal levels. In contrast, 300 µM BzATP (panel C), a relatively potent agonist at the P2X7 receptor, or a high concentration of ATP (1 mM) (panel D) induced the initial P2Y-mediated rise of [Ca2+]i, and successive applications led to sustained Ca2+ elevations. Cells were bathed in standard Na+ solution.

Sensitivity of Activity-dependent [Ca²⁺]_i Elevations to P2 Antagonists-- P2 antagonists were used to investigate which receptor subtype mediates the activity-dependent rise of [Ca²⁺]_i. 100 µM oxidized ATP, which inhibited the activity-dependent current, also caused a gradual reduction of the BzATP-induced rise of [Ca²⁺]_i (Fig. 8A). Such a blockade was observed in 7 of 8 cells tested, with no reversal seen on washout of antagonist. We next tested PPADS, which is reported to inhibit P2X₇, but not P2Y₂ or P2X₄ receptors (6). 100 µM PPADS did not block the initial rise of [Ca²⁺]_i but did prevent the activity-dependent component (Fig. 8B, 5 of 5 cells).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effects of P2 receptor antagonists on BzATP-induced [Ca2+]i elevations. Rabbit osteoclasts were loaded with fura-2, and [Ca2+]i was monitored. Test agents were delivered via pressure ejection from micropipettes every 30 s, indicated by bars below the traces. Panel A, successive stimulation with 150 µM BzATP led to the development of [Ca2+]i elevations that were irreversibly reduced by the P2X7 receptor antagonist, oxidized ATP (oxATP; 100 µM). Panel B, 100 µM PPADS, which is a blocker of several P2 receptors, including P2X7, inhibited the activity-dependent [Ca2+]i elevations when applied to the cell concurrently with 150 µM BzATP.

BzATP-induced Elevation of [Ca²⁺]_i Is Dependent on Extracellular Ca²⁺ but Not Na⁺-- We next examined the source of Ca²⁺ mediating the activity-dependent changes of [Ca²⁺]_i. After successive stimulation with BzATP, the progressive rise of [Ca²⁺]_i was abolished upon removal of extracellular Ca²⁺ (Fig. 9A, in 6 of 6 osteoclasts studied). This effect was fully reversible on readdition of Ca²⁺ (Fig. 9A, right), consistent with BzATP-induced influx of Ca²⁺. To test the possibility that the BzATP-induced rise of [Ca²⁺]_i was dependent on extracellular Na⁺, we studied the response of osteoclasts bathed in solutions in which NMG⁺ was substituted for Na⁺. Even in the absence of Na⁺, BzATP continued to induce elevations of [Ca²⁺]_i in 7 out of 7 cells tested (Fig. 9B), ruling out a requirement for extracellular Na⁺. We note that in Na⁺-free solutions, the BzATP-mediated rise of [Ca²⁺]_i developed rapidly. Even when studied in solutions containing Na⁺, this rapid development was observed in 22 of 52 osteoclasts studied, whereas the response developed gradually in the remaining cells. The rapid response, like the gradually developing response, was dependent on extracellular Ca²⁺. Thus, although the initial nucleotide-induced rise of [Ca²⁺]_i is caused by the release of Ca²⁺ from intracellular stores (20), the activity-dependent rise of [Ca²⁺]_i represents a Ca²⁺ influx pathway that has hitherto not been demonstrated in osteoclasts.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   The BzATP-induced rise of [Ca2+]i is dependent on Ca2+o but not Na+o. [Ca2+]i was measured in individual rabbit osteoclasts in response to BzATP, applied every 30 s, as indicated by the bars below the trace. Panel A, cells were bathed in Na+ solution with or without 1 mM Ca2+ as indicated above the trace. 100 µM BzATP elicited an initial transient rise of [Ca2+]i followed by development of responses that were dependent on bath Ca2+. Panel B, successive stimulation with BzATP in Na+-free solution containing NMG+ and Ca2+ led to development of Ca2+ transients.

BzATP Activates a Ca²⁺ Influx Pathway-- Patch clamp recording allowed us to evaluate the link between activity-dependent elevations of [Ca²⁺]_i and membrane current. A rise of [Ca²⁺]_i has previously been shown to activate intermediate conductance Ca²⁺-dependent K⁺ channels in osteoclasts (21). When studied using a K⁺-containing electrode solution, ATP elicited a biphasic response, with an initial inward P2X₄ current followed by outward K⁺ current caused by the P2Y receptor-mediated release of Ca²⁺ from stores (Fig. 10A). The I-V relationships confirm the different selectivity of the early and late currents (Fig. 10A, right). Subsequent stimulations with BzATP evoked an inward current that reversed near 0 mV, but no outward current, indicating desensitization of P2Y receptors (Fig. 10B). A striking change was observed with further stimulations, where we observed progressive development of Ca²⁺-dependent K⁺ current (4 of 6 cells, Fig. 10C). This channel provided us with a physiological indicator of a rise of [Ca²⁺]_i, confirming activity-dependent elevation of [Ca²⁺]_i in osteoclasts.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Activity-dependent development of Ca2+-dependent K+ current. Whole cell currents were recorded from a rabbit osteoclast at 30 mV. The pipette contained K+ electrode solution to allow recording of K+ currents. 100 µM ATP or 300 µM BzATP was applied for the times indicated by the bars above the current traces. To identify currents, voltage-ramp commands (0.6 V/s) were used to obtain I-V relationships. Panel A, initially, ATP induced an inward current (i) that reversed close to 0 mV, consistent with P2X4. This was followed by an outward current (ii) that reversed at 60 mV, caused by activation of a Ca2+-dependent K+ conductance. Panel B, upon subsequent stimulation of the same cell with BzATP, BzATP induced an inward current (iii) that reversed at 0 mV, but there was no later outward K+ current (iv). Panel C, after 12 stimulations, BzATP evoked the inward current (v), which was now followed by an outward current (vi) reversing at more negative membrane potentials, consistent with the reappearance of the Ca2+-dependent K+ conductance.

We next used combined patch clamp and fluorescence techniques to investigate the relationship between activity-dependent current and changes of [Ca²⁺]_i. The BzATP-induced current was closely associated with graded elevations of [Ca²⁺]_i (Fig. 11A). This relationship was observed in 10 of 10 osteoclasts that exhibited the activity-dependent current and provided a firm link between the changes in [Ca²⁺]_i and the P2X currents. When bath Ca²⁺ was removed, there was augmentation of the inward current (as shown above in Fig. 4), but notably the rise of [Ca²⁺]_i was abolished (Fig. 11B, 5 of 5 cells). This effect was fully reversible upon reintroduction of Ca²⁺ and provides direct evidence for a Ca²⁺ influx pathway in osteoclasts.

View larger version (21K): [in this window] [in a new window]   Fig. 11.   Activation of P2X7 receptors allows Ca2+ influx. Whole cell currents and [Ca2+]i were recorded simultaneously in single fura-2-loaded rabbit osteoclasts. Cells were superfused with Na+ solution with or without 1 mM Ca2+ as indicated. Panel A, 100 µM BzATP activated an inward current (bottom, recorded at 100 mV) that increased in amplitude with successive stimulations, accompanied a by progressive increase of [Ca2+]i (upper trace). Two stimulations occurred before the responses displayed. Panel B, in another cell, removal of bath Ca2+ reversibly abolished the rise of [Ca2+]i, even though the inward current persisted, indicating that BzATP induced Ca2+ influx. Breaks in the trace represent 2 min for bath exchange. Three stimulations occurred before the responses displayed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Osteoclasts Respond to Nucleotides with an Activity-dependent P2X₇ Current-- In this study, we examined the behavior of endogenously rather than heterologously expressed P2 receptors. Rabbit osteoclasts responded to initial stimulation with ATP and certain analogs with an initial inward P2X₄ current followed by an outward K⁺ current, mediated by P2Y receptor signaling. We demonstrate here that successive stimulation of osteoclasts leads to development of an inward current, with features identifying it as a P2X₇ current. Simultaneous patch clamp and fluorescence recording revealed that P2X₇ channels mediate Ca²⁺ influx in the absence of pore formation.

Pharmacological characteristics helped to resolve the receptor subtype underlying the activity-dependent current. This current was activated by successive stimulations with BzATP or a high concentration of ATP but not low concentrations of ATP. In this regard, ATP concentrations at least 10-fold higher than those of BzATP are required to activate P2X₇ receptors expressed in HEK293 cells (9). In contrast, P2X₄ receptors in rabbit osteoclasts are activated by low concentrations of ATP as well as BzATP, as reported previously for heterologously expressed P2X₄ receptors (31). P2X₂ receptors, which have been identified in rat osteoclasts using immunocytochemistry (16), are activated by low concentrations of ATP, so our data are inconsistent with these receptors mediating the sustained inward current. Antagonist blockade distinguishes the initial and activity-dependent currents. PPADS reduced the activity-dependent current, but had no effect on the initial P2X₄ current, consistent with previous studies showing that P2X₄ receptors are insensitive to this antagonist (32).

In response to prolonged or repeated nucleotide application, P2X₂, P2X₄, and P2X₇ receptors can exhibit features associated with formation of membrane pores (9-11). The activity-dependent channel in osteoclasts remains impermeable to the large cation NMG⁺, arguing against pore formation. The ability of P2X₇ receptors to induce pore formation appears to vary among species. For example, pore formation requires higher concentrations of agonists and is more sensitive to divalent cations in human than in rat cells (33). Differences have also been reported in the ability of P2X₇ receptors to induce pore formation in different cell types from the same species (34), leading to the suggestion that the P2X₇ receptor and the cytolytic pore are distinct entities (35).

P2X channels exhibit differences in their rates of inactivation. Initial nucleotide stimulation of rabbit osteoclasts activates the P2X₄ current that inactivates during agonist application, as described for P2X₄ receptors heterologously expressed in HEK293 cells (36). In contrast, the kinetics of the activity-dependent current are consistent with those of P2X₇ channels expressed in these cells, which deactivate slowly after agonist removal (9).

P2X channels vary in their sensitivity to divalent cations. Zn²⁺ at micromolar concentrations potentiates the P2X₄ current in rabbit osteoclasts (22), as shown for P2X₂ and P2X₄ receptors heterologously expressed in other systems (29, 37). In contrast, the activity-dependent inward current in rabbit osteoclasts was inhibited by Zn²⁺, as described previously for P2X₇ currents (38). Although the activity-dependent current develops in the presence of Ca²⁺ and Mg²⁺, a greater number of stimulations are required to elicit this inward current, and the current is reduced in amplitude. It is notable that ATP-induced pore formation has been reported in murine osteoclasts studied in the absence of divalent cations (39). To facilitate examination of the current (and Ca²⁺ fluorescence studies), our experiments were carried out in bath solutions containing 1 mM Ca²⁺, and Mg²⁺ was usually omitted.

Taken together, these pharmacological and biophysical data point to the conclusion that the activity-dependent current in osteoclasts is caused by P2X₇ receptors. Using immunocytochemistry, we confirmed that P2X₇ was expressed in multinucleated rat osteoclasts, consistent with a report from Hoebertz and co-workers (16). It is possible that native P2X channels in osteoclasts behave differently from previously characterized heterologously expressed receptors. Further, functional P2X channels are each composed of at least three subunits (7). Some P2X subtypes combine to form heteromultimeric channels, with functional evidence for P2X_2/3, P2X_1/5, P2X_4/6, and P2X_2/6 receptors (5, 8). When the P2X₇ receptor was coexpressed with P2X subtypes in HEK293 cells, the P2X₇ receptor did not coimmunoprecipitate with any other P2X subtypes, leading the authors to propose that functional P2X₇ channels are homomeric (40).

P2X₇ Receptors Mediate Ca²⁺ Influx-- Successive stimulation with BzATP or high concentrations of ATP led to the development of [Ca²⁺]_i elevations that were closely associated with inward current. These [Ca²⁺]_i elevations were dependent on extracellular Ca²⁺, indicating Ca²⁺ permeability of the channels. Many P2X receptors exhibit permeability to Ca²⁺ and could mediate the nucleotide-induced rise of [Ca²⁺]_i (31). However, P2X₄ receptors do not mediate a detectable Ca²⁺ influx in osteoclasts (20). Furthermore, activity-dependent elevation of [Ca²⁺]_i was prevented by PPADS, an antagonist at the P2X₇ but not P2X₄ receptors (6). Based on these characteristics, we conclude that P2X₇ receptors mediate the observed rise of [Ca²⁺]_i.

Other mechanisms of Ca²⁺ elevation must be considered. P2X channels in osteoclasts are permeable to Na⁺, and it is possible that a P2X-mediated rise of [Na⁺]_i could lead to an elevation of [Ca²⁺]_i via a number of mechanisms. First, a rise of [Ca²⁺]_i may be elicited by cell swelling caused by an accumulation of intracellular Na⁺. In chicken osteoclasts, cell swelling activates ion channels that are permeable to Ca²⁺ (24). Second, the nucleotide-induced elevation of [Ca²⁺]_i could arise because of decreased activity of Ca²⁺-ATPases. Cells respond to an increase in [Na⁺]_i with activation of the Na⁺,K⁺-ATPase. Increased activity of the Na⁺,K⁺-ATPase could lead to depletion of intracellular ATP, in turn leading to decreased activity of Ca²⁺-ATPases. Third, in some cells the Na⁺/Ca²⁺ exchanger can reverse direction with a rise of cytosolic Na⁺, leading to elevation of [Ca²⁺]_i (41). However, we demonstrate that replacing Na⁺ with NMG⁺, which does not permeate the activity-dependent P2X channel, did not abolish the activity-dependent rise of [Ca²⁺]_i, arguing against a mechanism involving elevation of [Na⁺]_i. Thus, our study provides the first evidence for a nucleotide-induced, P2X₇-mediated Ca²⁺ entry pathway in osteoclasts.

Possible Physiological Role of P2X₇ Receptor Signaling in Osteoclasts-- Osteoclasts are exposed to nucleotides from a number of sources. For example, nucleotides are released from the cytoplasm of damaged cells or in response to mechanical stimulation (3, 42, 43). In this regard, ATP is released from chondrocytes in response to mechanical stimulation and may act on cells in the bone microenvironment (44). Osteoblasts and osteoclasts respond to mechanical stimulation with increased [Ca²⁺]_i, and it is possible that this effect is mediated by efflux of nucleotides.

Nucleotides exert diverse effects on osteoclasts. Whereas low concentrations stimulate osteoclast formation and resorption, higher concentrations inhibit osteoclast formation (13, 14). It is possible that the effects of low concentrations of nucleotides are mediated through P2Y receptors, which induce large Ca²⁺ transients. On the other hand, the inhibitory effects of high concentrations of ATP may be mediated by P2X₇ receptors, which give rise to a sustained elevation of [Ca²⁺]_i. Although both P2Y and P2X₇ signaling lead to increases in [Ca²⁺]_i, divergent responses may arise because of the differences in amplitude and duration of the Ca²⁺ signal, as established in other systems (45). Alternately, differing effects of P2Y and P2X₇ receptors may reflect selective activation of other signaling systems, such as the mitogen-activated protein kinase or nuclear factor-B pathways.

Inhibitory effects mediated by P2X₇ receptors could result in fewer osteoclasts, providing a mechanism for suppressing bone resorption. In this regard, decreased cancellous and cortical bone mass has recently been found in mice lacking the P2X₇ receptor,² supporting a key role for P2X₇ receptors in the control of bone turnover. P2X₇ receptors regulate cell survival in several systems, for example triggering apoptosis in thymocytes, microglial cells, and dendritic cells (46-48). Coexpression of multiple P2 receptor subtypes may allow osteoclasts to respond appropriately to nucleotides, depending on the types of nucleotide present, their concentration, and the duration of exposure.

	ACKNOWLEDGEMENTS

We thank Dr. A. Frederik Weidema (Department of Cell Physiology, University of Nijmegen, The Netherlands) for assistance with preliminary studies and Dr. Graham Wagner and Mark Paciga (Department of Physiology, University of Western Ontario) for helpful advice on immunocytochemistry.

	FOOTNOTES

^* This work was supported by The Arthritis Society and the Canadian Arthritis Network.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.

To whom correspondence should be addressed. Tel.: 519-661-3768; Fax: 519-661-3827; E-mail: stephen.sims@fmd.uwo.ca.

Published, JBC Papers in Press, August 8, 2001, DOI 10.1074/jbc.M105881200

² Chidsey-Frink, K. L., Qi, H., Crawford, D. T., Simmons, H. A., Audoly, L., Gabel, C. A., Thompson, D. D., and Ke, H. Z. (2001) J. Bone Miner. Res. 16, S378

	ABBREVIATIONS

The abbreviations used are: [Ca²⁺]_i, cytosolic free Ca²⁺ concentration; ADPS, adenosine 5'-O-(2-thiodiphosphate); BzATP, 2'- & 3'-O-(4-benzoylbenzoyl)-ATP; NMG⁺, N-methyl-D-glucamine; PBS, phosphate-buffered saline; PPADS, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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