Mechanism and role of high-potassium-induced reduction of intracellular Ca2+ concentration in rat osteoclasts

Hiroshi Kajiya, Fujio Okamoto, Hidefumi Fukushima, Keisuke Takada, and Koji Okabe

Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka 814-0193, Japan

Submitted 21 January 2003 ; accepted in final form 3 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteoclasts are multinucleated, bone-resorbing cells that show structural and functional differences between the resorbing and nonresorbing (motile) states during the bone resorption cycle. In the present study, we measured intracellular Ca2+ concentration ([Ca2+]i) in nonresorbing vs. resorbing rat osteoclasts. Basal [Ca2+]i in osteoclasts possessing pseudopodia (nonresorbing/motile state) was around 110 nM and significantly higher than that in actin ring-forming osteoclasts (resorbing state, around 50 nM). In nonresorbing/motile osteoclasts, exposure to high K+ reduced [Ca2+]i, whereas high K+ increased [Ca2+]i in resorbing state osteoclasts. In nonresorbing/motile cells, membrane depolarization and hyperpolarization applied by the patch-clamp technique decreased and increased [Ca2+]i, respectively. Removal of extracellular Ca2+ or application of 300 µM La3+ reduced [Ca2+]i to ~50 nM in nonresorbing/motile osteoclasts, and high-K+-induced reduction of [Ca2+]i could not be observed under these conditions. Neither inhibition of intracellular Ca2+ stores or plasma membrane Ca2+ pumps nor blocking of L- and N-type Ca2+ channels significantly reduced [Ca2+]i. Exposure to high K+ inhibited the motility of nonresorbing osteoclasts and reduced the number of actin rings and pit formation in resorbing osteoclasts. These results indicate that in nonresorbing/motile osteoclasts, a La3+-sensitive Ca2+ entry pathway is continuously active under resting conditions, keeping [Ca2+]i high. Changes in membrane potential regulate osteoclastic motility by controlling the net amount of Ca2+ entry in a "reversed" voltage-dependent manner, i.e., depolarization decreases and hyperpolarization increases [Ca2+]i.

membrane depolarization; resorbing and motile activities; bone resorbing cycle


OSTEOCLASTS ARE tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells that alter their structure and function during the bone resorption cycle. In the resorbing state, osteoclasts adhere to the bone surface via integrin receptors and form ruffled borders and clear zones with a highly polarized cytoplasmic organization. They release protons and secrete lysosomal enzymes across the membrane of this ruffled border and digest both inorganic and organic components of the bone matrix (1, 5, 17). This process increases local Ca2+ concentration in the resorbed area and, subsequently, intracellular Ca2+ concentration ([Ca2+]i). As a result, resorbing activity gradually declines and the osteoclasts turn into nonresorbing/motile cells, which migrate and settle on a new surface area of the bone.

Thus elevation and decline of [Ca2+]i is very closely related with osteoclastic function. Bizzarri et al. (4) and Moonga et al. (22) demonstrated that calcitonin and interleukin-4, known inhibitors of bone resorption, increase [Ca2+]i in mouse and rat osteoclasts. Extracellular high-potassium (high K+) or extracellular high-calcium (high Ca2+) solution was also reported to increase [Ca2+]i (2, 4, 8, 18, 21, 34), suggesting the importance of cell membrane potential and Ca2+ transport across the membrane for [Ca2+]i regulation. On the other hand, Shankar et al. (27) reported conflicting results, in that both membrane depolarization induced by high-K+ solution and hyperpolarization induced by valinomycin increased [Ca2+]i. Furthermore, Miyauchi et al. (21) demonstrated that high-K+ solution reduced [Ca2+]i in osteoclasts attached to bone particles.

These reports lead us to hypothesize that the change in [Ca2+]i may be state dependent (i.e., differing in resorbing vs. nonresorbing osteoclasts). To test this hypothesis, we examined the effect of high-K+ solution in resorbing vs. nonresorbing osteoclasts, because high-K+ stimulation was demonstrated to induce both increases and decreases of [Ca2+]i (4, 21).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteoclast preparation. The procedure for isolation and culture of osteoclasts was slightly modified from the original methods published by Chambers et al. (6). Briefly, neonatal Wistar rats (1–2 days old) were anesthetized with diethyl ether and killed by cervical dislocation. The femora and tibiae were isolated. The procedures were approved by the Council on Animal Care of the Fukuoka Dental College. Adherent soft tissues were removed, and the bones were cut across their epiphyses in culture medium [{alpha}-minimum essential medium ({alpha}-MEM), GIBCO BRL, Grand Island, NY] containing 15% heat-inactivated fetal bovine serum (FBS; GIBCO BRL) and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). Osteoclasts were mechanically disaggregated by curetting bone fragments into {alpha}-MEM and agitating the suspension with a pipette. The supernatant (105–106 cells/coverslip) was then dispersed onto 5 x 5-mm glass coverslips, and cells were allowed to settle and attach for 30 min at 37°C. In some experiments, the supernatant was dispersed onto dentine slices (4-mm diameter) to assess bone resorption activity. Contaminating cells were removed by rinsing the coverslips with {alpha}-MEM. Osteoclasts were easily identified by their large size and multinucleated shape under a phase-contrast microscope. The cells were incubated with culture medium in 5% CO2-95% air at 37°C (pH 7.4). Under these conditions, >60% of osteoclasts formed actin rings on glass coverslips or dentine slices after culture for 4 h. All experiments were performed within 4–12 h after cell isolation.

[Ca2+]i measurements. [Ca2+]i was measured in single osteoclasts by a dual excitation ratiometric method using the fluorescent calcium indicator fura 2. Osteoclasts adhering to glass coverslips were incubated for 40 min at 25°C with 2 µM fura 2-AM in a physiological salt solution (PSS) containing (in mM) 134 NaCl, 6 KCl, 10 N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2.5 CaCl2, 0.5 MgCl2, and 10 glucose, pH adjusted to 7.3 with Tris. The coverslips were then washed with PSS for at least 5 min before experiments were started. Excitation wavelengths of 340 and 380 nm were applied via an inverted microscope equipped with a x40 fluor objective lens (Nikon, Tokyo, Japan). Emitted fluorescence was deflected to pass through a dicroic mirror (400 nm), and the transmitted light was filtered at 510 nm and monitored by a photomultiplier (SPEX Industries, Edison, NJ) and an ICCD camera (Hamamatsu Photonics, Hamamatsu, Japan). Coverslips were mounted on a temperature-controlled chamber (volume of 1 ml) and superfused continuously with 1 ml/min of PSS. To alter membrane potential, high-K+ solution was prepared by replacement of NaCl in PSS with an equimolar amount of KCl. All experiments were performed at 33–34°C. Fura 2 fluorescence was calibrated to [Ca2+]i at the end of each experiment, exposing the cells to 10 µM ionomycin to assess the Ca2+-saturated ratio (Rmax), followed by 5 mM EGTA to determine the nominally Ca2+-free ratio (Rmin). Finally, MnCl2 (5 mM) was added to estimate autofluorescence, which was subtracted from the experimental values. [Ca2+]i was calculated using the method published by Grynkiewicz et al. (11). At the end of the experiments, all cells were stained with TRAP to conclusively identify them as osteoclasts using an acid phosphatase kit (Sigma Chemical, St. Louis, MO).

Electrophysiological experiment. For recording membrane potential and currents, a conventional whole cell patch-clamp method was used. Cells were superfused continuously (1 ml/min) with PSS. The patch electrode was filled with standard electrode solution containing (in mM) 140 KCl, 3 MgCl2, 2 ATP (disodium salt), 0.3 EGTA, and 10 HEPES, adjusted to pH 7.3 with Tris. In the experiments shown in Fig. 6, membrane currents were recorded in K+-free extracellular solution prepared by replacing K+ in the PSS with Na+ (Na+-rich solution), using high-Cs+ electrode solution containing (in mM) 100 Cs aspartate, 45 CsCl, 3 MgCl2, 2 ATP (disodium salt), 0.3 EGTA, and 10 HEPES, adjusted to pH 7.3 with Tris. Ca2+-rich (Na+-free) solution contained (in mM) 71.8 CaCl2, 10 glucose, 70 mannitol, and 10 HEPES, adjusted to pH 7.3 with Tris. To eliminate outwardly rectifying Cl- current, a Cl- channel blocker, 5-nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB, 50 µM) or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 300 µM) was added extracellularly. The osmolality of all solutions, checked with a freezing-point depression osmometer (Osmometer Automatic, Knauer, Berlin, Germany), ranged from 287 to 302 mosmol/kgH2O. Membrane currents were recorded with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA), filtered at 1 kHz, and digitized at a sampling frequency of 2–5 kHz using pCLAMP 8.0 software (Axon Instruments). The resistance of the patch electrode ranged from 4 to 5 M{Omega} when filled with electrode solution. Seal resistances were always >5 G{Omega}. Leak subtraction was not made. An Ag-AgCl reference electrode was connected to the bath solution through 1 M KCl-agar to minimize changes in liquid junctional potentials.



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Fig. 6. La3+ inhibits Na+- and Ca2+-permeable pathways in osteoclasts in the nonresorbing/motile state. Aa: current-voltage (I-V) relationships of the background currents in Na+-rich solution (Na+-rich), Ca2+-rich, Na+-free solution (Ca2+-rich), and Ca2+-rich solution with added 300 µM LaCl3 (Ca2+-rich + La3+). To eliminate inward K+ and outward Cl- currents, Cs+ solution in the pipette and 50 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) as a Cl- channel blocker were used throughout the experiments. The I-V relationships were determined by application of voltage ramps ranging from -100 to +80 mV applied over 2 s (holding potential 0 mV). The equilibrium potential of Cl- (ECl) was set to -26 mV. Ab: I-V relationship of the La3+-sensitive current component. This current was obtained by subtraction of the current in presence of LaCl3 from that in Ca2+-rich solution without LaCl3. B: effects of LaCl3 on the current recorded in the Ca2+-rich solution elicited by a voltage ramp from -100 to 0 mV. Conductance values are expressed normalized to cell capacitance. Membrane capacitances of osteoclasts in our experiments ranged from 84 to 106 pF. Data were obtained from 3 cells. **P < 0.01.

 

[Ca2+]i and membrane potential measurements. For simultaneous recording of [Ca2+]i and membrane potential, both fura 2 and a voltage-sensitive dye were loaded in the same osteoclasts. After cells were loaded with fura 2, osteoclasts were incubated with 100 nM bis-(1,3-dibutyl barbituric acid)trimethine oxonol [DiBAC4(3)] in PSS for 10 min at 25°C. The coverslips were then washed with PSS for 5 min, and experiments were started. Fluorescence was measured in single cells excited with wavelengths of 340 and 493 nm for fura 2 and DiBAC4(3), respectively. Emitted fluorescence was deflected to pass through a dicroic mirror (510 nm), and the transmitted light was filtered at 520–560 nm detected using photomultiplier (SPEX Industries). The fluorescence intensities at 340 and 493 nm excitation were displayed. At the end of the experiments, cells were stained by TRAP to conclusively identify them as osteoclasts.

Actin microfilament and TRAP assay. Actin microfilaments were detected by binding of rhodamine-conjugated phalloidin (R-PHD) to F-actin. Osteoclasts, cultured in {alpha}-MEM on coverslips (5 x 5 mm) for 6 h after isolation, were incubated for 15 min in PSS or test solution. Osteoclasts on coverslips were washed with PSS and fixed with 10% formaldehyde at room temperature for 20 min. After being washed in PSS for 3 min, cells were permeabilized with 0.1% Triton X-100 for 10 min and rinsed with PSS.

To positively identify osteoclasts, cytochemical staining for TRAP was performed. TRAP-stained osteoclasts were then incubated for another 20 min at 37°C with 5 U/ml R-PHD to stain for F-actin. Coverslips were then washed and inspected under a fluorescence microscope (Nikon, Tokyo, Japan). To obtain rhodamine fluorescence, the emitted fluorescence was deflected to pass through a dicroic mirror (550 nm), and the transmitted light was filtered at 580 nm. The number of osteoclasts having actin rings was expressed relative to the total number of TRAP-positive multinucleated cells (MNCs; cells with three or more nuclei).

Bone resorption assay. Resorption activity was assessed by counting the pit formations on dentine slices. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2-95% air. To examine the effect of high K+ on resorption activity, 50 mM KCl was added to the culture medium. After 48 h, the osteoclasts on the dentine slices were fixed with 10% formaldehyde and stained with TRAP, and the number of MNCs was counted. The cells were then peeled from the dentine slices by ultrasonication in 0.25 M ammonium hydroxide, and resorption pits were stained with Mayer's hematoxylin solution. Finally, the number of pits was counted under a phase-contrast microscope. The ratio of resorption pits per total number of MNCs was used as an indicator of osteoclast resorption activity.

Measurement of osteoclast motility. Coverslips with adherent osteoclasts were placed in a temperature-controlled chamber (volume of 1 ml) and superfused continuously at 1 ml/min with {alpha}-MEM with 5 mM HEPES, adjusted to pH 7.3 with Tris. To assess cellular motility, cell surface areas were recorded on video using a charge-coupled device camera (CS8310; Tokyo Electronic Industry, Tokyo, Japan) mounted to a phase-contrast microscope (TMD-300; Nikon, Tokyo, Japan). Images were stored in a time-lapse videorecorder (KV-7168, Toshiba, Japan) and digitized to a computer (Macintosh Computer 8500/120; Apple Japan) at 2-min intervals over a 60-min period. NIH Image software was used for image acquisition, processing, and measurement of cell surface area. Cell motility was assessed as relative changes in membrane surface area. Membrane surface area A(t), at time t (in minutes), was quantitated by tracing the outlines of every osteoclast in the viewing field. Surface area was normalized to the value before high-K+ stimulation (100%; Fig. 3B; t = -10 min). A second measurement was taken 2 min after intervention, A(t + 2). The nonoverlapping area A(t + 2) - A(t) between these two successive surface areas was also measured, and the relative change in areas [A(t + 2) - A(t)]/A(t) was used as an index of cell motility. The effect of membrane depolarization on cell motility was tested by exposure to high-K+ medium (addition of 50 mM KCl in {alpha}-MEM) and assessed by comparing the motility before, during, and after application of high-K+ solution. In some experiments, to prevent the hyperosmolality caused by addition of 50 mM KCl, {alpha}-MEM with high K+ was prepared by replacing 50 mM NaCl in {alpha}-MEM with an equimolar amount of KCl using a made-to-order salt-deficient {alpha}-MEM (Nikken Biomedical Laboratories, Kyoto, Japan).



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Fig. 3. Effects of high-K+ solution on osteoclast motility. A: video images before (a) and during (b) application of 50 mM K+ solution. B and C: time courses of changes in cellular area (B) and motility (C) before and during application and after removal of high-K+ solution. Each symbol indicates means ± SE obtained from 4 preparations. In B and C, a and b mark time of recorded video images of Aa and Ab, respectively.

 

Chemicals. Fura 2-AM was obtained from Dojindo (Kumamoto, Japan). Ionomycin was purchased from Calbiochem-Novabiochem International (San Diego, CA). DiBAC4(3) was obtained from Molecular Probes (Eugene, OR). All other chemicals were obtained from Sigma Chemical.

Drugs were dissolved in dimethyl sulfoxide (DMSO) and later diluted to final concentration in the PSS. Final DMSO concentration was 0.1% or less.

Statistics. Data are expressed as means ± SE. The level of statistical significance was estimated by ANOVA, and P values <0.05 were considered as statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Morphological properties of resorbing and nonresorbing osteoclasts and the effect of high-K+ solution on bone resorption activity. There are two types of MNCs and TRAP-positive cells adherent to glass coverslips after 6 h of cultivation (Fig. 1Aa). These two types of cells are distinguished by their shape and ability to form actin rings (Fig. 1Ab). One type has a large, flat, and round shape and displays F-actin distinctly organized in so-called actin rings (Fig. 1Ab, white arrowhead). The other type has a more bulgy shape with serrated edges, and actin rings are not observed (Fig. 1Ab).



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Fig. 1. A: morphological characteristics of resorbing and nonresorbing/motile state rat osteoclasts. a: tartrate-resistant acid phosphatase (TRAP) activity of osteoclasts on glass. b: the same cell as in Aa stained with rhodamine-conjugated phalloidin (R-PHD). Arrowheads indicate actin ring formation. B: effect of high K+ on bone resorption activity. a: effect of high K+ in the presence (2.5 mM) or absence (0.1 mM EGTA) of Ca2+ and ionomycin (5 µM) on actin ring formation. Ordinate expresses the percentage of TRAP-positive multinucleated cells (MNCs) displaying actin ring from total number of MNCs. b: relationship between number of actin rings and pits formed in TRAP-positive MNCs. MNCs were cultured for 48 h on dentine slices. In Ba and Bb, each column indicates means ± SE, and number of experiments for each condition is shown in parenthesis. **P < 0.01.

 

There was an average of 71.9 ± 1.7 (means ± SE) TRAP-positive MNCs per coverslip (73 coverslips examined). Without stimulation (i.e., control), 61.9 ± 2.5% (average 43 cells/coverslip) of these TRAP-positive MNCs possessed an actin ring (Fig. 1Ba). Two days of cell cultivation on dentin slices, instead of glass coverslips, decreased both the number of TRAP-positive MNCs and the proportion of TRAP-positive MNCs displaying actin rings: 41.7 ± 3.8 TRAP-positive MNCs and 13.6 ± 3.1 TRAP-positive MNCs possessed actin rings on average, and 22.4 ± 3.5 pits were counted (10 dentine slice preparations, Fig. 1Bb, left). Exposure to 100 mM K+ solution abolished the ring formation within a few minutes, and only 28.4 ± 3.4% of MNCs remained actin ring positive after 15 min (glass coverslips, P < 0.05; Fig. 1Ba, middle). Actin rings were reorganized after 30- to 60-min superfusion with PSS, and percent values were restored to initial levels.

When the cells were exposed to Ca2+-free high-K+ solution, 50.6 ± 3.4% of TRAP-positive MNCs possessed actin rings, a value similar to control and significantly higher than that observed in Ca2+-containing high-K+ solution (P < 0.05). Application of 5 µM ionomycin, a Ca2+ ionophore, completely inhibited actin ring formation (0.16 ± 0.51% of TRAP-positive MNCs; P < 0.01; Fig. 1Ba). When MNCs were cultured in {alpha}-MEM with 50 mM KCl, the number of rings and pits was reduced to 2.75 ± 1.2 and 5.38 ± 2.6 (n = 19), respectively (P < 0.01). Because this solution has increased osmolality, we tested the effect of 50 mM NaCl added and found that cultivation of MNCs within {alpha}-MEM plus 50 mM NaCl did not alter actin ring or pit formation. Thus the inhibition of resorbing activity in MNCs was due to increased extracellular K+ ([K+]o) and not caused by hyperosmolality.

For the remainder of this paper, we will refer to TRAP-positive MNCs as osteoclasts.

Relationships between basal [Ca2+]i and high K+-induced [Ca2+]i change. Measurement of basal [Ca2+]i of rat osteoclasts in PSS showed two populations of cells: one with basal [Ca2+]i around 50 nM and the other with basal [Ca2+]i around 110 nM (Fig. 2A). Comparison with the morphological features revealed that flat- and round-shaped osteoclasts had mostly low basal [Ca2+]i (43 cells out of 50 examined round-shaped cells), whereas bulgy cells with serrated edges had a high basal [Ca2+]i (31 cells out of 33 examined bulgy cells). When osteoclasts were exposed to high-K+ solution, both increases and decreases in [Ca2+]i were recorded, depending on the basal [Ca2+]i levels. As shown in Fig. 2B, all osteoclasts with basal [Ca2+]i <60 nM elevated [Ca2+]i during high-K+ stimulation (39 cells out of 50 examined round-shaped cells). On the other hand, most osteoclasts with basal [Ca2+]i >90 nM decrease it during high-K+ stimulation (31 cells out of 33 examined bulgy cells).



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Fig. 2. Effects of high-K+ solution on intracellular calcium concentration ([Ca2+]i). A: distribution of basal [Ca2+]i in osteoclasts. B: relationship between basal [Ca2+]i level and changes in [Ca2+]i. C: effects of 100 mM K+ on [Ca2+]i in 2 types of osteoclasts having low and high [Ca2+]i under basal conditions. Video images were recorded before (a) and during (b) application of 100 mM K+ solution. Each color corresponds to [Ca2+]i as indicated at right. D: time course of [Ca2+]i changes induced by 100 mM K+ solution in the 2 types of cells. In D, a and b mark time of recorded video images of Ca and Cb, respectively.

 

Figure 2C illustrates typical results for [Ca2+]i changes induced by high-K+ solution in the two types of osteoclasts. Video images clearly showed that round, resorbing state osteoclasts (cell 1) increased, whereas serrated-edged, nonresorbing state osteoclasts (cell 2) decreased [Ca2+]i during superfusion of 100 mM K+ solution (Fig. 2, Ca and Cb). The time course of [Ca2+]i change in the two types of osteoclasts is shown in Fig. 2D. Changes in [Ca2+]i were reversible.

Effect of high-K+-induced [Ca2+]i reduction on osteoclast function. As shown in Fig. 1, round osteoclasts with actin rings were associated with pit formation and elevated [Ca2+]i, suggesting that [Ca2+]i elevation might shift the osteoclasts from the resorbing state to the nonresorbing/motile state. Thus, to investigate the role of [Ca2+]i in bulgy osteoclasts with serrated edges, we assessed motile activity, estimated by measuring the two-dimensional change in cell surface area. Replacement of culture medium ({alpha}-MEM with 20 mM NaHCO3, pH 7.3) to {alpha}-MEM with 5 mM HEPES (pH 7.3) had no effect on cell area and motility of rat osteoclasts (control; Fig. 3, B and C, -10~0 min), and pseudopodia activity continued within 2.5 h after exposure to the medium. Exposure to high-K+ medium (50 mM K+ {alpha}-MEM) caused retraction of the pseudopodia (Fig. 3, Aa and Ab), reduced cellular area by 10% (Fig. 3B), and reduced osteoclast motility (Fig. 3C) with maximal effect apparent after 5–10 min. This response was reversible and returned to control values.

Because the osmolality of high-K+ medium (addition of 50 mM K+ to {alpha}-MEM) is significantly increased, we also tested the effect of {alpha}-MEM with 50 mM KCl replaced for NaCl on osteoclastic cell area and motility. The inhibitory effects of {alpha}-MEM with high K+ replaced for NaCl were similar to that of the {alpha}-MEM with 50 mM KCl added (area changed to 93% of control; motility changed from 7 to 0.3%; n = 2). We also tested whether addition of 50 mM NaCl to {alpha}-MEM would affect cell area or motility. We found that this high-Na+ medium caused a slight cell shrinking (area changed to 95% of control) but had no effect on cell motility (n = 2). These results indicate that the inhibition of cell motility in osteoclasts was due to increased [K+]o and not caused by hyperosmolality.

The relationship between membrane depolarization and [Ca2+]i reduction in nonresorbing osteoclasts. To determine whether the high-K+-induced [Ca2+]i reduction was caused by membrane depolarization, we simultaneously recorded membrane potential and [Ca2+]i with a dual-probe method (Fig. 4A; see MATERIALS AND METHODS). Application of high-K+ solution to a nonresorbing/motile state osteoclast produced a rapid increase and decrease of fluorescence intensities at 493 (upper trace) and 340 (lower trace) nm, respectively (4 of 5 examined nonresorbing/motile cells). Although absolute values of the membrane potential and [Ca2+]i cannot be obtained simultaneously, these results indicate that there is a clear relationship between membrane depolarization and reduction of [Ca2+]i by high-K+ solution.



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Fig. 4. A: typical trace (4 out of 5 examined nonresorbing/motile cells) of membrane potential (upper trace) and [Ca2+]i (lower trace) in response to 100 mM K+ solution in an osteoclast in the nonresorbing/motile state. Membrane potential and [Ca2+]i were recorded simultaneously. B: effects of changing membrane potential on [Ca2+]i of an osteoclast in the nonresorbing/motile state. The cell was voltage clamped to 0 mV, and voltage steps to -50 and -70 mV were applied at the times indicated in C (upper trace). C: [Ca2+]i changes induced by membrane hyperpolarization and depolarization (3 out of 3 examined nonresorbing/motile cells). In C, ac show the times of recorded video images in Ba, Bb, and Bc, respectively.

 

To obtain direct evidence that membrane depolarization indeed caused this [Ca2+]i reduction, changes in [Ca2+]i were recorded under voltage-clamp conditions (patch-clamp technique). Membrane potential was kept at 0 mV after the whole cell patch-clamp configuration was established. Next, membrane hyperpolarization to -50 mV increased [Ca2+]i (Fig. 4, Ba and C), and subsequent return of the membrane to 0 mV reduced [Ca2+]i back to the original value (Fig. 4, Bb and C). Hyperpolarization of the membrane to -70 mV produced even greater increases in [Ca2+]i (Fig. 4Bc and C).

Dependence of high-K+-induced [Ca2+]i reduction on [Ca2+]o. Figure 5A shows that [Ca2+]i in nonresorbing osteoclasts decreased with elevation of [K+]o in a concentration-dependent manner. Application of 0.5 mM BaCl2, a K+ channel inhibitor, also reduced [Ca2+]i (data not shown).



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Fig. 5. Effects of extracellular Ca2+, La3+, thapsigargin, nicardipine, vanadate, and carbonyl cyanide-m-chlorophenylhydrazone (CCCP) on changes in [Ca2+]i induced by 100 mM K+ solution. A: extracellular K+ concentration-dependent reduction of [Ca2+]i in osteoclasts in the nonresorbing/motile state (n = 3). B and C: effects of Ca2+-free solution (n = 20) and 300 µM La3+ (n = 12) on changes in [Ca2+]i induced by 100 mM K+ solution. D: effects of 3 µM thapsigargin, 1 µM nicardipine, 500 nM vanadate, and 100 nM CCCP on the changes in [Ca2+]i induced by 100 mM K+ solution. Control indicates the high-K+-induced [Ca2+]i decrease (-50 nM in average) before pretreatment with inhibitors and was normalized to 100% for each cell (n = 5; dotted line in D). Cells were then exposed to drugs for 10 min before application of high-K+ solution. The change in high-K+-induced responses in the presence of each drug was expressed relative to control (percent of control). Each column indicates means ± SE, and the number of experiments under each condition is shown in parentheses.

 

Removal of [Ca2+]o or application of 300 µM La3+ reduced [Ca2+]i, and application of high-K+ solution did not further reduce [Ca2+]i, suggesting that [Ca2+]o is essential for maintaining the high [Ca2+]i levels in osteoclasts during the nonresorbing/motile state (Fig. 5, B and C). However, application of Ca2+ channel blockers nicardipine or {omega}-conotoxin GVIA had no effect on basal [Ca2+]i or the high-K+-induced response (Fig. 6D). Next, we tested whether Ca2+ uptake from the cytoplasm into intracellular Ca2+ stores might be involved in the high-K+-induced [Ca2+]i decrease. Cells were pretreated with each drug for 10 min before application of high-K+ solution. Osteoclasts were preincubated with thapsigargin, an inhibitor of intracellular Ca2+ store refilling, and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation in mitochondria. Preincubation of thapsigargin and CCCP produced a small increase in basal [Ca2+]i (about 20 nM on average). Neither thapsigargin (3 µM, applied for 10 min) nor 500 nM CCCP (applied for 10 min) affected the high-K+-induced [Ca2+]i decrease. Vanadate, an inhibitor of the plasma membrane Ca2+ pump, also slightly increased basal [Ca2+]i but had no effect on the [Ca2+]i decrease.

These results clearly indicate that reduction of [Ca2+]i induced by high-K+ solution is caused by inhibition of Ca2+ entry via La3+-sensitive and DHP-insensitive Ca2+ influx and not due to Ca2+ uptake into Ca2+ stores or extrusion into the extracellular space.

[Ca2+]i in resorbing-state osteoclasts was increased with elevation of [K+]o in a concentration-dependent manner. Although the removal of [Ca2+]o in resorbing-state osteoclasts abolished high-K+-induced [Ca2+]i increases, neither L-type or N-type Ca2+ channel blockers nor La3+ modified the high-K+-induced [Ca2+]i response (data not shown). Preincubation with thapsigargin and CCCP also had no effect on the high-K+-induced [Ca2+]i responses.

Possible pathway of Ca2+ entry in osteoclasts during the nonresorbing/motile state. Electrophysiological studies have shown that several ion channels are present in osteoclasts; that is, an inwardly rectifying K+, outwardly rectifying K+, and outwardly rectifying Cl- channels (1, 26, 28, 32). In addition, recent evidence showed the presence of nonselective cation conductances in rat and rabbit osteoclasts (9, 13, 23). Among these ionic conductances, the nonselective cation channels have been considered to be pathways for Ca2+ flux, because electrophysiological experiments did not show evidence for voltage-dependent Ca2+ selective channels (25, 26, 28).

In the present study, the predominant membrane current in osteoclasts in the nonresorbing/motile state was the inwardly rectifying K+ current, which was abolished by Cs+ in the electrode solution or omission of K+ in PSS (data not shown). Eight of fifteen cells total exhibited an outwardly rectifying Cl- current, which was eliminated by addition of NPPB (50 µM) or DIDS (100 µM) to the bath (data not shown). Voltage-dependent ionic currents such as outwardly rectifying K+ and voltage-dependent Ca2+ currents were not detected. When both the inwardly rectifying K+ current and outwardly rectifying Cl- currents were eliminated, a remaining background current with slight outward rectification was seen (Fig. 6Aa, labeled Na+-rich). Following application of Ca2+-rich Na+-free solution, inward current was reduced with no apparent effect on the outward current (labeled Ca2+-rich). Application of LaCl3 (300 µM) produced further inhibition of inward current (Fig. 6Aa; labeled Ca2+-rich + La3+), whereas nicardipine (10 µM) did not inhibit this current (data not shown). The current-voltage (I-V) relationship of the La3+-sensitive current exhibited outward rectification with a reversal potential close to 0 mV (0.8 ± 2.1 mV, n = 3) (Fig. 6Ab). Slope conductance of the inward current (measured between -100 and 0 mV) in Ca2+-rich solution (4.04 ± 0.16 pS/pF) was significantly inhibited by addition of La3+ (2.42 ± 0.13 pS/pF, n = 3, P < 0.01) (Fig. 6B), demonstrating that La3+-sensitive Ca2+ entry pathways indeed exist in nonresorbing/motile rat osteoclasts.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
[Ca2+]i mobilization by high-K+-induced depolarization in osteoclasts during resorbing and nonresorbing states. Conflicting results regarding the effects of membrane depolarization and exposure to high K+ on [Ca2+]i and Ca2+ entry in osteoclasts have been published (2, 4, 8, 21, 27). It was reported that exposure to high K+ increases [Ca2+]i in osteoclast-like cells [avian (21), mouse (4), and rat (27)]. In contrast, Miyauchi et al. (21) reported that high-K+ solution reduced [Ca2+]i in avian osteoclasts after 5 h cultivation if the cells were incubated in the presence of bone particles. Valinomycin, a K+-channel ionophore, increased [Ca2+]i in rat osteoclasts (27). These observations indicate that membrane potential changes do not uniformly alter [Ca2+]i in osteoclasts.

It is well known that osteoclasts differ in their morphological and functional properties between the resorbing and nonresorbing/motile states of the bone resorption cycle (15, 17, 18). Resorbing osteoclasts exhibit actin rings without pseudopodia, whereas nonresorbing/motile osteoclasts have pseudopodia but not actin rings. In our present experiments, 61% of MNCs possessed actin rings when cultured on glass coverslip. As shown in Fig. 2, resorbing state osteoclasts (possessing actin rings) showed low [Ca2+]i, and nonresorbing/motile state osteoclasts showed high [Ca2+]i at rest. High-K+ solution increased [Ca2+]i in resorbing osteoclasts but reduced [Ca2+]i in nonresorbing osteoclasts. These results demonstrate the state dependence of basal [Ca2+]i and Ca2+ response.

Possible mechanisms of high-K+-induced [Ca2+]i mobilization in nonresorbing osteoclasts. In nonresorbing osteoclasts, high K+ produced membrane depolarization and at the same time decreased [Ca2+]i. Because this decrease of [Ca2+]i could be observed simultaneously with membrane depolarization and mimicked by direct membrane depolarization via whole cell patch-clamp method (Fig. 4), a hyperpolarization-dependent Ca2+ entry pathway is likely to be present in osteoclasts during the nonresorbing/motile state.

Miyauchi et al. (20) and Yamakawa et al. (33) reported that Arg-Gly-Asp reduced [Ca2+]i in avian osteoclast precursors. This reduction was suppressed by vanadate, a plasma Ca2+ pump inhibitor, but not by modulators of intracellular Ca2+ stores. However, in our present study, neither vanadate, thapsigargin, nor CCCP modified the high-K+-induced [Ca2+]i reduction, suggesting either species differences or stimulation-dependent differences in Ca2+ mobilization. In rat osteoclasts, either removal of extracellular Ca2+ or application of La3+, a nonspecific Ca2+ influx blocker, inhibited high-K+-induced reduction in [Ca2+]i. These results indicate that in nonresorbing osteoclasts, membrane depolarization switches off a La3+-sensitive Ca2+ influx pathway rather than activating Ca2+ transport to the extracellular space or into intracellular Ca2+ stores.

As shown in Fig. 6, a voltage-dependent Ca2+ channel could not be detected, but a La3+-sensitive current component was identified in the background current of our cells. In a preliminary report, mRNAs for TRP-like channels (TRP3 and 6) have been detected in mouse osteoclasts (3). In TRP channel-expressing mammalian cells, TRP conductances in general are nonselective, permeable to Ca2+ (or Mg2+) > monovalent cations, show linear (or slightly outwardly rectified) I-V relationship under physiological conditions, and are inhibited by La3+ (19). If this background current were mostly carried through TRP channels, then the reversal potential should be more positive because Ca2+ was the main charge-carrying cation under the conditions of our experiments. Therefore, the background current seems to be due to other divalent cation conductances. Whereas TRP channels may be latent in the background current, other channels such as La3+-sensitive-Ca2+-permeable conductance, or possibly other divalent cation conductances including TRP channel variant phenotypes, are more likely to play a role in the hyperpolarization-induced [Ca2+]i entry mechanism. Additional experiments, ideally using single-channel analysis, will be required to clarify these questions.

Bizzarri et al. (4) and Miyauchi et al. (21) demonstrated that high-K+ solution increased [Ca2+]i by increasing Ca2+ influx via a DHP-sensitive pathway. However, our previous reports showed no evidence for the presence of DHP-sensitive Ca2+ channels in rat osteoclasts (25, 26, 28), and the resting membrane potential of resorbing state osteoclasts was high (around -25 mV; Okamoto F, Kajiya H, and Okabe K, unpublished observation). Furthermore, Bizzarri et al. (4) showed that valinomycin, a K+ ionophore, reduced [Ca2+]i when the membrane of osteoclasts was depolarized by high K+; under these conditions, valinomycin would not be expected to hyperpolarize the membrane because the K+ equilibrium potential was kept high by high-K+ external solution. It is uncertain whether depolarization induced by high-K+ solution is sufficient to elevate [Ca2+]i in resorbing state osteoclasts. In these cells, both membrane surfaces are polarized by accumulation of various specific proteins, such as vacuolar H+-ATPase and ClC7, and create a unique extracellular environment (very high [Ca2+]o and very low pH) underneath the osteoclastic cell membrane. Thus the intracellular milieu does not simply depend on the extracellular milieu.

We cannot speculate about possible mechanisms for [Ca2+]i elevation in resorbing-state osteoclasts; however, this process does not appear to utilize the same Ca2+ entry pathway employed by nonresorbing osteoclasts, because elevation of [Ca2+]i by high-K+ solution in resorbing osteoclasts is abolished by superfusion of Ca2+-free solution, but not La3+ (H. Kajiya, unpublished observations). Further studies are required to clarify these issues.

Effects of membrane depolarization on osteoclastic resorbing activity and motility. Many investigators have reported that osteoclasts exhibit actin rings during the bone-resorbing state (14, 15, 30, 35). Actin ring formation in osteoclasts is regulated by signaling pathways mediated by PKA (16, 29), phosphatidylinositol 3-kinase (PI 3-kinase) (24), and intracellular Ca2+ (14, 21, 31). In our experiments, high-K+ stimulation inhibited actin ring and pit formation on dentine slices. This disruption of ring formation induced by high K+ was prevented by using a Ca2+-free extracellular solution. Furthermore, ionomycin, a Ca2+ ionophore, also completely inhibited the ring formation in a [Ca2+]o-dependent manner. Therefore, we conclude that high-K+ solution depolarizes the membrane and inhibits actin ring formation due to elevation of [Ca2+]i through activation of Ca2+ entry.

On the other hand, exposure to high K+ in nonresorbing/motile osteoclasts reduced motility. It is likely that high-K+-induced reduction of [Ca2+]i contributes to suppressing motility. Changes in [Ca2+]i induced by hormones and cytokines regulate cytoskeletal structure, chemotactic migration, and motility of osteoclasts: Grano et al. (10) reported that hepatocyte growth factor increases [Ca2+]i and stimulates chemotactic migration in osteoclast-like cells obtained from human giant cell tumors. Colucci et al. (7) reported that soluble laminin-2 (merosin), which is expressed in basement membranes and binds integrin receptors, increases [Ca2+]i and stimulates the chemotactic migration of human osteoclast-like cells. Miyauchi et al. (20) reported that reduction of cytosolic Ca2+ favors actin ring formation. In nonresorbing/motile osteoclasts, higher basal [Ca2+]i keeps motile activity high, and inhibition of the La3+-sensitive Ca2+ influx by membrane depolarization promotes anchoring of the cells onto the bone surface and formation of actin rings.

In conclusion, membrane depolarization in nonresorbing/motile state osteoclasts decreases [Ca2+]i via inhibition of La3+-sensitive Ca2+ influx. Nonselective cation channels are good candidates for this Ca2+ entry system.


    DISCLOSURES
 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education (Frontier Research Grant).


    ACKNOWLEDGMENTS
 
We thank Dr. A. Carl for valuable comments and English editing. We also thank Dr. K. Kitamura for valuable comments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. Kajiya, Dept. of Physiological Science and Molecular Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka Japan, 814-0193 (E-mail address: kajiya{at}college.fdcnet.ac.jp).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Arkett SA, Dixon SJ, and Sims SM. Substrate influences rat osteoclast morphology and expression of potassium conductance. J Physiol 458: 633–653, 1992.[Abstract]

2. Bax CM, Shankar VS, Moonga BS, Huang CL, and Zaidi M. Is the osteoclast calcium "receptor" a receptor-operated calcium channel? Biochem Biophys Res Commun 183: 619–625, 1992.[ISI][Medline]

3. Bennett BD, Zhu MX, and Hruska KA. Trp channels in osteoclast calcium sensing (Abstract). J Bone Miner Res 15: S517, 2000.

4. Bizzarri C, Shioi A, Teitelbaum SL, Ohara J, Harwalkar VA, Erdmann JM, Lacey DL, and Civitelli R. Interleukin-4 inhibits bone resorption and acutely increases cytosolic Ca2+ in murine osteoclasts. J Biol Chem 269: 13817–13824, 1994.[Abstract/Free Full Text]

5. Blair HC, Kahn AJ, Crouch EC, Jeffrey JJ, and Teitelbaum SL. Isolated osteoclasts resorb the organic and inorganic components of bone. J Cell Biol 102: 1164–1172, 1986.[Abstract]

6. Chambers TJ, Fuller K, and Darby JA. Hormonal regulation of acid phosphatase release by osteoclasts disaggregated from neonatal rat bone. J Cell Physiol 132: 92–96, 1987.

7. Colucci S, Giannelli G, Grano M, Faccio R, Quaranta V, and Zallone-Zambonin A. Human osteoclast-like cells selectively recognize laminin isoforms, an event that induces migration and activates Ca2+ mediated signals. J Cell Sci 109: 1527–1535, 1996.[Abstract/Free Full Text]

8. Datta HK, MacIntyre I, and Zaidi M. Intracellular calcium in the control of osteoclast function. I. Voltage-insensitive and lack of effects of nifidipine, BAY K 8644 and diltiazem. Biochem Biophys Res Commun 167: 183–188, 1990.[ISI][Medline]

9. Espinosa L, Itstein C, Cheynel H, Delmas PD, and Chenu C. Active NMDA glutamate receptors are expressed by mammalian osteoclasts. J Physiol 518: 291–303, 1999.[Abstract/Free Full Text]

10. Grano M, Galimi F, Zambonin G, Colucci S, Cottone E, and Zallone-Zambonin A. Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro. Proc Natl Acad Sci USA 93: 7644–7648, 1996.[Abstract/Free Full Text]

11. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of calcium indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract]

12. Harteneck C, Plant TD, and Schultz G. From worm to man: three subfamilies of TRP channels. Trends Neurosci 23: 159–166, 2000.[ISI][Medline]

13. Kai Y, Ikemoto Y, Abe K, and Oka M. Two type K+ currents underlying inward rectification of rat osteoclast membrane: a single-channel analysis. Jpn J Physiol 46: 231–241, 1996.[ISI][Medline]

14. Kajiya H, Okabe K, Okamoto F, Tsuzuki T, and Soeda H. Protein tyrosine kinase inhibitors increase cytosolic calcium and inhibit actin organization as resorbing activity in rat osteoclasts. J Cell Physiol 183: 83–90, 2000.[ISI][Medline]

15. Kanehisa J, Yamanaka T, Doi S, Turksen K, Heersche JNM, Aubin JE, and Takeuchi H. A band of F-actin containing podosomes is involved in bone resorption by osteoclasts. Bone 11: 287–293, 1990.[ISI][Medline]

16. Lakkakorpi PT and Väänänen HK. Calcitonin, prostaglandin E2 and dibutyryl cyclic adenosine 3',5'-monophospahte disperse the specific microfilament structure in resorbing osteoclasts. J Histochem Cytochem 38: 1487–1493, 1990.[Abstract]

17. Lakkakorpi PT and Väänänen HK. Kinetics of the osteoclasts cytoskeleton during the resorption cycle in vitro. J Bone Miner Res 6: 817–826, 1991.[ISI][Medline]

18. Lakkakorpi PT, Lehenkari PP, Rautiala TJ, and Väänänen HK. Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in presence of high extracellular calcium. J Cell Physiol 168: 668–677, 1996.[ISI][Medline]

19. Minke B and Cook B. TRP channels proteins and signal transduction. Physiol Rev 82: 429–472, 2002.[Abstract/Free Full Text]

20. Miyauchi A, Hruska KA, Greenfield EM, Duncan R, Alvares J, Barattolo R, Colucci S, Zambonin-Zallone A, Teitelbaum SL, and Teti A. Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly bone resorption. J Cell Biol 111: 2543–2552, 1990.[Abstract]

21. Miyauchi A, Alvarez J, Greenfield EM, Teti A, Grano M, Colucci S, Zambonin-Zallone A, Ross FP, Teitelbaum SL, Cheresh D, and Hruska KA. Recognition of osteopontin and related peptides by an {alpha}v{beta}3 integrin stimulated immediate cell signals in osteoclasts. J Biol Chem 266: 20369–20374, 1991.[Abstract/Free Full Text]

22. Moonga BS, Alam AS, Bevis PJ, Avaldi F, Soncini R, Huang CL, and Zaidi M. Regulation of cytosolic free calcium in isolated rat osteoclasts by calcitonin. J Endocrinol 132: 241–249, 1992.[Abstract]

23. Naemsch LN, Dixon SJ, and Sims SM. Activity-dependent development of P2X7 current and Ca2+ entry in rabbit osteoclasts. J Biol Chem 276: 39107–39114, 2001.[Abstract/Free Full Text]

24. Nakamura I, Takahashi N, Sasaki T, Tanaka S, Udagawa N, Murakami H, Kimura K, Kabuyama Y, Kurokawa T, Suda T, and Fukui Y. Wortmannin, a specific inhibitor of phosphatidylinositol-3 kinase, blocks osteoclastic bone resorption. FEBS Lett 361: 79–84, 1995.[ISI][Medline]

25. Okabe K, Okamoto F, Kajiya H, Takada K, and Soeda H. Estrogen directly acts on osteoclasts via inhibition of inward rectifier K+ channels. Arch Pharm (Weinheim) 361: 610–620, 2000.

26. Ravesloot JH, Ypey DL, Vrijheid-Lammers T, and Nijweide PJ. Voltage-activated K+ conductances in freshly isolated embryonic chicken osteoclasts. Proc Natl Acad Sci USA 86: 6821–6825, 1989.[Abstract]

27. Shankar VS, Huang CL, Adebanjo O, Simon B, Alam AS, Moonga BS, Pazianas M, Scotto RH, and Zaidi M. Effects of membrane potential on surface Ca2+ receptor activation in rat osteoclasts. J Cell Physiol 162: 1–8, 1995.[ISI][Medline]

28. Sims SM, Kelly ME, and Dixon SJ. K+ and Cl- currents in freshly isolated rat osteoclasts. Pflügers Arch 419: 358–370, 1991.[ISI][Medline]

29. Suzuki H, Nakamura I, Takahashi N, Ikuhara T, Matsuzaki K, Isogai Y, Hori M, and Suda T. Calcitonin-induced changes in the cytoskeleton are mediated by a signal pathway associated with protein kinase A in osteoclast. Endocrinology 137: 4685–4690, 1996.[Abstract]

30. Tanaka S, Takahashi N, Udagawa N, Murakami H, Nakamura I, Kurokawa T, and Suda T. Possible involvement of focal adhesion kinase, p125FAK, in osteoclastic bone resorption. J Cell Biochem 58: 424–435, 1995.[ISI][Medline]

31. Teti A, Colucci S, Grano M, Argentino L, and Zambonin-Zallone A. Protein kinase C affects microfilaments, bone resorption, and [Ca2+]o sensing in cultured osteoclasts. Am J Physiol Cell Physiol 263: C130–C139, 1992.[Abstract/Free Full Text]

32. Yamashita N, Ishii T, Ogata E, and Matsumoto T. Inhibition of inwardly rectifying K+ current by external Ca2+ ions in freshly isolated osteoclasts. J Physiol 480: 217–224, 1994.[Abstract]

33. Yamakawa K, Duncan R, and Hruska KA. An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism. Am J Physiol Renal Fluid Electrolyte Physiol 266: F651–F657, 1994.[Abstract/Free Full Text]

34. Zaidi M, Alam AS, Huang CL, Pazianas M, Bax CM, Bax BE, Moonga BS, Bevis PJ, and Shankar VS. Extracellular Ca2+ sensing by osteoclast. Cell Calcium 14: 271–277, 1993.[ISI][Medline]

35. Zhang D, Udagawa N, Nakamura I, Murakami H, Saito S, Yamasaki K, Shibasaki Y, Morii N, Narumiya S, Takahashi N, and Suda T. The small GTP-binding protein, rho p21, is involved in bone resorption by regulating cytoskeletal organization in osteoclasts. J Cell Sci 108: 2285–2292, 1995.[Abstract/Free Full Text]