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
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ABSTRACT |
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membrane depolarization; resorbing and motile activities; bone resorbing cycle
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).
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MATERIALS AND METHODS |
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[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 3334°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
25 kHz using pCLAMP 8.0 software (Axon Instruments). The resistance of
the patch electrode ranged from 4 to 5 M when filled with electrode
solution. Seal resistances were always >5 G
. 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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[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 520560 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 -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 -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
-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,
-MEM with high
K+ was prepared by replacing 50 mM NaCl in
-MEM with an
equimolar amount of KCl using a made-to-order salt-deficient
-MEM
(Nikken Biomedical Laboratories, Kyoto, Japan).
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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.
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RESULTS |
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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 -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
-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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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 (-MEM with 20 mM NaHCO3, pH 7.3) to
-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+
-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 510 min.
This response was reversible and returned to control values.
Because the osmolality of high-K+ medium (addition of 50 mM
K+ to -MEM) is significantly increased, we also tested the
effect of
-MEM with 50 mM KCl replaced for NaCl on osteoclastic cell
area and motility. The inhibitory effects of
-MEM with high
K+ replaced for NaCl were similar to that of the
-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
-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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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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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 -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.
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DISCUSSION |
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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.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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