Prostaglandin E2 activates outwardly rectifying Cl channels via a cAMP-dependent pathway and reduces cell motility in rat osteoclasts

Fujio Okamoto, Hiroshi Kajiya, Hidefumi Fukushima, Eijiro Jimi, and Koji Okabe

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

Submitted 4 December 2003 ; accepted in final form 17 March 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined changes in electrical and morphological properties of rat osteoclasts in response to prostaglandin (PG)E2. PGE2 (>10 nM) stimulated an outwardly rectifying Cl current in a concentration-dependent manner and caused a long-lasting depolarization of cell membrane. This PGE2-induced Cl current was reversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and tamoxifen. The anion permeability sequence of this current was I > Br {approx} Cl > gluconate. When outwardly rectifying Cl current was induced by hyposmotic extracellular solution, no further stimulatory effect of PGE2 was seen. Forskolin and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) mimicked the effect of PGE2. The PGE2-induced Cl current was inhibited by pretreatment with guanosine 5'-O-2-(thiodiphosphate) (GDP{beta}S), Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89), and protein kinase A inhibitors. Even in the absence of nonosteoclastic cells, PGE2 (1 µM) reduced cell surface area and suppressed motility of osteoclasts, and these effects were abolished by Rp-cAMPS or H-89. PGE2 is known to exert its effects through four subtypes of PGE receptors (EP1–EP4). EP2 and EP4 agonists (ONO-AE1-259 and ONO-AE1-329, respectively), but not EP1 and EP3 agonists (ONO-DI-004 and ONO-AE-248, respectively), mimicked the electrical and morphological actions of PGE2 on osteoclasts. Our results show that PGE2 stimulates rat osteoclast Cl current by activation of a cAMP-dependent pathway through EP2 and, to a lesser degree, EP4 receptors and reduces osteoclast motility. This effect is likely to reduce bone resorption.

prostanoid receptor agonists; electrophysiology; motile activity; bone resorption


PROSTAGLANDIN (PG)E2 is an important regulator of many physiological and pathological processes of bone metabolism and is generally believed to modulate both bone formation and bone resorption (30). Yoshida et al. (39) found that PGE2 acts on osteoblast precursors, inducing osteoblasts for bone formation, but also acts on mature osteoblasts, inducing osteoclasts for bone resorption.

PGE2 exerts its effects through specific receptors that consist of four subtypes designated EP1, EP2, EP3, and EP4 (25), and osteoblasts have been shown to express several of these PGE2 receptor subtypes (32, 33). Recent studies using EP receptor-knockout animals and selective EP receptor agonists demonstrated that EP4 and/or EP2 mediate both anabolic (37, 39) and catabolic (24, 33) actions of PGE2.

Evidence is mounting that PGE2 also has a direct effect on osteoclasts and negatively regulates their bone-resorbing activity (5, 6, 10, 14, 22). Chambers and coworkers (5, 6, 10) demonstrated that PGE2 causes cytoplasmic retraction and reduction of motility (i.e., movement of the peripheral cell membrane lamellipodia) in isolated osteoclasts, thereby inhibiting bone resorption. Kaji et al. (14) reported that PGE2 inhibits bone resorption in the absence of osteoblasts but stimulates bone resorption in the presence of osteoblasts. A study using highly purified rabbit osteoclasts showed that osteoclasts express EP4 and EP2 receptors and that both receptors participate in the inhibitory effect of PGE2 on osteoclast activity (22).

Several types of ion channels have been identified in osteoclasts, and their physiological roles in the activities of osteoclasts have been reported (19). Recently, we demonstrated (15) that membrane depolarization induced by exposing osteoclasts to high-K+ solution decreases intracellular Ca2+ concentration ([Ca2+]i) and causes cytoplasmic retraction and reduction of motility. This result demonstrates the importance of cell membrane potential for controlling [Ca2+]i and morphology in osteoclasts. Because membrane potential is regulated by the activities of several ion channel types expressed in osteoclasts, activation and deactivation of these channels is likely to determine osteoclast morphology. Several agents that modulate bone resorption have also been shown to modulate ion channels in osteoclasts (20, 27, 29). Therefore, to understand the role of PGE2 in osteoclast physiology and pathology, an understanding of its actions on ion channels is desirable. There is no previous report on PGE2-mediated effects on osteoclast ion channels to our knowledge.

In the present study, we applied the whole cell patch-clamp technique to isolated rat osteoclasts and examined the effects of PGE2 on ion channel activity and the role of these channels in PGE2-induced morphological changes. We found that PGE2 acts directly on rat osteoclasts and stimulates outwardly rectifying Cl channels via a cAMP-dependent mechanism through EP2 and EP4 receptors. This pathway contributes to reduction of cell area and loss of osteoclast motility.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Osteoclast preparation. All procedures involving animals were approved by the Council on Animal Care at the Fukuoka Dental College. Osteoclasts were obtained from the femur and tibia of neonatal Wistar rats (1–2 days old). Rats were anesthetized with diethyl ether and killed by decapitation. The femur and tibia were separated from adhering soft tissues. The bones were finely chopped into culture medium [{alpha}-minimum essential medium ({alpha}-MEM); Invitrogen, Grand Island, NY] containing 15% heat-inactivated fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin G, 0.15 mg/ml streptomycin sulfate, and 20 mM NaHCO3. The resulting suspension was gently agitated with a bore-tipped pipette to hasten dissociation of osteoclasts from the bone surface. This suspension was seeded on glass coverslips (5 mm x 5 mm) and incubated in the culture medium with 5% CO2-95% air at 37°C. After 30 min, the coverslips were gently washed with fresh culture medium to remove nonadherent cells and debris. Adherent cells were reincubated in fresh culture medium at 37°C. We obtained a preparation of mixed bone cells by this procedure. As we reported previously (15), two types of multinucleated cells adhering to glass coverslips can be distinguished by their shape and their ability to form a distinctive organization of F-actin, called "actin rings." One cell type had a flat, round shape and displayed an actin ring when stained with rhodamine-conjugated phalloidin after cell fixation with formaldehyde (15). The other cell type showed a more bulgy shape with serrated edges and did not display actin rings. For electrophysiological and morphological analysis, we selected only multinucleated cells having a bulgy shape with serrated edges (40- to 90-µm diameter). Identity of the selected cells was confirmed by tartrate-resistant acid phosphatase (TRAP) stain at the end of the electrophysiological recordings, as described previously (27). In all morphological and some electrophysiological experiments, we used a pure osteoclast preparation that was obtained by the following procedure. The preparation of mixed bone cells, obtained as described above, was placed in a 35-mm tissue culture dish filled with culture medium. The culture dish was mounted on an inverted microscope, and cells with one or two nuclei were removed manually from the coverslips by means of a cell scraper (made of a thin silicone rubber) with a micromanipulator (MMW-23; Narishige Scientific Instrument Laboratory, Tokyo, Japan). Multinucleated cells remaining on the coverslip were positively stained by TRAP. All experiments were done within 8 h after cell isolation.

Electrophysiological measurements. The coverslip with adherent cells was placed in a recording chamber (volume 1 ml) mounted on an inverted microscope (TE 300; Nikon, Tokyo, Japan) and continuously superfused (1 ml/min) with standard extracellular solution (HEPES-buffered solution) containing (in mM) 134 NaCl, 6 KCl, 10 glucose, 0.5 MgCl2, 1.25 CaCl2, and 10 HEPES, adjusted to pH 7.3 with Tris. In some preliminary experiments a bicarbonate-buffered solution was used, which was prepared by replacing HEPES in the standard extracellular solution with 14.5 mM NaHCO3. This solution was gassed with 5% CO2-95% O2 and had a pH of 7.3. The patch pipette solution for recording of membrane potential and whole cell currents (shown in Fig. 1A) contained (in mM) 140 KCl, 3 MgCl2, 2 ATP (disodium salt), 0.3 EGTA, and 10 HEPES, adjusted to pH 7.3 with Tris. For experiments designed to eliminate K+ currents, a K+-free solution was prepared by replacing K+ in the extracellular and patch pipette solutions with equimolar amounts of Na+ and Cs+, respectively.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. Effects of prostaglandin (PG)E2 on whole cell currents in rat osteoclasts. A: whole cell currents recorded from a cell bathed in standard extracellular solution (HEPES-buffered solution) with the patch pipette solution containing KCl. a: Effect of 100 nM PGE2 on the whole cell currents evoked by voltage steps. Membrane potential was held at –50 mV, and square pulses (300-ms duration) were applied from –120 to 90 mV in 30-mV increments. Currents were recorded before (control) and 5 min after application of PGE2. Dashed line indicates zero-current level. b: Current-voltage (I-V) relationships recorded by voltage ramps (2-s duration) from –120 to 100 mV from a holding potential of –50 mV. The I-V relationships obtained before (control), 4 min after application of PGE2 (100 nM PGE2), and 5 min after cumulative application of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 100 nM PGE2 + 100 µM DIDS) to the extracellular solution are superimposed. Recordings in a and b were obtained from the same cell. K+ and Cl concentrations in extracellular ([K+]o and [Cl]o, respectively) and patch pipette ([K+]p and [Cl]p, respectively) solutions are also indicated. B: effects of 100 nM PGE2 on whole cell currents and its inhibition by DIDS recorded in bicarbonate-buffered extracellular solution. The results were obtained in the same experimental conditions as the recordings shown in A except for the buffer system.

 
To examine the Cl dependence of the current, the extracellular concentration of Cl ([Cl]o) was altered by replacing NaCl with equimolar amounts of sodium gluconate. To examine anion permeability, NaCl in the extracellular K+-free solution was replaced entirely with the same concentration of NaI, NaBr, or sodium gluconate. The ionic composition of the patch pipette solution used in the experiments for Cl dependence and anion permeability was (in mM) 140 CsCl, 3 MgCl2, 2 ATP (disodium salt), 0.3 EGTA, and 10 HEPES, adjusted to pH 7.3 with Tris. In some experiments, a patch pipette solution with the following ionic composition was used (in mM): 95 cesium aspartate, 47.5 CsCl, 3 MgCl2, 2 ATP (disodium salt), 0.3 EGTA, and 10 HEPES, adjusted to pH 7.3 with Tris.

Osmolarity of all solutions was measured with a freezing-point depression osmometer (Osmometer Automatic; Knauer, Berlin, Germany) and adjusted to 290–300 mosM with mannitol. Hypotonic solution (210 mosM) was made by reducing NaCl concentration in the extracellular K+-free solution to 100 mM. Low-NaCl (100 mM) isotonic solution (290 mosM) was prepared by addition of 80 mM mannitol to the hypotonic solution for control experiments. In some experiments, 100 mM NaCl in the hypotonic solution was replaced entirely with the same concentration of NaI, NaBr, or sodium gluconate.

Membrane potential and currents were recorded with the whole cell configuration patch-clamp technique (12) with an amplifier (Axopatch 200A; Axon Instruments, Foster City, CA). Currents were filtered at 1 kHz and digitized at a sampling frequency of 2–5 kHz. Data acquisition and analysis were performed with pCLAMP 8.0 software (Axon Instruments). All electrophysiological experiments were performed at 26–27°C.

Patch pipettes were prepared with a pipette puller (P-97; Sutter Instrument, Novato, CA), and their tips were heat polished with a microforge (MF83; Narishige Scientific Instrument Laboratory). Values of electrode resistance of the patch pipette filled with the patch pipette solution were in the range of 2.5–5 M{Omega}. Series resistance (60–80%) was compensated to minimize the clamp speed. An Ag-AgCl reference electrode was connected to the extracellular solution through a 3 M KCl-agar salt bridge to minimize changes in liquid junction potentials. Membrane potential was then corrected for liquid junction potentials as previously described (17). The zero-current potential before formation of the gigaseal was set to 0 mV.

Morphological analysis. The pure osteoclast preparation was placed in a plastic petri dish filled with {alpha}-MEM containing 5 mM HEPES, adjusted to pH 7.3 with Tris. In some preliminary experiments, we used {alpha}-MEM containing 14.5 mM NaHCO3, equilibrated with 5% CO2-95% O2 to maintain a pH of 7.3, as the bath solution. In these experiments, FBS was omitted from {alpha}-MEM (serum-free culture medium) to avoid any influence by this ingredient. The petri dish was then placed in a temperature-controlled incubation chamber (Tokai Hit, Japan) mounted on an inverted phase-contrast microscope (TMS; Nikon) and incubated at 37°C. The microscope was connected through a charge-coupled device (CCD) camera (CS8310; Tokyo Electronic Industry, Tokyo, Japan) to a time-lapse video recorder (KV 7168; Toshiba, Tokyo, Japan). Osteoclasts were preincubated with serum-free culture medium for ~1 h before the recordings were started. PGE2 or other test agents were added directly to the serum-free culture medium. Images of osteoclasts were digitized at intervals of 2 min and stored in a Macintosh G4 computer (Apple). Image analysis software (NIH Image, version 1.62) was used for measurement of osteoclast planar area. The planar area A(t) of an osteoclast at time t (in minutes) was quantitated by tracing the outlines of individual cells and normalized to 100% at the beginning of the recording. Osteoclasts continuously move their membrane periphery by protracting and retracting lamellipodia. This phenomenon has been described in terms of motility (1), and we assessed this motility on the basis of the method originally described by Alam et al. (1). An outline of each cell obtained at time t was overlaid on the subsequent outline at time t + 2. The nonoverlapping area ({Delta}A) was measured with NIH Image and expressed relative to A(t). The change in {Delta}A/A(t) was used as an index of cell motility.

Drugs. PGE2 receptor agonists, ONO-DI-004 (EP1 agonist), ONO-AE1-259 (EP2 agonist), ONO-AE-248 (EP3 agonist), and ONO-AE1-329 (EP4 agonist), were kindly supplied by Ono Pharmaceutical (Osaka, Japan). PGE2, forskolin, guanosine 5'-O-(2-thiodiphosphate) (GDP{beta}S) trilithium salt, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), phorbol 12-myristate 13-acetate (PMA), Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS), and tamoxifen were purchased from Sigma (St. Louis, MO). N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89), ionomycin, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) were obtained from Calbiochem (La Jolla, CA). The above drugs were dissolved in dimethyl sulfoxide (DMSO; Sigma) and later diluted with extracellular solutions or the patch pipette solutions to reach final concentration, which resulted in a final DMSO concentration of <0.05%. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; Sigma) was dissolved directly in the extracellular solution. All other chemicals were obtained from Sigma.

Statistical analysis. Data are expressed as means ± SE for n observations. Statistical differences were analyzed with Student's t-test, and a value of P < 0.05 was considered to be significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Stimulatory effects of PGE2 on outward rectifier Cl current in rat osteoclasts. Figure 1Aa shows the effect of PGE2 on whole cell currents evoked by voltage step pulses (from –120 to 90 mV in 30-mV increments) applied from a holding potential of –50 mV. In the standard extracellular solution (HEPES-buffered solution), hyperpolarizing pulses elicited an inwardly rectifying current due to inward rectifier K+ channels, whereas depolarizing pulses elicited a much smaller outwardly rectifying current (Fig. 1Aa), as described previously (27, 29). Application of PGE2 (100 nM) to the bath for 5 min augmented the outward current, whereas the inward current was not altered. Figure 1Ab shows the current-voltage (I-V) relationships in the absence (control) and presence of either PGE2 (100 nM) alone or PGE2 plus the Cl channel blocker DIDS (100 µM), a stilbene derivative, with a voltage ramp from –120 to 100 mV. The I-V curves in the presence and absence of PGE2 crossed near 0 mV, right at the calculated equilibrium potential for Cl (0 mV) but far from that for K+ (–79 mV) under our experimental conditions. Cumulative application of DIDS inhibited the outward current induced by PGE2 without visible effect on the inward current (Fig. 1Ab; 100 nM PGE2 + 100 µM DIDS). The effects of PGE2 on whole cell currents were also examined in bicarbonate-buffered extracellular solution, and the same results were observed: PGE2 (100 nM) augmented the outward current with minor effect on the inward current, and cumulative application of DIDS abolished the augmentation of the outward current (Fig. 1B). Therefore, we assumed that HEPES-buffered solution is an adequate experimental condition to elucidate the effects of PGE2 on osteoclast ion channels, and all subsequent electrophysiological experiments were performed with HEPES-buffered solutions. When K+ in the extracellular and pipette solutions was replaced by Na+ or Cs+, the inward rectifier K+ current was completely abolished (see Figs. 1Aa and 2Aa). Application of PGE2 (1 µM) augmented the outwardly rectifying currents with slow inactivation kinetics at strong positive potentials (Fig. 2Aa). Figure 2Ab shows outwardly rectifying current elicited by a voltage ramp from –70 to 100 mV and the currents obtained before application, during application, and after removal of PGE2. The reversal potential of the PGE2-stimulated current was about –18 mV, very close to the predicted equilibrium potential for Cl [–24 mV; [Cl]o and Cl concentration of pipette solution ([Cl]p) were 143.5 and 53.5 mM, respectively]. The stimulatory effect reached a maximum within 5 min after application of PGE2 and was sustained for >10 min with only slight current reduction. Removal of PGE2 abolished the stimulatory effect. PGE2 stimulated current at concentrations of 10 nM and above. The relationship between PGE2 concentration and current augmentation is shown in Fig. 2Ac.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2. Outward rectifier current stimulated by PGE2 and its Cl dependence. A: whole cell currents recorded in K+-free solutions. a: Effect of 1 µM PGE2 on whole cell currents evoked by voltage step pulses (from –70 to 110 mV in 30-mV increments, holding potential –50 mV). Currents were recorded before (control), 5 min after application of PGE2, and 10 min after removal of PGE2 (washout). b: I-V relationships recorded before (control), 4 min after application of PGE2, and 9 min after removal of PGE2 (washout) obtained from the recordings shown in Ba. I-V relationships were obtained by voltage ramps (2 s) from –70 to 100 mV (holding potential –50 mV). c: Relationship between PGE2 concentration and current augmentation. Amplitudes of the net current stimulated by PGE2 (measured at 80 mV) were measured 5 min after application of PGE2 and expressed as a current density by normalizing to cell capacitance. The net current is the difference current between control (before application) and after application of PGE2. Each point indicates mean ± SE of n = ~8–16 cells. B: Cl dependence of the PGE2-induced outward rectifier current. a: I-V relationships of the net current stimulated by 1 µM PGE2 obtained at 143.5, 73.5, and 33.3 mM [Cl]o. [Cl]p was fixed at 146 mM. I-V relationships obtained by voltage ramps (2 s) from –70 to 100 mV (holding potential –50 mV) are superimposed. [Cl]o was altered after the current was maximally stimulated by PGE2 (1 µM). b: Semilogarithmic plot of the reversal potential of net current stimulated by 1 µM PGE2 vs. [Cl]o. Straight line indicates a least-squares fit to these data. Numbers of cells studied are indicated in parentheses.

 
Similar stimulatory effects of PGE2 on Cl current were also recorded from a pure osteoclast preparation (osteoclasts cultured without nonosteoclastic cells). Amplitudes of current in the presence of 1 µM PGE2 at a membrane potential of 80 mV were the same in osteoclasts prepared from pure osteoclast or mixed-cell preparations [23.5 ± 5.8 pA/pF (n = 8) for pure osteoclast preparations and 25.7 ± 4.2 pA/pF (n = 12) for mixed-cell preparations]. This indicates the presence of a direct stimulatory pathway for PGE2 on outwardly rectifying current in osteoclasts.

The Cl dependence of the current stimulated by 1 µM PGE2 was determined by varying [Cl]o. As shown in Fig. 2Ba, reversal potentials of the PGE2-stimulated outwardly rectifying current shifted with [Cl]o. Mean reversal potentials were 1.1 ± 1.2 (n = 5), 16.3 ± 1.8 (n = 3) and 33.8 ± 2.8 (n = 3) mV for 143.5, 73.5, and 33.3 mM [Cl]o, respectively (Fig. 2Bb), and these values were close to the predicted reversal potentials for these [Cl]o. A least-squares fit for the reversal potentials against [Cl]o on a semilogarithmic scale had a slope of 44 mV per 10-fold change in [Cl]o, suggesting that the stimulated current by PGE2 is mainly carried by Cl.

Anion permeability of Cl channels stimulated by PGE2. Relative anion permeability of the Cl channels stimulated by PGE2 was determined (Fig. 3). Values for the relative membrane permeability of each anion against Cl (PX/PCl) were estimated from the reversal potential changes by the following equation derived from the Goldman-Hodgkin-Katz equation:

(1)
where X denotes I, Br, or gluconate; [Cl]o-control is the extracellular concentration of Cl in the control solution; [X]o-test and [Cl]o-test are extracellular concentrations of X and Cl in the test solution; {Delta}Erev is the difference between reversal potentials measured in the control solution (143.5 mM [Cl]o-control) and test solutions (140 mM [X]o-test, 3.5 mM [Cl] o-test); and F, R, and T are the Faraday constant, the gas constant, and the absolute temperature.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Anion permeability of the current stimulated by PGE2. A: I-V relationships of the current stimulated by PGE2 in Cl-substituted solutions. Voltage ramps (2 s) from –70 to 100 mV (holding potential –50 mV) were applied. NaCl (140 mM) in the extracellular solution was replaced by an equimolar amount of NaI (I, top), NaBr, or sodium gluconate (Br or gluconate, bottom). B: permeability ratios of various anions relative to Cl (PX/PCl). A value of PX/PCl for each anion was calculated from the shift in reversal potential caused by anion replacement using Eq. 1 (see RESULTS). Numbers of cells studied are indicated in parentheses.

 
Replacement of Cl by I shifted the reversal potential toward more negative potentials (Fig. 3A, top). In contrast, replacement of Cl by gluconate, but not by Br, shifted the reversal potential in the positive direction (Fig. 3A, bottom). The estimated PX/PCl was 1.50 ± 0.09 (n = 4) for I, 1.09 ± 0.09 (n = 5) for Br, and 0.29 ± 0.05 (n = 4) for gluconate (Fig. 3B), giving an anion permeability sequence of I > Br {approx} Cl > gluconate.

Sensitivity of PGE2-stimulated Cl current to Cl channel blockers. To examine the effects of the Cl channel blockers DIDS, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and NPPB on the PGE2-stimulated Cl current (Fig. 4, A and B), these blockers were applied to the bath after the current was maximally stimulated by PGE2. DIDS (30 µM) reversibly inhibited the PGE2-induced Cl current in a voltage-dependent manner, being more effective on the outward current than on the inward current (Fig. 4A; 44 ± 5% inhibition at 70 mV vs. 20 ± 6% inhibition at –70 mV; n = 5). The following application of 100 µM DIDS caused further suppression of the PGE2-stimulated current. Another stilbene derivative, SITS, also inhibited the PGE2-induced Cl current in a voltage-dependent manner (data not shown). NPPB (30 µM), an arylaminobenzoate, also inhibited the PGE2-induced Cl current and affected outward and inward currents equally (Fig. 4B; 60 ± 4% inhibition at 70 mV vs. 55 ± 6% inhibition at –70 mV; n = 5). Figure 4C shows the effect of tamoxifen, an inhibitor of the swelling-activated Cl channels (26), on the PGE2-stimulated Cl current. Application of tamoxifen (10 µM) caused only weak inhibition of the PGE2-stimulated Cl current. The following application of a higher concentration of tamoxifen (100 µM) further inhibited the PGE2-stimulated Cl current in a voltage-independent manner (Fig. 4C; 89 ± 8% inhibition at 70 mV vs. 82 ± 6% inhibition at –70 mV; n = 5).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4. Effects of Cl channel blockers and hypotonic solution on PGE2-stimulated Cl current. A–C: inhibitory effects of Cl channel blockers DIDS (A) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; B) and the antiestrogen tamoxifen (C) on the Cl current stimulated by PGE2. I-V relationships obtained before (control), 4 min after application of 1 µM PGE2, and after addition of DIDS, NPPB, or tamoxifen to the extracellular solution are superimposed. Two concentrations of each drug (30 and 100 µM) were successively applied. A voltage ramp (2 s) from –70 to 100 mV was used (holding potential –50 mV). D: osmolarity of the extracellular solution was reduced from 290 mosM (control) to 210 mosM (Hypo). I-V relationships were obtained before (control), 9 min after application of the hypotonic solution (Hypo), and 5 min after addition of 1 µM PGE2 (Hypo + PGE2). In this experiment low-NaCl (100 mM) isotonic solution was used for control. I-V relationships were obtained by voltage ramps (2 s) from –70 to 100 mV (holding potential –50 mV).

 
Effects of PGE2 on Cl current under hyposmotic conditions. Outwardly rectifying Cl current has also been elicited by reduction of extracellular osmolarity in rabbit osteoclasts (17) and mice osteoclasts (31). Therefore, we examined the effects of osmotic changes on the PGE2-stimulated Cl current (Fig. 4D). When extracellular solution was changed from isotonic (290 mosM) to hypotonic solution (210 mosM), outwardly rectifying current was augmented. The activation of this current reached a maximum within 10 min after perfusion with hypotonic solution (Fig. 4D; Hypo). Addition of PGE2 (1 µM) did not further enhance this current (Hypo, 22.5 ± 6.0 pA/pF at 70 mV; Hypo+PGE2, 23.7 ± 5.8 pA/pF at 70 mV; n = 3). The hyposmotically induced current was inhibited by DIDS (100 µM), NPPB (100 µM), and tamoxifen (100 µM), and the difference currents (control current – current in presence of drug) showed reversal potentials of –17 ± 2 (n = 3), –15 ± 3 (n = 3), and –14 ± 3 (n = 3) mV, respectively. The estimated PX/PCl of the hyposmotically induced current was 1.66 ± 0.11 (n = 5) for I, 1.03 ± 0.05 (n = 3) for Br, and 0.23 ± 0.08 (n = 3) for gluconate, showing the same anion permeability sequence as the current stimulated by PGE2 (Fig. 3B).

Effect of PGE2 on osteoclast membrane potential. Because activation of Cl channels in osteoclasts causes membrane depolarization (17), we examined the effect of PGE2 on the membrane potential of osteoclasts bathed in standard extracellular solution. Seven of ten cells examined possessed a resting membrane potential between –65 and –73 mV (–68 ± 2 mV; n = 7). Three cells showed a resting potential of –5 to –38 mV. In cells possessing membrane potential of around –70 mV, application of PGE2 (1 µM) produced a long-lasting depolarization to 0 ± 3 mV (n = 4) with a latency of 1–2 min. Simultaneous application of DIDS (100 µM) suppressed the PGE2-induced membrane depolarization (n = 4).

Receptor subtypes and intracellular signal transduction pathways involved in PGE2-induced stimulation of Cl current. To determine the PGE2 receptor subtypes (EPs) involved in the PGE2 stimulation of Cl current, the stimulatory effects of EP1, EP2, EP3, and EP4 receptor agonists on Cl current were examined. Figure 5A shows dose-response relationships between amplitude of outwardly rectifying current recorded at 80 mV and concentration of various EP agonists. The EP2 agonist ONO-AE1-259 stimulated the outwardly rectifying current in a concentration-dependent manner (Fig. 5A). The stimulatory effect was comparable to that of PGE2. The EP4 agonist ONO-AE1-329 also stimulated this current (Fig. 5A), but the maximal effect was much smaller than that of PGE2 or the EP2 agonist. Reversal potentials of the ONO-AE1-259 (1 µM)- and ONO-AE1-329 (1 µM)-induced currents were shifted to positive potentials by lowering [Cl]o, and least-squares fits against [Cl]o on a semilogarithmic scale had slopes of 47 (n = 3) and 45 (n = 3) mV per 10-fold change in [Cl]o, respectively. The estimated PX/PCl values of the ONO-AE1-259 (1 µM)-induced current were 1.50 ± 0.08 (n = 4) for I, 1.05 ± 0.04 (n = 3) for Br, and 0.25 ± 0.09 (n = 3) for gluconate. Those of the ONO-AE1-329 (1 µM)-induced current were 1.46 ± 0.08 (n = 3) for I, 1.01 ± 0.02 (n = 3) for Br, and 0.28 ± 0.11 (n = 3) for gluconate. Thus currents induced by ONO-AE1-259 and ONO-AE1-329 displayed the same anion permeability sequence of I > Br {approx} Cl. DIDS (30 µM) and NPPB (30 µM) reversibly suppressed the ONO-AE1-259 (1 µM)-induced current in voltage-dependent (50 ± 5% inhibition at 70 mV vs. 16 ± 3% inhibition at –70 mV; n = 3) and -independent (55 ± 6% inhibition at 70 mV vs. 51 ± 4% inhibition at –70 mV; n = 3) manners, respectively. The ONO-AE1-329 (1 µM)-induced current was also inhibited by DIDS (50 µM) in a voltage-dependent manner (60 ± 5% inhibition at 70 mV vs. 28 ± 5% inhibition at –70 mV; n = 3). Thus electrophysiological and pharmacological properties of currents induced by ONO-AE1-259 and ONO-AE1-329 were very similar to those of currents induced by PGE2. On the other hand, no significant stimulatory effects on Cl current were observed with EP1 and EP3 agonists ONO-DI-004 and ONO-AE-248 (Fig. 5A).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5. Effects of selective agonists for PGE2 receptor subtypes (EP agonists) and modulators of intracellular signal transduction pathways. A: relationships between concentration of ONO-AE1-259 (EP2 agonist), ONO-AE1-329 (EP4 agonist), ONO-AE-248 (EP3 agonist), and ONO-DI-004 (EP1 agonist) and the amplitude of the net Cl currents stimulated by these agonists. The concentration-response curve for PGE2 is shown by a dotted line. Each point indicates mean ± SE of n = ~4–12 cells. B: a: Effects of forskolin (10 and 30 µM), dibutyryl cAMP (DBcAMP; 300 µM), phorbol 12-myristate 13-acetate (PMA; 10 nM), and ionomycin (5 µM) on the Cl current. Data were obtained 5 min after application of each signal modulator. b: Effects of guanosine 5'-O-2-(thiodiphosphate) (GDP{beta}S), Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS), and H-89 on the PGE2-induced stimulation of Cl current. Data were obtained 5 min after application of 1 µM PGE2 in the absence (control) or presence of the intracellular signal modulators. GDP{beta}S (1 mM) or Rp-cAMPS (300 µM) was added to the patch pipette solution and allowed to diffuse into the cell for ~10 min before application of PGE2. H-89 (100 nM) was applied extracellularly for ~10 min before application of PGE2. *P < 0.01 vs. control. In A and B, amplitudes of the net currents were measured at a membrane potential of 80 mV and expressed as current density by normalizing to cell capacitance. Numbers of cells examined are indicated in parentheses.

 
PGE2, via EP2 and EP4 subtypes, is known to stimulate adenylate cyclase and increase intracellular cAMP levels (25). Extracellular application of forskolin (10 and 30 µM), an activator of adenylate cyclase, reversibly augmented the outwardly rectifying current in a concentration-dependent manner (Fig. 5Ba). DIDS (30 µM) and NPPB (30 µM) reversibly suppressed the forskolin (30 µM)-induced current in voltage-dependent (52 ± 4% inhibition at 70 mV vs. 19 ± 3% inhibition at –70 mV; n = 5) and -independent (59 ± 5% inhibition at 70 mV vs. 56 ± 4% inhibition at –70 mV; n = 4) manners, respectively. The estimated PX/PCl values of the forskolin (30 µM)-induced current were 1.45 ± 0.05 (n = 5) for I, 1.01 ± 0.02 (n = 3) for Br, and 0.28 ± 0.05 (n = 3) for gluconate. DBcAMP (300 µM), a membrane-permeant analog of cAMP, also stimulated the Cl current with properties similar to those of the PGE2- and forskolin-induced currents, such as the voltage dependence and independence of block by DIDS and NPPB and the anion permeability sequence of I > Br {approx} Cl > gluconate (data not shown). In contrast, extracellularly applied PMA (10 nM), an activator of PKC, had no stimulatory effect on the Cl current. Elevation of [Ca2+]i by ionomycin (5 µM) had very little effect, either. GDP{beta}S (1 mM) inhibited the outward rectifying current stimulated by 1 µM PGE2, demonstrating an involvement of GTP binding proteins in the PGE2-mediated activation of current (Fig. 5Bb). Rp-cAMPS (300 µM) and H-89 (100 nM), inhibitors of PKA, also inhibited the stimulatory effect of PGE2 on the outwardly rectifying current. In control experiments, these signal modulators in the absence of PGE2 did not significantly affect outwardly rectifying current. Additionally, the stimulatory effects of ONO-AE1-259 (1 µM) and ONO-AE1-329 (1 µM) on the Cl current were greatly diminished by pretreatment with GDP{beta}S (1 mM in the pipette solution) or Rp-cAMPS (300 µM in the pipette solution) (data not shown).

Effects of PGE2 and EP agonists on cell planar area and motility of isolated rat osteoclasts. To assess the action of PGE2 on osteoclast morphology, we examined PGE2 effects on cell planar area and motility with pure osteoclast preparations (see MATERIALS AND METHODS). The morphological changes were observed in HEPES-buffered medium to match the experimental conditions of our electrophysiological experiments. Figure 6A represents time-lapse video images of bulgy serrated osteoclasts recorded before application, during application, and after removal of 1 µM PGE2. Although osteoclasts showed intense motility, ceaselessly protruding and retracting lamellipodia before application of PGE2, a sustained decrease or increase in cell planar area did not occur. DMSO (0.05%) did not change averaged values of osteoclast planar area or motility. Application of 1 µM PGE2 produced a progressive decrease in planar area (19 ± 5% inhibition at 15 min; n = 5), reduced osteoclast motility (21 ± 3% inhibition at 15 min; n = 5) (Fig. 6B), and resulted in a spiny appearance of the cell periphery (Fig. 6A, center). Effects of PGE2 on cell planar area and motility reached a maximum within 10 min after application of PGE2 and were sustained during the application (Fig. 6B). Removal of PGE2 reversed both effects (Fig. 6A, right). The effects of PGE2 on cell planar area and motility were also examined in bicarbonate-buffered medium, and results qualitatively similar to those in HEPES-buffered medium were obtained: PGE2 (1 µM) reduced osteoclast planar area (14 ± 5% inhibition at 15 min; n = 4) and inhibited motility (21 ± 3% inhibition at 15 min; n = 4). These results suggest that PGE2-induced morphological changes are relevant to osteoclast physiology. Because the preparation contained only osteoclasts (pure osteoclast preparation), these results also confirm that PGE2 acts directly on osteoclasts.



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 6. Effects of PGE2 and EP agonists on cell planar area and motility of osteoclasts. A: time-lapse video images before (control), 10 min after application of PGE2 (1 µM), and 20 min after washout. B: time course of the effects of PGE2 (1 µM) on cell planar area (top) and motility (bottom). Planar area was normalized to the value at the beginning of the recording (100% at t = –10 min). Motility was assessed by the method described by Alam et al. (Ref. 1; see also MATERIALS AND METHODS). Values are means ± SE of n = 5 observations. C: effects of PGE2 (0.1 and 1 µM), 10 µM ONO-DI-004, 1 µM ONO-AE1-259, 10 µM ONO-AE-248, and 10 µM ONO-AE1-329 on planar area and cell motility. Data were obtained 15 min after application of each drug. Dimethyl sulfoxide (DMSO; 0.05%) was applied as control. Each column indicates mean ± SE, and numbers of observations are given in parentheses. *P < 0.05 compared with control.

 
We next determined the EPs contributing to PGE2-induced reduction of cell planar area and motility. Effects of PGE2 and EP agonists on osteoclast planar area and motility are summarized in Fig. 6C. The EP2 agonist ONO-AE1-259 (1 µM) reduced cell planar area and motility with effects comparable to those of 1 µM PGE2. A higher concentration of the EP4 agonist ONO-AE1-329 (10 µM) also caused a decrease of cell planar area but showed no significant effect on cell motility. In contrast, neither the EP1 agonist ONO-DI-004 nor the EP3 agonist ONO-AE-248 elicited any significant changes in osteoclast planar area or motility when used in concentrations up to 10 µM.

Signal transduction pathways involved in PGE2-induced morphological changes. Application of forskolin (10 µM) reduced osteoclast planar area and motility (Fig. 7A). DBcAMP (1 mM) also reduced cell planar area and motility. Pretreatment with Rp-cAMPS (50 µM) for 30 min inhibited the PGE2-induced reduction of osteoclast planar area and motility (Fig. 7B). Figure 7C summarizes the effects of PKA inhibitors on 1 µM PGE2-induced reduction of cell planar area and motility. Pretreatment of osteoclasts with H-89 (1 µM) also reduced the PGE2 effects on cell planar area and motility.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 7. Effects of intracellular signal modulators on cell planar area and motility of osteoclasts. A: effects of PGE2 (1 µM), forskolin (10 µM), and DBcAMP (1 mM) on osteoclast planar area (top) and motility (bottom). Data were obtained 15 min after application of each drug. DMSO (0.05%) was applied as control. B: effects of 1 µM PGE2 in the presence of 50 µM Rp-cAMPS on osteoclast planar area (top) and motility (bottom). Values are means ± SE of n = 3 observations. Rp-cAMPS was continuously applied for at least 30 min before and during the application of PGE2. C: effects of Rp-cAMPS (50 µM) and H-89 (1 µM) on the PGE2-induced changes in planar area (top) and motility (bottom). Effects on planar area and motility were measured 15 min after application of 1 µM PGE2. Control indicates the effects of 1 µM PGE2 in the absence of Rp-cAMPS and H-89. Rp-cAMPS and H-89 were applied extracellularly for at least 30 min before and during the application of PGE2. In A and C, values are means ± SE and numbers of observations are given in parentheses. *P < 0.05 compared with control.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mechanisms of action of PGE2 in rat osteoclasts. Osteoblasts have been reported to be the main target for PGE2 action on bone metabolism (14, 34). The present study shows that PGE2 activates outwardly rectifying Cl channels in bulgy, serrated rat osteoclasts, independent of the presence of osteoblasts or other nonosteoclastic cells. PGE2 reduced osteoclast planar area associated with inhibition of their motility even in the absence of nonosteoclastic cells; thus actions of PGE2 were independent of soluble factors secreted from nonosteoclastic cells. This demonstrates that PGE2 has a direct action on osteoclasts.

PGE2 exerts its effects through specific membrane receptors coupled to GTP-binding proteins (25). Four subtypes of PGE2 receptors with different intracellular signaling pathways have been identified. The EP1 subtype induces [Ca2+]i mobilization, both EP2 and EP4 subtypes stimulate adenylate cyclase, and the EP3 subtype mainly inhibits adenylate cyclase (25). Among specific agonists for each of the four subtypes of PGE2 receptors, ONO-AE1-259, an EP2 agonist, was most effective in stimulating the Cl current and reducing cell planar area and motility. Our results are most consistent with the presence of EP2 receptor on osteoclasts: 1) GDP{beta}S, Rp-cAMPS, and H-89 inhibit the PGE2-induced current, and 2) forskolin and DBcAMP mimic the PGE2-induced current.

ONO-AE1-329, an EP4 agonist, also had a stimulatory effect on Cl current and caused a reduction of cell planar area, but both effects were much weaker than those of ONO-AE1-259. It thus appears that PGE2-induced changes in osteoclast electrophysiology and morphology are mediated predominantly by the EP2 subtype and to a lesser degree by the EP4 subtype. In contrast to our findings, Mano et al. (22) reported that direct action of PGE2 on rabbit osteoclasts was mediated by the EP4 subtype and that EP4 mRNA was abundantly expressed in rabbit osteoclasts, whereas the other types of EP mRNAs, including EP2 mRNA, were detected only in minor amounts. Although this discrepancy may be attributed to differences in animal species, further experiments at the molecular or protein level are required to elucidate the different mechanisms of PGE2 action in rats and rabbits.

Properties of PGE2-stimulated Cl current in osteoclasts. The PGE2-stimulated Cl current examined in this study was characterized by outward rectification, rapid activation, time-dependent inactivation at strong depolarizing potentials (Fig. 2), an anion permeability sequence of I > Br {approx} Cl > gluconate (Fig. 3), and block by DIDS, NPPB, and tamoxifen (Fig. 4, A–C). The Cl current stimulated by forskolin also showed characteristics similar to those of the PGE2-stimulated Cl current. The electrophysiological and pharmacological properties of the PGE2-stimulated Cl current examined in our study also resemble those of the current induced by hyposmotic stimulation (swelling-activated Cl currents) demonstrated previously in a variety of cells (23, 26, 36, 38) including mouse osteoclasts (31). In addition, any further stimulatory effect of PGE2 on the current was abolished after currents were induced by hyposmotic solution (Fig. 4D), indicating that PGE2-induced current and hyposmotically activated current share a common pathway. The presence of such a common pathway for cAMP- and swelling-activated Cl currents has also been suggested in rat carotid body cells (4).

It is well known that cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels are activated by a cAMP-dependent pathway (13). However, the electrophysiological properties of CFTR Cl channels, showing a linear I-V relationship in symmetrical Cl concentration, insensitivity to extracellular DIDS, and an anion permeability sequence of Cl > I (2, 7, 13), are unlike those of the PGE2-stimulated current demonstrated in our study. Therefore, the channel responsible for the PGE2-stimulated current in rat osteoclasts differs from the CFTR Cl channel. A recent report by Diewald et al. (8) shows that CLC-7 Cl channels expressed in Xenopus oocytes display outwardly rectifying properties similar to the Cl current described in the present study. However, the electrophysiological and pharmacological properties of CLC-7 Cl channels have not yet been established. Thus it is presently uncertain whether or not the Cl current stimulated by PGE2 is carried by CLC-7 Cl channels.

Possible contribution of Cl channels to PGE2-induced changes in osteoclast morphology. Osteoclasts alternate between resorptive and motile states (16), both of which are necessary for effective bone resorption. Cl channels have been documented to play important roles in bone resorption during the resorptive state of osteoclasts. During bone resorption, osteoclasts transport H+ into the resorption lacuna through vacuolar H+ pumps located in the ruffled border membrane (35). Because this H+ pump is electrogenic (9), countermovement of appropriate ions is required to maintain the activity of the H+ transport at a constant level and to prevent the accumulation of electric charges (3). Thus Cl channels act as a charge transport pathway for electrical neutralization against the charge accumulation associated with the H+ transport. For example, the Cl channel blocker DIDS inhibits bone resorption (11, 18). Also, disruption of the CLC-7 Cl channel in mice leads to severe osteopetrosis that is due to failure of osteoclasts to secrete H+ (21). These studies demonstrate that the CLC-7 Cl channel provides a Cl-conductive pathway that acts in conjunction with the H+ pump for electroneutral HCl secretion into the resorption lacuna by resorptive-state osteoclasts. In addition, Cl efflux through Cl channels would also serve to maintain the inward Cl gradient that generates the driving force for the Cl/HCO3 exchange mechanism.

In contrast, little is known about the roles of Cl channels in nonresorbing/motile-state osteoclasts. Recently, we (15) demonstrated that bulgy, serrated osteoclasts maintain higher basal [Ca2+]i compared with flat, round osteoclasts and that exposure of these cells to a high-K+ solution, which causes cell membrane depolarization, reduces [Ca2+]i, cell planar area, and motility. Both inhibition of inwardly rectifying K+ channels and activation of outwardly rectifying Cl channels may induce membrane depolarization in osteoclasts (17, 27). Our results demonstrate that the PGE2-induced depolarization in osteoclasts is due to activation of outwardly rectifying Cl channels.

In many cell types including osteoclasts, it has been suggested that Cl channels also contribute to cell volume regulation and that their activation leads to the loss of cytoplasmic osmolytes and water, resulting in a cell volume decrease (28, 31). The physiological consequences of this effect for osteoclast function and bone resorption remain to be investigated.

Electrophysiological and morphological analyses of PGE2 actions were performed with HEPES-buffered (or in some experiments bicarbonate buffered) solutions. We found a good correlation between electrophysiological and morphological changes induced by PGE2 regarding time course, reversibility, and cAMP dependence. These results suggest a correspondence between electrophysiological and morphological effects of PGE2. Qualitatively similar results were also observed in our experiments using bicarbonate-buffered solutions, indicating that the electrophysiological and morphological changes induced by PGE2 are of physiological significance.

It is likely that activation of the Cl channel by PGE2 will lower [Ca2+]i levels by depolarizing the membrane potential of bulgy, serrated osteoclasts and thereby reduce osteoclast motility as observed in this study. Because there is a good correlation between osteoclast motility and bone-resorbing activity (40), suppression of osteoclast motility by PGE2 via EP2 receptor activation is likely to reduce bone resorption.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Japan (15591988 to F. Okamoto and Frontier Research Grant).


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


    FOOTNOTES
 

Address for reprint requests and other correspondence: F. Okamoto, Dept. of Physiological Science and Molecular Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, Japan 814-0193 (E-mail: fujipi{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
 GRANTS
 REFERENCES
 
1. Alam ASMT, Moonga BS, Bevis PJR, Huang CL, and Zaidi M. Amylin inhibits bone resorption by a direct effect on the motility of rat osteoclasts. Exp Physiol 78: 183–196, 1993.[Abstract]

2. Anderson MP, Gregory RJ, Thompson S, Souza DW, Paul S, Mulligan RC, Smith AE, and Welsh MJ. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253: 202–205, 1991.[ISI][Medline]

3. Blair HC, Teitelbaum SL, Ghiselli R, and Gluck S. Osteoclastic bone resorption by a polarized vacuolar proton pump. Science 245: 855–857, 1989.[ISI][Medline]

4. Carpenter E and Peers C. Swelling- and cAMP-activated Cl currents in isolated rat carotid body type I cells. J Physiol 503: 497–511, 1997.[Abstract]

5. Chambers TJ and Ali NN. Inhibition of osteoclastic motility by prostaglandins I2, E1, E2 and 6-oxo-E1. J Pathol 139: 383–397, 1983.[ISI][Medline]

6. Chambers TJ, McSheehy PM, Thomson BM, and Fuller K. The effect of calcium-regulation hormones and prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones. Endocrinology 116: 234–239, 1985.[Abstract]

7. Dawson DC, Smith SS, and Mansoura MK. CFTR: mechanism of anion conduction. Physiol Rev 79, Suppl: S47–S75, 1999.[Medline]

8. Diewald L, Rupp J, Dreger M, Hucho F, Gillen C, and Nawrath H. Activation by acidic pH of CLC-7 expressed in oocytes from Xenopus laevis. Biochem Biophys Res Commun 291: 421–424, 2002.[CrossRef][ISI][Medline]

9. Forgac M. Structure and function of vacuolar class of ATP-driven proton pumps. Physiol Rev 69: 765–796, 1989.[Free Full Text]

10. Fuller K and Chambers TJ. Effect of arachidonic acid metabolites on bone resorption by isolated rat osteoclasts. J Bone Miner Res 4: 209–215, 1989.[ISI][Medline]

11. Hall TJ and Chambers TJ. Optimal bone resorption by isolated rat osteoclasts requires chloride/bicarbonate exchange. Calcif Tissue Int 45: 378–380, 1989.[ISI][Medline]

12. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.[ISI][Medline]

13. Hume JR, Duan D, Collier ML, Yamazaki J, and Horowitz B. Anion transport in heart. Physiol Rev 80: 31–81, 2000.[Abstract/Free Full Text]

14. Kaji H, Sugimoto T, Kanatani M, Kukase M, Kumegawa M, and Chihara K. Prostaglandin E2 stimulates osteoclast-like cell formation and bone-resorbing activity via osteoblasts: role of cAMP-dependent protein kinase. J Bone Miner Res 11: 62–71, 1996.[ISI][Medline]

15. Kajiya H, Okamoto F, Fukushima H, Takada K, and Okabe K. Mechanism and role of high-potassium-induced reduction of intracellular Ca2+ concentration in rat osteoclasts. Am J Physiol Cell Physiol 285: C457–C466, 2003.[Abstract/Free Full Text]

16. Kanehisa J and Heersche JN. Osteoclastic bone resorption: in vitro analysis of the rate of resorption and migration of individual osteoclasts. Bone 9: 73–79, 1988.[ISI][Medline]

17. Kelly ME, Dixon SJ, and Sims SM. Outwardly rectifying chloride current in rabbit osteoclasts is activated by hyposmotic stimulation. J Physiol 475: 377–389, 1994.[Abstract]

18. Klein-Nulend J and Raisz LG. Effects of two inhibitors of anion transport on bone resorption in organ culture. Endocrinology 125: 1019–1024, 1989.[Abstract]

19. Komarova SV, Dixon SJ, and Sims SM. Osteoclast ion channels: potential targets for antiresorptive drugs. Curr Pharm Des 7: 637–654, 2001.[ISI][Medline]

20. Komarova SV, Pilkington MF, Weidema AF, Dixon SJ, and Sims SM. RANK ligand-induced elevation of cytosolic Ca2+ accelerates nuclear translocation of nuclear factor {kappa}B in osteoclasts. J Biol Chem 278: 8286–8293, 2003.[Abstract/Free Full Text]

21. Kornak U, Kasper D, Bosl MR, Kaiser E, Schweizer M, Schulz A, Friedrich W, Delling G, and Jentsch TJ. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104: 205–215, 2001.[ISI][Medline]

22. Mano M, Arakawa T, Mano H, Nakagawa M, Kaneda T, Kaneko H, Yamada T, Miyata K, Kiyomura H, Kumegawa M, and Hakeda Y. Prostaglandin E2 directly inhibits bone-resorbing activity of isolated mature osteoclasts mainly through the EP4 receptor. Calcif Tissue Int 67: 85–92, 2000.[CrossRef][ISI][Medline]

23. Meng XJ and Weinman SA. cAMP- and swelling-activated chloride conductance in rat hepatocytes. Am J Physiol Cell Physiol 271: C112–C120, 1996.[Abstract/Free Full Text]

24. Miyaura C, Masaki I, Suzawa T, Sugimoto Y, Ushikubi F, Ichikawa A, Narumiya S, and Suda T. Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice. J Biol Chem 275: 19819–19823, 2000.[Abstract/Free Full Text]

25. Narumiya S, Sugimoto Y, and Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 79: 1193–226, 1999.[Abstract/Free Full Text]

26. Nilius B, Prenen J, Voets T, Eggermont J, and Droogmans G. Activation of volume-regulated chloride currents by reduction of intracellular ionic strength in bovine endothelial cells. J Physiol 506: 353–361, 1998.[Abstract/Free Full Text]

27. Okabe K, Okamoto F, Kajiya H, Takada K, and Soeda H. Estrogen directly acts on osteoclasts via inhibition of inward rectifier K+ channels. Naunyn Schmiedebergs Arch Pharmacol 361: 610–620, 2000.[CrossRef][ISI][Medline]

28. Okada Y. Volume expansion-sensing outward-rectifier Cl channel: fresh start to the molecular identity and volume sensor. Am J Physiol Cell Physiol 273: C755–C789, 1997.[Abstract/Free Full Text]

29. Okamoto F, Okabe K, and Kajiya H. Genistein, a soybean isoflavone, inhibits inward rectifier K+ channels in rat osteoclasts. Jpn J Physiol 51: 501–509, 2001.[ISI][Medline]

30. Pilbeam CC, Harrison JR, and Raisz LG. Prostaglandins and bone metabolism. In: Principles of Bone Biology (2nd ed.), edited by Bilezikian JP, Raisz LG, and Rodan GA. San Diego, CA: Academic, 2002, vol. 2, pt. I, chapt. 54, p. 979–994.

31. Sakai H, Nakamura F, and Kuno M. Synergetic activation of outwardly rectifying Cl currents by hypotonic stress and external Ca2+ in murine osteoclasts. J Physiol 515: 157–168, 1999.[Abstract/Free Full Text]

32. Suda M, Tanaka K, Natsui K, Usui T, Tanaka I, Fukushima M, Shigeno C, Konishi J, Narumiya S, Ichikawa A, and Nakao N. Prostaglandin E receptor subtypes in mouse osteoblastic cell line. Endocrinology 137: 1698–1705, 1996.[Abstract]

33. Suzawa T, Miyaura C, Inada M, Maruyama T, Sugimoto Y, Ushikubi F, Ichikawa A, Narumiya S, and Suda T. The role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and EP4) in bone resorption: an analysis using specific agonists for the respective EPs. Endocrinology 141: 1554–1559, 2000.[Abstract/Free Full Text]

34. Udagawa N, Takahashi N, Jimi E, Matsuzaki K, Tsurukai T, Itoh K, Nakagawa N, Yasuda H, Goto M, Tsuda E, Higashio K, Gillespie MT, Martin TJ, and Suda T. Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colony-stimulating factor: receptor activator of NF-{kappa}B ligand. Bone 25: 517–523, 1999.[CrossRef][ISI][Medline]

35. Väänänen HK, Karhukorpi EK, Sundquist K, Wallmark B, Roininen I, Hentunen T, Tuukkanen J, and Lakkakorpi P. Evidence for the presence of a proton pump of the vacuolar H+-ATPase type in the ruffled borders of osteoclasts. J Cell Biol 111: 1305–1311, 1990.[Abstract]

36. Von Weikersthal SF, Barrand MA, and Hladky SB. Functional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells. J Physiol 516: 75–84, 1999.[Abstract/Free Full Text]

37. Weinreb M, Grosskop A, and Shir N. The anabolic effect of PGE2 in rat bone marrow cultures is mediated via the EP4 receptor subtype. Am J Physiol Endocrinol Metab 276: E376–E383, 1999.[Abstract/Free Full Text]

38. Yamazaki J, Duan D, Janiak R, Kuenzli K, Horowitz B, and Hume R. Functional and molecular expression of volume-regulated chloride channels in canine vascular smooth muscle cells. J Physiol 507: 729–736, 1998.[Abstract/Free Full Text]

39. Yoshida K, Oida H, Kobayashi T, Maruyama T, Tanaka M, Katayama T, Yamaguchi K, Segi E, Tsuboyama T, Matsushita M, Ito K, Ito Y, Sugimoto Y, Ushikubi F, Ohuchida S, Kondo K, Nakamura T, and Narumiya S. Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation. Proc Natl Acad Sci USA 99: 4580–4585, 2002.[Abstract/Free Full Text]

40. Zaidi M. Modularity of osteoclast behaviour and "mode-specific" inhibition of osteoclast function. Biosci Rep 10: 547–556, 1990.[ISI][Medline]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
287/1/C114    most recent
00551.2003v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Okamoto, F.
Articles by Okabe, K.
Articles citing this Article
PubMed
PubMed Citation
Articles by Okamoto, F.
Articles by Okabe, K.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.