Differences in regulation of pHi in large (>= 10 nuclei) and small (=< 5 nuclei) osteoclasts

Rita L. Lees1 and Johan N. M. Heersche1,2

1 Department of Pharmacology, Faculty of Medicine, and 2 Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5G 1G6


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Osteoclasts are multinucleated cells that resorb bone by extrusion of protons and proteolytic enzymes. They display marked heterogeneity in cell size, shape, and resorptive activity. Because high resorptive activity in vivo is associated with an increase in the average size of osteoclasts in areas of greater resorption and because of the importance of proton extrusion in resorption, we investigated whether the activity of the bafilomycin A1-sensitive vacuolar-type H+-ATPase (V-ATPase) and amiloride-sensitive Na+/H+ exchanger differed between large and small osteoclasts. Osteoclasts were obtained from newborn rabbit bones, cultured on glass coverslips, and loaded with the pH-sensitive indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Intracellular pH (pHi) was recorded in single osteoclasts by monitoring fluorescence. Large (>= 10 nuclei) and small (<= 5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pHi, their pHi was decreased by bafilomycin A1 addition or removal of extracellular Na+, and the realkalinization upon readdition of Na+ was bafilomycin A1 sensitive. After acid loading, a subpopulation of large osteoclasts (40%) recovered by V-ATPase activity alone, whereas all small osteoclasts recovered by Na+/H+ exchanger activity. Interestingly, in 60% of the large osteoclasts, pHi recovery was mediated by both the Na+/H+ exchanger and V-ATPase activity. Our results show a striking difference between pHi regulatory mechanisms of large and small osteoclasts that we hypothesize may be associated with differences in the potential resorptive activity of these cells.

vacuolar-type hydrogen-adenosinetriphosphatase; sodium/hydrogen exchanger; nuclear number; intracellular pH


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

OSTEOCLASTS ARE MULTINUCLEATED cells responsible for bone resorption. Osteoclastic bone resorption is a complex process starting with migration and attachment of osteoclasts to the bone surface. This is followed by the development of a sealing zone, the formation of a ruffled border, and the subsequent extrusion of protons, Cl-, and proteolytic enzymes in the extracellular resorption zone between the ruffled border and the bone surface (52). Proton extrusion is accomplished by a vacuolar-type H+-ATPase (V-ATPase) localized on the ruffled border (8, 53). Maintenance of intracellular pH (pHi) is believed to be controlled mainly by Na+/H+ exchangers and Cl-/HCO3- exchangers on the basolateral membrane (21, 35, 49).

Osteoclasts from humans, rats, and rabbits can under normal circumstances contain anywhere from 2 to 30 nuclei, with an average of 3 to 10 nuclei. However, in diseases characterized by increased bone resorption such as Paget's disease, end-stage renal disease, periodontal disease, and rheumatoid arthritis, osteoclast size is generally increased (1, 28, 32, 45). In Paget's disease, osteoclasts have been reported to reach a diameter of 100 µm and to contain up to 100 nuclei (45).

Osteoclasts become multinucleated as a result of cell fusion, either with other multinuclear osteoclasts or with mononuclear osteoclast precursors (17, 25), and certain enzymes and receptors are up- or downregulated upon formation of multinuclear osteoclasts from their mononuclear precursors (14, 56). However, it is not known whether increased multinuclearity in osteoclasts is associated with changes in gene expression, as has been reported for other multinucleated cells (15, 16, 34, 38, 54). Piper et al. (39) studied the relationship between osteoclast size (as determined by the number of nuclei) and the resorptive ability of these cells and found a positive correlation between the size of an osteoclast and the volume of the resorption pit made. They also found that the volume resorbed per nucleus tended to decrease with increasing nuclear number. However, their observations included only osteoclasts associated with a resorption pit and did not take into account the possibility that osteoclasts can form several pits within a given period of time (27). Thus they may have underestimated the activity of their osteoclasts. We recently found that, in cultures with significantly greater numbers of large osteoclasts, both pit size and pit number were increased, indicating that large osteoclasts were not only resorbing a larger surface area per pit but also formed more pits than smaller osteoclasts (30).

One striking characteristic of osteoclasts is the marked heterogeneity observed in their behavior and responsiveness to inhibitory and stimulatory agents. Kanehisa (26) investigated calcitonin-induced inhibition of osteoclast function and found that, although the majority of cultured rabbit osteoclasts exposed to calcitonin stopped migrating and started contracting, others showed no discernible changes in cytoplasmic motility or general morphology. In addition, although osteoclasts at 9 of 23 separate resorption sites regained resorptive activity due to spontaneous escape from calcitonin-induced contraction, the remaining 14 did not. Another example was reported by Owens and Chambers (37), who found that although macrophage colony stimulating factor increased the percentage of migrating rat osteoclasts from 10 to 60%, the remaining 40% of osteoclasts did not respond to this cytokine. Equally striking were the observations of Hall et al. (22), who found that although 75% of rat osteoclasts required continued mRNA and protein synthesis to resorb bone when cultured in vitro, some cells still actively resorbed in the presence of the inhibitors actinomycin D and cycloheximide. In addition, Yu and Ferrier (58) reported that only 27% of osteoclasts in 1-day cultures responded to interleukin-1alpha with an increase in intracellular Ca2+ concentration, whereas 84% responded in 3-day cultures. Heterogeneity has also been observed with regard to pHi regulation; under conditions of chronic extracellular acidosis and induced acid load, 40% of osteoclasts recovered by V-ATPase activity, whereas the remaining 60% did not (36, 40). Although some investigators have associated variable responses with osteoclast size or shape (2, 40), the reasons for the variable responses have not been elucidated.

Proton extrusion and pHi regulation have a pivotal role in the resorptive process. Interestingly, heterogeneity of osteoclasts with regard to activity of the proton pumps has also been noted (36, 40). Therefore, we sought to determine in this study whether small (2-5 nuclei) and large (>= 10 nuclei) osteoclasts differed with regard to V-ATPase and Na+/H+ exchanger activities. We found that basal pHi in large and small osteoclasts differed significantly, as did the responses to Na+ removal and to the addition of the V-ATPase inhibitor bafilomycin A1. Upon acid loading, small osteoclasts recovered by an Na+-dependent and amiloride-sensitive mechanism, whereas large osteoclasts consisted of the following two groups: an Na+-dependent group that was maximally inhibited by the addition of both amiloride and bafilomycin A1 and an Na+-independent group that was sensitive only to bafilomycin A1. These results clearly show that large and small osteoclasts differ with regard to the activity of proton extrusion mechanisms.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials. Medium 199, alpha -minimum essential medium (alpha -MEM), FCS, and antibiotics (penicillin G, gentamicin, fungizone) were obtained from GIBCO-BRL (Burlington, ON). HEPES-buffered HCO3--free RPMI, bafilomycin A1, and HEPES were purchased from Sigma. 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and nigericin were from Molecular Probes (Eugene, OR). Amiloride was obtained from ICN Biochemicals (Montreal, PQ). All other chemicals were purchased from Sigma or Fisher.

Osteoclast isolation and identification. Osteoclasts were isolated from rabbit long bones as described previously (3, 27). Briefly, the femurs, tibiae, humeri, and radii of 1-day-old New Zealand White rabbits were dissected out. After removal of adherent soft tissues, the shafts were placed in a sterile petri dish containing medium 199 and cut longitudinally, and the interior surfaces were curetted to release the bone cells. The medium containing cells and bone fragments was agitated by pipetting to release additional cells attached to the bone fragments. These bone marrow preparations containing osteoclasts were centrifuged at 200 g for 10 min, and the cells obtained from the bones of one rabbit were resuspended in 8 ml of alpha -MEM (pH 7.4) with 10% FCS and antibiotics (100 µg/ml penicillin G, 0.5 µg/ml gentamicin, and 0.3 µg/ml fungizone). The osteoclast-containing cell suspension was plated on glass coverslips in six-well tissue culture dishes (100 µl/coverslip) and allowed to attach overnight in alpha -MEM containing 10% FCS and antibiotics in humidified air (37°C and 5% CO2). The cells were then cultured for 3-8 h in 25 mM HEPES-buffered, HCO3--free RPMI with 10% serum at pH 7.4 and 37°C in an air incubator before measurement of pHi. In some cases, the cells were cultured for 24 h under these conditions, but the results were pooled as no differences were found between short- and long-term culture in HEPES-buffered HCO3--free medium.

Osteoclasts were identified as cells containing two or more nuclei (as seen by phase-contrast microscopy). A few of the cells in the initial experiments were also fixed and stained for tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts. For staining, cells were fixed with 4% neutral buffered Formalin for 10 min and were washed with PBS. A solution of Michaelis veronal acetate buffer (pH 5) containing naphthol AS-MX phosphate as substrate, hexazonium pararosanilin as coupler, and 20 mM L-(+)-tartaric acid was then added to the cells for 5 min at room temperature. TRAP-positive cells stained red. Because all of the cells used in the initial pH measurements were TRAP positive, TRAP staining was omitted in subsequent experiments.

pHi measurements. The pHi was determined using the microfluorometric technique described previously for osteoclasts (35). Briefly, coverslips with cells were placed in a Leiden coverslip dish and were maintained at 37°C for the remainder of the experiment. The cells were loaded with the H+-sensitive fluorescent dye BCECF by incubation with 1 µM of the parent acetoxymethyl ester for 10 min and then were washed with RPMI and incubated in Na+-containing medium (in mM: 140 NaCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 20 HEPES, pH 7.4). For incubation in Na+-free conditions, Na+ was replaced with either K+ or N-methyl-D-glucamine (NMG+). Test solutions were applied to the cells by pipetting 3 ml of the desired medium (with or without inhibitors) into the chamber. Previously used culture medium was removed by aspiration. In some experiments, treatments were applied sequentially to the same cells (see Figs. 2 and 3). Because the effects of amiloride are reversible, it was added first and then was washed out with Na+-containing medium alone, after which bafilomycin A1 was added. In the experiments shown in Figs. 5 and 6, treatments with transport blockers were all independent observations on different cells; however, a few cells in Fig. 6 (19 out of a total of 131) received sequential treatment as in the experiments shown in Figs. 2 and 3. No statistical difference was seen between the results of sequential and independent addition of inhibitors in the experiments shown in Fig. 6, and thus the results were pooled. The rate of pHi recovery shown in Figs. 5 and 6 is the initial maximal rate of change.

Fluorescence intensity in single cells was measured using a Nikon TMD-Diaphot microscope attached to an M-series dual-wavelength illumination system from Photon Technologies. Illumination was on for 2 s and off for 8 s (to minimize photobleaching), and the photometric data were recorded at a rate of 5 points/s. The excitation light was directed to the cells via a 510-nm dichroic mirror. Fluorescence emission collected by a Nikon Fluor ×40 oil-immersion objective traversed a 542 ± 64-nm band-pass filter.

Acid loading of osteoclasts. For acid loading, we used the NH4+ "prepulse" method (47). The adherent cells were first exposed to 40 mM NH4Cl for 5-8 min, during which time there was a rapid rise in pHi as NH3 entered the cell and combined with H+ to form NH4+, followed by a slow decline in pHi due to influx of NH4+, which dissociated to form NH3 and H+. Osteoclasts were then transferred to NH4+-free medium, resulting in a rapid loss of NH3, subsequent dissociation of internal NH4+ into NH3 and H+, and a decrease in pHi to a level much lower than the starting pHi. Recovery of osteoclast pHi was then monitored in the cells in the presence of different media and inhibitors of pHi regulatory mechanisms.

pHi calibration. Calibration of the fluorescence ratio vs. pH of each individual osteoclast was performed using the K+/H+ ionophore nigericin. Upon completion of the pH experiments, the cells were equilibrated in K+ medium (140 mM) of varying pH in the presence of 5 µM nigericin, and calibration curves were constructed by plotting the extracellular pH [pHo; which is assumed to be identical to pHi (50)] against the corresponding fluorescence ratio. Additionally, some cells were calibrated by the null-point method, as originally described by Eisner et al. (13), to verify the values found with the nigericin technique. The principle of this method is to find the mixture of permeant acid (A) and base (B) that, upon entering the cell, produces no change in pHi. The pHi can then be calculated from the composition of this mixture, according to the following equation: pHi = pHo - 0.5log([A]/[B]), where brackets denote concentration. Thus, with pHo of 7.4, we prepared Na+-containing solutions of pH 6.8-7.8 using butyric acid as the weak acid and trimethylamine as the weak base.

Statistics. Data were analyzed statistically by one-way ANOVA and with an unpaired, double-sided Student's t-test. All results are expressed as means ± SE for n number of cells.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Size distribution of osteoclasts. The osteoclast-containing cell cultures obtained from newborn rabbit long bones were comprised of a variety of stromal cell types and osteoclasts. Osteoclasts were defined as cells having two or more nuclei under phase-contrast microscopy and staining positively for TRAP after fixation. We have reported previously that cells so identified bind calcitonin and resorb bone when plated on bone slices (43, 51). The size and nuclear number of the osteoclasts ranged from cells with a diameter of 30 µm and 2 nuclei to cells with a diameter of 90 µm and 23 nuclei. We studied the following two groups of osteoclasts: small osteoclasts that contained 2-5 nuclei (60% of the total number of osteoclasts) and large osteoclasts that contained 10 or more nuclei (10% of the osteoclasts); we investigated the pHi changes in these cells. This choice was arbitrary and was made to decrease the likelihood of overlap for parameters that might increase or decrease in value gradually.

Determination of pHi in osteoclasts by two independent methods of calibration. BCECF was uniformly distributed in the cytoplasmic compartment. Small osteoclasts maintained a steady resting pHi of 7.34 ± 0.02 (n = 94) when cultured on glass coverslips for 2-6 h in HCO3--free HEPES-buffered RPMI with 10% FCS, as determined by the nigericin calibration technique. In contrast, large osteoclasts had a significantly higher pHi of 7.70 ± 0.02 (n = 156) under the same conditions (P < 0.001). This difference in steady-state pHi between large and small osteoclasts was confirmed when another calibration technique (the null-point method) was used (n = 12, Fig. 1A). pHi tracings for a single small osteoclast and a single large osteoclast, calibrated by both the null-point method and then the nigericin method (y-axis), are shown in Fig. 1, B and C. The pHi of the representative small osteoclast in Fig. 1B decreased when a null solution of pH 7.05 was added but increased after addition of a null solution of pH 7.45, indicating that this osteoclast had a resting pHi between these values, i.e., ~7.2. A similar procedure applied to the large osteoclast shown in Fig. 1C indicates a resting pHi of ~7.7. Because of the excellent agreement between the two calibration methods and the greater ease of performing nigericin calibration, all future calibrations were performed with the nigericin technique.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Basal pHi of small (2-5 nuclei) and large (>= 10 nuclei) osteoclasts. Osteoclasts were cultured on glass coverslips in Na+-containing HEPES-buffered medium, and pHi was recorded. Two independent methods of pHi calibration, the nigericin technique and the null-point calibration method, gave almost identical results. A: large osteoclasts have a resting pHi of 0.4 pH units above that of small osteoclasts. Representative trace of a small osteoclast (B) and a large osteoclast (C) calibrated first with the null-point technique and then the nigericin technique (calibration not shown). The ordinate axis represents the results of the nigericin method, and the horizontal bars represent the addition of the indicated null-point solutions. Results represent means ± SE of 12 observations.

Effect of amiloride and bafilomycin A1 on osteoclast resting pHi. Upon addition of amiloride (an inhibitor of the Na+/H+ exchanger) to Na+-containing medium, the pHi of both small and large osteoclasts did not change significantly (Fig. 2A). Upon the removal of amiloride by washing and the addition of bafilomycin A1 (the specific and widely used inhibitor of the V-ATPase) in Na+-containing medium to these same cells, the pHi of the large osteoclasts decreased significantly from 7.59 ± 0.04 to 7.37 ± 0.05 at a rapid initial rate of -0.16 ± 0.01 pH units/min, whereas the pHi of small osteoclasts did not change. When these same cells were treated with both amiloride and bafilomycin A1, there was a significant drop in pHi of small osteoclasts from 7.34 ± 0.03 to 7.19 ± 0.05, whereas that of large osteoclasts remained at about the same level as seen with bafilomycin A1 alone. Representative traces of a small (Fig. 2B) and a large (Fig. 2C) osteoclast treated with amiloride and then with bafilomycin A1 are shown.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of amiloride (Amil) and bafilomycin A1 (Baf) addition on osteoclast pHi. A: amiloride (1 mM, an inhibitor of the Na+/H+ exchanger) had no significant effect on pHi of small osteoclasts (n = 11) or large osteoclasts (n = 8) cultured in Na+ medium. There was no significant effect of bafilomycin A1 (200 nM, an inhibitor of the V-ATPase) on pHi of small osteoclasts cultured in Na+ medium (n = 11), whereas the pHi of large osteoclasts decreased significantly (n = 9). The addition of both amiloride and bafilomycin A1 together resulted in a significant decrease in pHi of both small (n = 11) and large (n = 7) osteoclasts. Representative trace of a small osteoclast (B) and of a large osteoclast (C) treated first with amiloride and then with bafilomycin. Results represent means ± SE. # P < 0.01 and omega  P < 0.05.

Cl- channels and V-ATPase activity in small and large osteoclasts. Because V-ATPase-mediated proton transport is often charge coupled to passive Cl- permeability, we wished to ascertain whether the observed differences in V-ATPase activity between small and large osteoclasts were due to differences in Cl- channels in these cells. To evaluate this, the Cl- channel inhibitor DIDS was added to Na+-containing medium in addition to bafilomycin A1. Once again, the pHi of small osteoclasts did not change significantly compared with control Na+-containing medium (Fig. 3A). Under the same conditions, the pHi of large osteoclasts decreased significantly from 7.55 ± 0.05 to 7.35 ± 0.06. Interestingly, however, DIDS alone raised the pHi of both large and small osteoclasts by 0.2 pH units. Representative traces of a small and a large osteoclast are shown in Fig. 3, B and C, respectively.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of DIDS on vacuolar-type H+-ATPase (V-ATPase) activity in small and large osteoclasts. A: DIDS (10-100 µM, an inhibitor of Cl- channels) significantly alkalinized both small (n = 6) and large (n = 8) osteoclasts to the same extent. Simultaneous addition of DIDS and bafilomycin A1 (200 nM) resulted in a significant acidification of large osteoclasts (n = 10) but had no effect on the pHi of small osteoclasts (n = 8). Representative trace of a small (B) and a large (C) osteoclast treated first with DIDS and then with both DIDS and bafilomycin A1. Results represent means ± SE. omega  P < 0.05 and # P < 0.01 compared with Na+ medium.

The importance of extracellular Na+ in maintaining high pHi in large osteoclasts. When Na+-containing medium was replaced by K+- or NMG+-containing medium, the pHi of small osteoclasts was not affected (Fig. 4, A and B). Similar treatment of large osteoclasts resulted in a decrease in pHi by 0.40 pH units. This drop in pHi of large osteoclasts occurred rapidly (within 20 s) and was maintained until Na+ was added back to the external medium (Fig. 4C).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of removal of extracellular Na+ on osteoclast pHi. Osteoclasts cultured on glass coverslips in Na+-containing medium were transferred to Na+-free medium. A: small osteoclasts (n = 20) had a very small drop in pHi, whereas large osteoclasts (n = 45) had a significant decrease in pHi. Representative trace of pHi changes in a small osteoclast (B) and a large osteoclast (C) after removal and readdition of Na+ medium. The pHi of large osteoclasts decreased immediately upon removal of Na+ and remained low until Na+ was reapplied, at which time recovery was very fast to basal levels. Results represent means ± SE. * P < 0.001.

Effect of amiloride and bafilomycin A1 on osteoclast pHi recovery. To determine which pH regulatory mechanisms were involved in the pH recovery of large osteoclasts after readdition of Na+, the effects of various inhibitors were evaluated independently in different cells (Fig. 5A). Under Na+-free conditions, recovery was negligible, averaging 0.01 pH units/min. The addition of Na+ alone resulted in immediate and rapid recovery at a rate of 0.20 pH units/min. Addition of amiloride resulted in a 25% inhibition in the initial maximal rate of Na+-induced pHi recovery. Addition of bafilomycin A1 resulted in a 70% reduction in the rate of pHi recovery of large osteoclasts in the presence of Na+. Complete inhibition of pHi recovery was seen with addition of both amiloride and bafilomycin A1, suggesting that both the Na+/H+ exchanger and the V-ATPase contribute to pHi recovery in the presence of Na+. Representative traces of three different large osteoclasts and their recovery in the presence of amiloride, bafilomycin A1, or amiloride and bafilomycin A1 are shown in Fig. 5, B-D, respectively.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Rate of pHi recovery of large osteoclasts after Na+ removal in the presence of various inhibitors of pHi regulatory mechanisms. A: under Na+-free conditions, recovery was negligible (n = 45). Addition of Na+ resulted in rapid recovery (n = 27). The addition of 1 mM amiloride (Am) to Na+ medium had a slight inhibitory effect on pHi recovery (n = 9) compared with Na+ alone. Bafilomycin A1 (200 nM) significantly decreased the recovery in Na+ medium (n = 7), whereas the combination of bafilomycin A1 and amiloride completely inhibited pHi recovery (n = 7). Representative traces of 3 different large osteoclasts recovering in the presence of amiloride (B), bafilomycin (C), or amiloride and bafilomycin (D). Results represent means ± SE. omega  P < 0.05 and * P < 0.001 compared with recovery in the presence of Na+. a P < 0.05 compared with bafilomycin only.

Effect of amiloride and bafilomycin A1 on osteoclast pHi recovery after acid load. Because the results shown above strongly suggested that both the Na+/H+ exchanger and V-ATPase activity operated to different degrees in large and small osteoclasts at physiological pHi, we decided to investigate whether under more acidic conditions similar differences would be detectable. We used the ammonium prepulse technique to decrease pHi. Small osteoclasts did not recover in the absence of Na+ but did recover in the presence of Na+ (Fig. 6A). Recovery of pHi in small osteoclasts in the presence of Na+ was completely inhibited by amiloride, indicating that the recovery was mediated by amiloride-sensitive Na+/H+ exchange.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 6.   Rate of pHi recovery of large and small osteoclasts after acid loading. NH4Cl was added for 5-8 min to osteoclasts cultured on glass coverslips. The isosmotic replacement of NH4+ with N-methyl-D-glucamine (NMG+) or K+ resulted in a rapid and immediate drop in pHi in both small and large osteoclasts. A: small osteoclasts recovered very slowly in the absence of Na+ (n = 35) and very rapidly in the presence of Na+. Recovery in the presence of Na+ was completely and reversibly inhibited by 1 mM amiloride (n = 27), whereas 200 nM bafilomycin A1 had no effect (n = 11). Large osteoclasts were divided into Na+-dependent cells, where pHi recovery occurred only in the presence of Na+ (n = 49), and Na+-independent cells, where pHi recovery occurred regardless of the presence of Na+ (n = 31). The pHi recovery of Na+-dependent large osteoclasts was only partially sensitive to amiloride (n = 20) or bafilomycin A1 (n = 23) but was inhibited to a much greater extent in the presence of both inhibitors (n = 13). The pHi recovery of Na+-independent cells was significantly inhibited by bafilomycin A1 (n = 15) but not by amiloride (n = 17). The addition of both bafilomycin A1 and amiloride did not enhance the inhibitory effect of bafilomycin A1 alone (n = 5). Results represent means ± SE. # P < 0.01 and * P < 0.001 compared with recovery in the presence of Na+. a P < 0.05 compared with amiloride only. Representative trace of a small osteoclast (B), a large Na+-dependent osteoclast (C), and a large Na+-independent osteoclast (D) recovering from an acid load in the presence of amiloride.

Interestingly, 60% of the 80 large cells studied behaved like small osteoclasts in that they did not recover from an acid load in the absence of Na+, although in contrast to small osteoclasts, the Na+-dependent recovery of these large cells was not completely inhibited by amiloride. These cells were categorized as large, Na+-dependent osteoclasts. However, in the remaining 40% of large osteoclasts, pHi increased at the same rate in the presence or absence of Na+ and was not inhibited by amiloride, suggesting that recovery here was not mediated by the Na+/H+ exchanger. These were categorized as large Na+-independent osteoclasts. Representative traces of a small osteoclast (Fig. 6B), a large Na+-dependent osteoclast (Fig. 6C), and a large Na+-independent osteoclast (Fig. 6D) are shown.

We next evaluated the role of V-ATPase in the recovery from acid load in Na+-containing medium for the three different categories of cells. In small cells, bafilomycin A1 had no effect, suggesting that the proton pump was not responsible for the recovery in pHi (Fig. 6A). In the large, Na+-dependent osteoclasts, addition of bafilomycin A1 alone reduced the recovery rate by ~25%, but this decrease was not significant with the number of osteoclasts measured (P < 0.17, n = 23). However, in these large cells, the addition of both amiloride and bafilomycin A1 resulted in complete inhibition of pHi recovery. The degree of inhibition was significantly different from that by amiloride alone, suggesting that both the Na+/H+ exchanger and the proton pump were active in these cells. In the large, Na+-independent cells, pHi recovery was inhibited by bafilomycin A1, and addition of amiloride did not enhance this effect, suggesting that the proton pump was solely responsible for the pHi recovery in these cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The production and extrusion of protons and the maintenance of pHi are essential processes in the function and activity of osteoclasts. It is widely accepted that V-ATPases found in the ruffled border of osteoclasts pump protons into the extracellular resorption zone, thus acidifying the bone surface and dissolving the mineral component of bone (8, 46, 53). Proton extrusion into the extracellular resorption zone is accompanied by passive Cl- flux through a Cl- channel. The driving force behind Cl- movement is the potential difference that arises from electrogenic proton transport across the ruffled membrane during bone resorption (7, 41, 44). With regard to maintenance of an appropriate pHi, Teti et al. (49) demonstrated that, during bone resorption, one of the mechanisms of alkaline export was an Na+-independent Cl-/HCO3- exchanger. Other reports indicate the presence and function of an amiloride-sensitive Na+/H+ exchanger (20, 21, 31, 35) and a proton conductance (35). However, in many cases, differences in pHi regulatory responses between osteoclasts of the same preparation have been reported, in particular with regard to Na+/H+ exchange and V-ATPase activities for which the investigators have had no explanation or attributed them to differences in substrate, stage of resorption cycle, or cell shape (20, 31, 35, 40, 59, 60).

The first striking finding in the present investigation was the existence of a significant difference (0.4 pH units) in steady-state pHi of small and large osteoclasts maintained in HEPES-buffered Na+-containing medium at pH 7.4. The value obtained for small osteoclasts was in good agreement with that observed in a number of other cell types and with other reports on osteoclasts cultured on glass in nominally HCO3--free, HEPES-buffered medium at pH 7.4 (35, 36, 40, 49, 60). However, large osteoclasts had an unusually high pHi of 7.70. Because Boyarsky and colleagues (10) recently reported that the nigericin technique to calibrate BCECF in vascular smooth muscle cells could lead to errors of up to 0.2 pH units in estimating steady-state pHi, we verified our results using the null-point method. This method has been reported to be more reliable than the nigericin method in some instances because it is independent of K+ concentration, thus eliminating the error involved in estimating intracellular K+ (4, 10, 11, 13, 57). The results of the two methods of calibration were in excellent agreement, indicating that the difference in pHi between large and small osteoclasts is not an artifact of the recording system. Interestingly, Ravesloot et al. (40) in their experiments with rat osteoclasts also found that a small percentage of the cells tested had high initial pHi values, but they did not attempt to associate this with a specific population of osteoclasts. Other reports on osteoclast pHi regulation do not mention the size of osteoclasts studied, although Nordstrom et al. (35) did specifically state that the majority of their studies were performed on compact osteoclasts containing three to five nuclei. The great abundance of small osteoclasts compared with large ones (60 vs. 10%) in rabbit, rat, and chick osteoclast preparations cultured on glass coverslips (our results and Refs. 3 and 39) and the greater ease of performing single-cell experiments with cells of a smaller diameter would suggest that other investigators may also have concentrated mainly on smaller osteoclasts.

The cause of the high basal pHi in large osteoclasts was further investigated. First, we excluded the possibility that the high pHi in large osteoclasts was a result of maintaining the cells in HEPES-buffered medium. Buckler et al. (11) had shown that in type I carotid body cells of neonatal rats the remarkably alkaline pHi (7.8) was due to the transfer of these cells from CO2-containing to HEPES-buffered (CO2-free) medium. They observed that, when measuring pHi within 1 h of transfer, the pHi was 7.8, but if the cells were cultured in HEPES-buffered medium for 24 h, the pHi was 7.3. However, this was not the case for osteoclasts in our study; large osteoclasts cultured for 24 h in HEPES-containing medium had the same alkaline pHi as those cultured for 2 h, whereas small osteoclasts had the same low pHi (7.3) at the two time points (results not shown). It is also relevant in this regard that Teti et al. (49) found that avian osteoclasts attached to bone and transferred to HCO3--free HEPES-containing medium became slightly acidified, not alkaline, within 2 min of transfer and then recovered to resting pHi within 20 min.

We next discovered that, although Na+ was required for large osteoclasts to maintain their alkaline pHi, the Na+/H+ exchanger was not the primary mechanism involved in this process, as addition of amiloride did not lower the resting pHi of large cells. However, amiloride added after Na+ removal slightly inhibited the recovery of these cells (25% inhibition) to their starting pHi upon readdition of Na+ to the extracellular medium, suggesting a small role for the Na+/H+ exchanger. Interestingly, the specific V-ATPase inhibitor bafilomycin A1 inhibited the Na+-dependent alkalinization by 70%, whereas the combination of bafilomycin A1 and amiloride completely inhibited the recovery. The fact that V-ATPase activity was Na+ dependent in these cells was surprising because V-ATPase activity is generally not linked to the presence of Na+ in the extracellular medium. However, Mernissi et al. (33) did report Na+-dependent ATPase activity in the rat nephron that was clearly not a Na+-K+-ATPase, K+-ATPase, or Ca2+-ATPase, but they did not look at pH regulation or bafilomycin sensitivity to determine whether it was a V-ATPase. Taken together, our findings suggest that both small and large osteoclasts cultured on glass contain active proton pumps but that their role in pHi regulation is much more important in large osteoclasts than in small osteoclasts. This was further supported by our observation that the addition of bafilomycin A1 resulted in a significant decrease in basal pHi in large osteoclasts but not in small ones.

One possible explanation for the difference in V-ATPase activities between large and small osteoclasts could be the variation in number or activities of Cl- channels in these cells. Because V-ATPase activity is often charge coupled to passive Cl- permeability, a lack of anion conductance due to insufficient Cl- channels may inhibit further V-ATPase activity due to the electrogenic nature of this pump (9, 44). Thus lower V-ATPase activity in small osteoclasts could be due to fewer Cl- channels in these cells compared with large osteoclasts and not directly as a result of fewer or less active V-ATPases. Various reports have indeed demonstrated the presence of Cl- channels in osteoclasts (7, 9, 29, 41, 44). Interestingly, Sims et al. (44) and Kelly et al. (29) reported that only 30-50% of their osteoclasts had a basal Cl- current; however, they used very high concentrations of the Cl- channel inhibitors DIDS and SITS (0.5-1 mM). Such high concentrations may have inhibited other ion currents as well, including V-ATPases (9). To study Cl- channel activity in our cells, we added 10-100 µM DIDS [shown by Blair et al. (9) to reversibly inhibit Cl- channels in osteoclasts without affecting other proton transport mechanisms] in addition to 200 nM bafilomycin A1. The pHi of small osteoclasts did not change significantly, whereas that of large osteoclasts did decrease, thus indicating that the differences in small and large osteoclast responses to bafilomycin A1 were not caused by differences in Cl- channel activity. Surprisingly, DIDS addition alone resulted in a rise in pHi in both large and small osteoclasts, likely due to inhibition of Cl-/HCO3- exchange activity in these cells. That this transporter continues to be active in our cells is possible, because significant accumulation of HCO3- by carbonic anhydrase activity may continue in osteoclasts maintained in nominally HCO3--free medium as a result of CO2 production in association with glycolysis and ATP generation in the mitochondria (6). The fact that both large and small osteoclasts exhibited a similar increase is further evidence that anion translocation is not different in these cells, only V-ATPase activity.

To further study the characteristics of the pHi regulatory mechanisms in large and small osteoclasts, we next examined the recovery of these cells from an acid load in the presence of various inhibitors. In agreement with previously reported data for osteoclasts (35, 60), the recovery of small osteoclasts from very low pHi was Na+ dependent, amiloride sensitive, and bafilomycin A1 resistant. All of these data suggest a classical Na+/H+ exchanger as the primary mechanism of realkalinization in these cells. The fact that this antiporter was very active in recovery from an acid load in small osteoclasts but was not very active at physiological pH in these cells is consistent with other reports that Na+/H+ exchangers only become active below a set-point pH of ~6.8 (5, 19, 23, 55).

The recovery from an acid load by large osteoclasts, however, was more complex. Surprisingly, 40% of the large osteoclasts recovered at a very fast rate from an acid load in the absence of Na+ (Na+ independent), whereas 60% of the large osteoclasts recovered from an acid load in an Na+-dependent manner (Na+ dependent). The Na+-independent cells did not respond to amiloride but were sensitive to inhibition by bafilomycin A1, indicating that V-ATPase was exclusively responsible for the realkalinization. Ravesloot et al. (40) also described a subpopulation of cells with active proton pumps in rat osteoclasts cultured on glass. Similar to our results, they found that these cells recovered from an acid load in the absence of Na+ and also had a higher mean initial pHi. They attempted to correlate the presence of high initial pHi and recovery from an acid load in the absence of Na+ with cell shape (round and well spread vs. polygonal with cytoplasmic extensions) but concluded that osteoclast shape was not sufficient to explain the differences. They did not report on the number of nuclei seen in their cells. We also examined the shape of our rabbit osteoclasts and found that the great majority of our cells were round and did not display irregularly shaped cytoplasmic extensions as described by Ravesloot et al. (40). This may be due to the fact that rat osteoclasts take on a stellar appearance when transferred to serum-free medium (18), whereas similarly treated rabbit osteoclasts do not (Lees and Heersche, unpublished observations). Ingber et al. (24) and Schwartz et al. (42) reported that spreading of bovine capillary endothelial cells and normal fibroblasts leads to rapid activation of the Na+-H+ antiporter and elevation of pHi. They also found that fully spread cells had a higher pHi than round cells and that local clustering of integrins led to activation of the Na+-H+ antiporter and hence the rise in pHi. The evidence available for osteoclasts suggests that this explanation for the difference in pHi of large and small osteoclasts is not likely, because pHi did not seem to correlate with the degree of spreading but rather with nuclear number.

In the Na+-dependent category of large osteoclasts, pHi recovery was only partially inhibited by amiloride and bafilomycin A1, but when amiloride and bafilomycin A1 were added together, pHi recovery was completely inhibited. The presence and activity of both a V-ATPase and Na+/H+ exchanger in the same cell has also been reported in other cell types, i.e., alveolar macrophages, rabbit outer medullary collecting duct cells, rat papillary collecting duct cells, and guinea pig pancreatic duct cells (5, 12, 23, 48). The findings of De Ondarza and Hootman (12) in guinea pig pancreatic duct cells were similar to ours in that neither amiloride nor bafilomycin A1 alone significantly inhibited pHi recovery from an acid load, whereas both inhibitors added together completely abolished this recovery.

Osteoclasts cultured on bone slices cycle between an actively resorbing phase and a migratory phase (27). Lehenkari et al. (31) reported that active, bone-resorbing osteoclasts regulate their pHi differently from osteoclasts cultured on glass or nonresorbing cells cultured on bone. The active osteoclasts on bone had greater V-ATPase activity than Na+/H+ exchange activity and had a slightly higher pHi than inactive cells. It is tempting to hypothesize that in our experiments the category of large Na+-independent cells that rely on V-ATPase activity exclusively for regulating their pHi represent the actively resorbing population. If large resorbing osteoclasts remain activated when transferred to our culture system, then this could account for our finding of two categories of large osteoclasts. This could imply that small osteoclasts and the category of large Na+-dependent osteoclasts are in an inactive or nonresorbing state. We are currently performing experiments using a system in which we can distinguish between actively resorbing and nonresorbing osteoclasts to test this hypothesis.


    ACKNOWLEDGEMENTS

We thank Dr. Lamara Shrode for technical assistance and Dr. Sergio Grinstein for valuable comments and critical review of the manuscript.


    FOOTNOTES

This work was supported by the Arthritis Society of Canada.

Portions of this work were presented in part as an abstract at the 19th Annual Meeting of the American Society for Bone and Mineral Research, Cincinnati, OH, 1997.

Address for reprint requests and other correspondence: J. N. M. Heersche, Faculty of Dentistry, Rm. 403, Univ. of Toronto, 124 Edward St., Toronto, Ontario, Canada M5G 1G6 (E-mail: johan.heersche{at}utoronto.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 24 November 1999; accepted in final form 27 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Aota, S, Nakamura T, Suzuki K, Tanaka Y, Okazaki Y, Segawa Y, Miura M, and Kikuchi S. Effects of indomethacin administration on bone turnover and bone mass in adjuvant-induced arthritis in rats. Calcif Tissue Int 59: 385-391, 1996[ISI][Medline].

2.   Arkett, SA, Dixon SJ, and Sims SM. Substrate influences rat osteoclast morphology and expression of potassium conductances. J Physiol (Lond) 458: 633-653, 1992[Abstract].

3.   Asotra, S, Gupta AK, Sodek J, Aubin JE, and Heersche JNM Carbonic anhydrase II mRNA expression in individual osteoclasts under "resorbing" and "nonresorbing" conditions. J Bone Miner Res 9: 1115-1122, 1994[ISI][Medline].

4.   Babcock, DF. Examination of the intracellular ionic environment and of ionophore action by null point measurements employing the fluorescein chromophore. J Biol Chem 258: 6380-6389, 1983[Abstract/Free Full Text].

5.   Bidani, A, Brown SES, and Heming TA. pHi regulation in alveolar macrophages: relative roles of Na+-H+ antiport and H+-ATPase. Am J Physiol Lung Cell Mol Physiol 266: L681-L688, 1994[Abstract/Free Full Text].

6.   Blair, HC. How the osteoclast degrades bone. Bioessays 20: 837-846, 1998[ISI][Medline].

7.   Blair, HC, and Schlesinger PH. Purification of a stilbene sensitive chloride channel and reconstitution of chloride conductivity into phospholipid vesicles. Biochem Biophys Res Commun 171: 920-925, 1990[ISI][Medline].

8.   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].

9.   Blair, HC, Teitelbaum SL, Tan H-L, Koziol CM, and Schlesinger PH. Passive chloride permeability charge coupled to H+-ATPase of avian osteoclast ruffled membrane. Am J Physiol Cell Physiol 260: C1315-C1324, 1991[Abstract/Free Full Text].

10.   Boyarsky, G, Hanssen C, and Clyne L. Inadequacy of high K+/nigericin for calibrating BCECF. I. Estimating steady-state intracellular pH. Am J Physiol Cell Physiol 271: C1131-C1145, 1996[Abstract/Free Full Text].

11.   Buckler, KJ, Vaughan-Jones RD, Peers C, and Nye PCG Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat. J Physiol (Lond) 436: 107-129, 1991[Abstract].

12.   De Ondarza, J, and Hootman SR. Confocal microscopic analysis of intracellular pH regulation in isolated guinea pig pancreatic ducts. Am J Physiol Gastrointest Liver Physiol 272: G124-G134, 1997[Abstract/Free Full Text].

13.   Eisner, DA, Kenning NA, O'Neill SC, Pocock G, Richards CD, and Valdeolmillos M. A novel method for absolute calibration of intracellular pH indicators. Pflügers Arch 413: 553-558, 1989[ISI][Medline].

14.   Elleder, M. Enzyme patterns in human endocytotic multinucleate giant cells---a histochemical study. Acta Histochem 79: 1-10, 1986[ISI][Medline].

15.   Enelow, RI, Sullivan GW, Carper HT, and Mandell GL. Cytokine-induced human multinucleated giant cells have enhanced candidacidal activity and oxidative capacity compared with macrophages. J Infect Dis 166: 664-668, 1992[ISI][Medline].

16.   Fais, S, Burgio VL, Silvestri M, Capobianchi MR, Pacchiarotti A, and Pallone F. Multinucleated giant cells generation induced by interferon-gamma. Changes in the expression and distribution of the intercellular adhesion molecule-1 during macrophage fusion and multinucleated giant cell formation. Lab Invest 71: 737-744, 1994[ISI][Medline].

17.   Fischman, DA, and Hay ED. Origin of osteoclasts from mononuclear leucocytes in regenerating newt limbs. Anat Rec 143: 329-334, 1962[ISI].

18.   Fuller, K, Owens JM, Jagger CJ, Wilson A, Moss R, and Chambers TJ. Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts. J Exp Med 178: 1733-1744, 1993[Abstract].

19.   Grinstein, S, and Furuya W. Characterization of the amiloride-sensitive Na+-H+ antiport of human neutrophils. Am J Physiol Cell Physiol 250: C283-C291, 1986[Abstract/Free Full Text].

20.   Gupta, A, Edwards JC, and Hruska KA. Cellular distribution and regulation of NHE-1 isoform of the Na-H exchanger in the avian osteoclast. Bone 18: 87-95, 1996[ISI][Medline].

21.   Hall, TJ, and Chambers TJ. Na+/H+ antiporter is the primary proton transport system used by osteoclasts during bone resorption. J Cell Physiol 142: 420-424, 1990[ISI][Medline].

22.   Hall, TJ, Schaeublin M, and Chambers TJ. The majority of osteoclasts require mRNA and protein synthesis for bone resorption in vitro. Biochem Biophys Res Commun 195: 1245-1253, 1993[ISI][Medline].

23.   Hays, SR, and Alpern RJ. Apical and basolateral membrane H+ extrusion mechanisms in inner stripe of rabbit outer medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 259: F628-F635, 1990[Abstract/Free Full Text].

24.   Ingber, DE, Prusty D, Frangioni JV, Cragoe EJJ, Lechene C, and Schwartz MA. Control of intracellular pH and growth by fibronectin in capillary endothelial cells. J Cell Biol 110: 1803-1811, 1990[Abstract].

25.   Jaworski, ZFG, Duck B, and Sekaly G. Kinetics of osteoclasts and their nuclei in evolving secondary Haversian systems. J Anat 133: 397-405, 1981[ISI][Medline].

26.   Kanehisa, J. Time course of "escape" from calcitonin-induced inhibition of motility and resorption of disaggregated osteoclasts. Bone 10: 125-129, 1989[ISI][Medline].

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

28.   Kaye, M, Zucker SW, Leclerc YG, Prichard S, Hodsman AB, and Barré P-E. Osteoclast enlargement in endstage renal disease. Kidney Int 27: 574-581, 1985[ISI][Medline].

29.   Kelly, MEM, Dixon SJ, and Sims SM. Outwardly rectifying chloride current in rabbit osteoclasts is activated by hyposmotic stimulation. J Physiol (Lond) 475: 377-389, 1994[Abstract].

30.   Lees, RL, and Heersche JNM Macrophage colony stimulating factor increases osteoclastic bone resorption in long-term osteoclast-containing rabbit bone marrow cultures by increasing osteoclast size. J Bone Miner Res 14: 937-945, 1999[ISI][Medline].

31.   Lehenkari, PP, Laitala-Leinonen T, Linna T-J, and Vaananen HK. The regulation of pHi in osteoclasts is dependent on the culture substrate and on the stage of the resorption cycle. Biochem Biophys Res Commun 235: 838-844, 1997[ISI][Medline].

32.   Makris, GP, and Saffar JL. Quantitative relationship between osteoclasts, osteoclast nuclei and the extent of the resorbing surface in hamster periodontal disease. Arch Oral Biol 27: 965-969, 1982[ISI][Medline].

33.   Mernissi, GE, Barlet-Bas C, Khadouri C, Marsy S, Cheval L, and Doucet A. Characterization and localization of ouabain-insensitive Na-dependent ATPase activities along the rat nephron. Biochim Biophys Acta 1064: 205-211, 1991[ISI][Medline].

34.   Nishii, H, Ashitaka Y, Maruo M, and Mochizuki M. Studies on the effect of thyroid hormone and epidermal growth factor on the cultured human cytotrophoblast. Endocrinol Jpn 38: 279-286, 1991[Medline].

35.   Nordstrom, T, Rotstein OD, Romanek R, Asotra S, Heersche JNM, Manolson MF, Brisseau GF, and Grinstein S. Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance. J Biol Chem 270: 2203-2212, 1995[Abstract/Free Full Text].

36.   Nordstrom, T, Shrode LD, Rotstein OD, Romanek R, Goto T, Heersche JNM, Manolson MF, Brisseau GF, and Grinstein S. Chronic extracellular acidosis induces plasmalemmal vacuolar type H+ ATPase activity in osteoclasts. J Biol Chem 272: 6354-6360, 1997[Abstract/Free Full Text].

37.   Owens, J, and Chambers TJ. Macrophage colony-stimulating factor (M-CSF) induces migration in osteoclasts in vitro. Biochem Biophys Res Commun 195: 1401-1407, 1993[ISI][Medline].

38.   Papadimitriou, JM, and Van Bruggen I. Evidence that multinucleate giant cells are examples of mononuclear phagocytic differentiation. J Pathol 148: 149-157, 1986[ISI][Medline].

39.   Piper, K, Boyde A, and Jones SJ. The relationship between the number of nuclei of an osteoclast and its resorptive capability in vitro. Anat Embryol (Berl) 186: 291-299, 1992[ISI][Medline].

40.   Ravesloot, JH, Eisen T, Baron R, and Boron WF. Role of Na-H exchangers and vacuolar H+ pumps in intracellular pH regulation in neonatal rat osteoclasts. J Gen Physiol 105: 177-208, 1995[Abstract].

41.   Schlesinger, PH, Blair HC, Teitelbaum SL, and Edwards JC. Characterization of the osteoclast ruffled border chloride channel and its role in bone resorption. J Biol Chem 272: 18636-18643, 1997[Abstract/Free Full Text].

42.   Schwartz, MA, Lechene C, and Ingber DE. Insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integrin alpha beta , independent of cell shape. Proc Natl Acad Sci USA 88: 7849-7853, 1991[Abstract].

43.   Shibutani, T, and Heersche JNM Effect of medium pH on osteoclast activity and osteoclast formation in cultures of dispersed rabbit osteoclasts. J Bone Miner Res 8: 331-336, 1993[ISI][Medline].

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

45.   Singer, FR, and Roodman GD. Paget's disease of bone. In: Principles of Bone Biology, edited by Bilezikian JP, Raisz LG, and Rodan GA.. San Diego, CA: Academic, 1996, p. 969-977.

46.   Sundquist, K, Lakkakorpi P, Wallmark b., and Vaananen K. Inhibition of osteoclast proton transport by bafilomycin A1 abolishes bone resorption. Biochem Biophys Res Commun 168: 309-313, 1990[ISI][Medline].

47.   Swallow, CJ, Grinstein S, and Rotstein OD. Regulation of cytoplasmic pH in resident and activated peritoneal macrophages. Biochim Biophys Acta 1022: 203-210, 1990[ISI][Medline].

48.   Takeda, K, Oohara T, Tabel K, and Asano Y. Dual regulatory mechanisms of proton transport in rat papillary collecting duct cells in culture. Jpn J Physiol 39: 397-410, 1989[ISI][Medline].

49.   Teti, A, Blair HC, Teitelbaum SL, Kahn AJ, Koziol C, Konsek J, Zambonin-Zallone A, and Schlesinger PH. Cytoplasmic pH regulation and chloride/bicarbonate exchange in avian osteoclasts. J Clin Invest 83: 227-233, 1989[ISI][Medline].

50.   Thomas, JA, Buchsbaum RN, Zimniak A, and Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18: 2210-2218, 1979[ISI][Medline].

51.   Turksen, K, Kanehisa J, Opas M, Heersche JNM, and Aubin JE. Adhesion patterns and cytoskeleton of rabbit osteoclasts on bone slices and glass. J Bone Miner Res 3: 389-400, 1988[ISI][Medline].

52.   Vaananen, K. Osteoclast function: biology and mechanisms. In: Principles of Bone Biology, edited by Bilezikian JP, Raisz LG, and Rodan GA.. San Diego, CA: Academic, 1996, p. 103-113.

53.   Vaananen, 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].

54.   Vignery, A, Wang F, Qian HY, Benz EJ, Jr, and Gilmore-Hebert M. Detection of the Na+-K+-ATPase alpha 3-isoform in multinucleated macrophages. Am J Physiol Renal Fluid Electrolyte Physiol 260: F704-F709, 1991[Abstract/Free Full Text].

55.   Wakabayashi, S, Fafournoux P, Sardet C, and Pouyssegur J. The Na+/H+ antiporter cytoplasmic domain mediated growth factor signals and controls "H+-sensing". Proc Natl Acad Sci USA 89: 2424-2428, 1992[Abstract].

56.   Woods, C, Domenget C, Solari F, Gandrillon O, Lazarides E, and Jurdic P. Antagonistic role of vitamin D3 and retinoic acid on the differentiation of chicken hematopoietic macrophages into osteoclast precursor cells. Endocrinology 136: 85-95, 1995[Abstract].

57.   Yamashiro, DJ, and Maxfield FR. Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells. J Cell Biol 105: 2713-2721, 1987[Abstract].

58.   Yu, H, and Ferrier J. Interleukin-1 alpha induces a sustained increase in cytosolic free calcium in cultured rabbit osteoclasts. Biochem Biophys Res Commun 191: 343-350, 1993[ISI][Medline].

59.   Yu, H, and Ferrier J. Osteoclast ATP receptor activation leads to a transient decrease in intracellular pH. J Cell Sci 108: 3051-3058, 1995[Abstract/Free Full Text].

60.   Zimolo, Z, Weslowski G, and Rodan GA. Acid extrusion is induced by osteoclast attachment to bone. Inhibition by alendronate and calcitonin. J Clin Invest 96: 2277-2283, 1995[ISI][Medline].


Am J Physiol Cell Physiol 279(3):C751-C761
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society