Expression of vacuolar H+-ATPase in osteoclasts and its role in resorption

By means of light- and electron-microscopic immunocytochemistry, we have demonstrated the expression of vacuolar H⁺-ATPase in mouse osteoclasts. In fully differentiated osteoclasts, intense immunolabeling was observed along the plasma membranes including those of ruffled borders and associated pale vesicles and vacuoles, whereas those of clear zones and basolateral cell surfaces were entirely free of immunoreaction. Specific expression of vacuolar H⁺-ATPase was also detected over polyribosomes and cisterns of the rough-surfaced endoplasmic reticulum. Multinucleated osteoclastic cells were suspended on dentine slices and cultured for 48 h in the presence or absence of either concanamycin B or bafilomycin A1, specific inhibitors of vacuolar H⁺-ATPase. Morphometric analysis of co-cultured dentine slices with backscattered electron microscopy revealed that both inhibitors strongly reduced the formation of resorption lacunae in a dose-dependent manner. These results suggest that vacuolar H⁺-ATPase is produced in the rough-surfaced endoplasmic reticulum, stored in the membrane vesicles, and transported into the ruffled border membranes of osteoclasts, and that this enzyme plays a key role in the creation of an acidic subosteoclastic microenvironment for the demineralization of co-cultered substrates.     

Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V(1) and V(0). The peripheral cytoplasmically oriented V(1) domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V(0) domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V(0) domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c', and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c', and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V(0) domain and efficient osteoclastic bone resorption.     

Osteoclast-independent bone resorption by fibroblast-like cells

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.     

Localization of osteopontin in resorption lacunae formed by osteoclast-like cells: a study by a novel monoclonal antibody which recognizes rat osteopontin

The characteristics of a monoclonal antibody produced against osteoclast-like multinucleated cells (MNCs) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. The in vitro immunization was performed using as immunogen the MNCs from rat bone marrow cell culture, which revealed many characteristics of osteoclasts. After screening and cloning of hybridomas, the monoclonal antibody HOK 1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNCs and their putative migratory traces on culture dishes. Immunofluorescent examination of paraffin sections revealed intense reactivity on the epithelium of the choroid plexus, the ileum and the proximal-convoluted tubules of the kidney, and also on bone cells such as osteocytes, osteoblasts, and osteoclasts. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK 1 was osteopontin. Positive HOK 1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNCs on human tooth slices and on the surface of osteoclasts. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts under a specific state might trap this protein on their cell surface.     

Immunohistochemical localization of membrane type 1-matrix metalloproteinase (MT1-MMP) in osteoclasts in vivo

Membrane type 1-matrix metalloproteinase (MT1-MMP) is capable of mediating proteolysis of extracellular matrix. The enzyme has been demonstrated in osteoclasts, in vitro. However, the precise localization in vivo, and therefore the function of the enzyme in osteoclasts, is still unclear. In this study, we immunohistochemically examined the localization of MT1-MMP in rat osteoclasts to clarify the role of MT1-MMP in osteoclastic bone resorption and bone turnover. The localization of MT1-MMP was visualized by the pre-embedding method using anti-MT1-MMP antibody and horseradish peroxidase (HRP) or gold-conjugated antibody. Immunoreactivity of anti-MT1-MMP was found in osteoclasts at the osteoclast-bone interface, but it was not uniform. Ultrastructurally, the immunoreactivity visualized by HRP was found in sealing zone. The plasma membrane at this site showed an irregular border and some invaginations. Immunoreactivity was also found on the surface of certain small vesicles in the cytoplasm. Enhanced silver granules were mainly associated with the sealing membrane. In this study, we demonstrated, for the first time, the localization of MT1-MMP in the sealing zone of osteoclast in vivo. Its distribution suggests that the enzyme modifies the bone surface to facilitate the migration and attachment of osteoclasts as well as scavenging the resorption lacunae.     

The vacuolar ATPase in bone cells: a potential therapeutic target in osteoporosis

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that couples ATP hydrolysis to proton pumping across membranes. Recently, there is increasing evidence that V-ATPase may contribute to the pathogenesis of bone resorption disorders due to it is predominantly expressed in osteoclasts also function in bone resorption making it a good candidate in a therapeutic target for osteoporosis. Osteoclasts are capable of generating an acidic microenvironment necessary for bone resorption by utilizing V-ATPases to pump protons into the resorption lacuna. In addition, it has been shown that therapeutic interventions have been proposed that specifically target inhibition of the osteoclast proton pump. Modulation of osteoclastic V-ATPase activity has been considered to be a suitable therapy for the treatment of osteoporosis. All theses findings suggest that V-ATPase have important biological effects in bone resorption that might be a promising therapeutic target for osteoporosis. In this review, we will briefly discuss the biological features of osteoporosis and summarize recent advances on the role of V-ATPase in the pathogenesis and treatment of osteoporosis.     

Immunohistochemical localization of cathepsins B,D and L in the rat osteoclast

Immunohistochemical localization of cathepsins B, D and L in the osteoclasts of rat alveolar and femoral bones was investigated by using the avidin-biotin-peroxidase complex method for semithin, 1-m-thick cryosections. Extracellular immunoreactivity for cathepsins B and L was clearly demonstrated along the bone resorption lacunae; the intensity of the extracellular immunoreactivity of cathepsin L was stronger than that of cathepsin B. However, the intracellular immunoreactivity of both cathepsins was weak compared with that of cathepsin D. The intracellular immunoreactivity of cathespin D in the osteoclasts was clearly observed in the granules and/or vacuoles, but extracellular cathepsin D immunoreactivity was either negligible or not detected along the resorption lacunae. In the adjacent sections stained with anti-cathepsin L or D, extensive extracellular deposition of cathepsin L was found along the bone resorption lacunae, with or without osteoclasts, although the intracellular reactivity of cathepsin L was weak. This is the first morphological study in which cathepsins B and L have been demonstrated to be produced in the osteoclasts and extensively secreted into resorption lacunae, and in which cathepsin D was found to be present in the cells but scantily secreted into the lacunae. These findings suggest that cathepsins B and L directly and effectively participate in the degradation of the bone matrix.     

Myosin II antibodies inhibit the resorption activity of isolated rat osteoclasts

We present microinjection data in support of an indirect approach by which cytoplasmic protein interactions important in the processes of bone resorption can be elucidated. Three polyclonal antibodies (M1, M3, M5) raised against myosin II from perfused rat liver differently affected the actin-activated Mg ATPase of myosin II. These antibodies microinjected into isolated rat osteoclasts affected osteoclast morphology and activity in bone resorption. M1, which completely inhibited myosin ATPase activity at a antibody:myosin ratio of 10:1, initially promoted the extension/retraction motility of lamellipodia but eventually reduced the spread area of osteoclasts along the substrate after 20 hr. M3, which inhibited ATPase activity by 70%, had similar effects; however, M5, which weakly inhibited ATPase activity, neither promoted extension/retraction nor reduced spread area of osteoclasts. Immunofluorescence showed that these antibodies removed myosin II from the majority of actin filaments in injected osteoclasts. Because antibodies that did not bind to a myosin II column had little effect on the extension/retraction of lamellipodia or the osteoclast spread area, these data suggest that myosin II participates in the stabilization of osteoclast lamellipodia along the substrate. M1 injection strongly inhibited injected osteoclasts from excavating resorption lacunae in bone slices, compared to control antibody. M3 and M5 were less effective but also inhibited bone resorption. These data show that myosin II is functionally important in bone resorption and that the osteoclast-differentiated activity of bone resorption is a more sensitive assay for myosin activity than lamellipodia motility or cell morphology.     

Osteoclast activation: Potent inhibition by the bisphosphonate alendronate through a nonresorptive mechanism

Alendronate, an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its mechanism of action is unknown. Because it localizes to bone surfaces, we compared the sensitivity of components of the resorptive process to incubation on alendronate-coated bone surfaces. We found that bone resorption by osteoclasts isolated from neonatal rat bone was unaffected by alendronate (10^-4 M). Osteoclast production in bone marrow cultures, as assessed by the production of calcitonin-receptor positive cells, was observed even at 10^-4 M, but bone resorption in these cultures was almost completely abolished by 10^-6 M alendronate. The greater sensitivity of osteoclast activation to inhibition by alendronate that these results suggest was supported by similar inhibition of osteoblast-mediated activation of osteoclasts from neonatal rat bone. Thus, activation of osteoclasts by osteoblastic/stromal cells is apparently the most sensitive component of the pathway whereby bone resorption is affected. Moreover, the ability of alendronate to suppress osteoclastic activation does not depend on resorption-mediated release of alendronate from bone surfaces. This ability extends the range of cell types and processes that might be affected by alendronate, beyond those in the immediate vicinity of resorbing cells, to include any cell that comes into contact with alendronate-coated bone surfaces. J. Cell. Physiol. 172:79–86, 1997. © 1997 Wiley-Liss, Inc.     

Minocycline impairment of both osteoid tissue removal and osteoclastic resorption in a synchronized model of remodeling in the rat

In addition to their antibacterial effects, tetracyclines may inhibit interstitial collagenase activity and bone resorption. These properties were assessed morphometrically using minocycline (25 and 50 mg/kg/day given by the IM route) in a rat model of synchronized remodeling in which osteoclastic resorption peaks 4 days after the activating event (the extractions of the upper molars) along the antagonist mandibular cortex, a zone undergoing physiologically active formation. During the first 2 days of activation, minocycline at the two doses impaired very significantly the disorganization of both the osteoid seam and the layer of osteoblasts, a prerequisite to give osteoclasts access to the mineralized bone surface. The number of readily identifiable osteoblasts decreased slightly during this period, suggesting that minocycline prevented their transformation into lining cells. Their synthetic activity, as estimated by the size of the cells and their nucleus, appeared relatively preserved too, mostly with the higher dose. At the peak of osteoclascia, the bone surfaces undergoing remodeling were significantly decreased in the minocycline-treated groups. The resorption surface was reduced (P < 0.0003) as well as the number of osteoclasts (P < 0.0007), which were also significantly smaller. Their resorbing activity was dramatically affected as well: they excavated lacunae whose area was significantly reduced by over 70%. In addition, formation was still a prominent activity in the treated animals. These data are compatible with the inhibition at the early stages of activation of an osteoblast-secreted collagenase whose action may be the elimination of the osteoid seam. The inhibition of an osteoclast collagenase and/or of a bone matrix bound-collagenase may be responsible for the reduction in lacunar size. A direct effect of minocycline on osteoclast resorptive activity may also participate in the low resorption profile, as tetracyclines are known to interfere with the intracellular [Ca²⁺]. © 1996 Wiley-Liss, Inc.     

Secretion of tartrate-resistant acid phosphatase by osteoclasts correlates with resorptive behavior

There have been dramatic advances recently in our understanding of the regulation of osteoclastic differentiation. However, much less is known of the mechanisms responsible for the induction and modulation of resorptive behavior. We have developed a strategy whereby osteoclasts can be generated in vitro and released into suspension in a fully-functional state. We now exploit this approach to show that tartrate-resistant acid phosphatase (TRAP) is released by osteoclasts during bone resorption. TRAP release was inhibited by the secretion-inhibitor Brefeldin A, and was not accompanied by LDH release. This suggests that TRAP release is due to secretion, rather than cell death. Consistent with this, TRAP secretion was stimulated by resorbogenic cytokines, was inhibited by the resorption-inhibitor calcitonin, and correlated with excavation of the bone surface. We found that, in contrast to incubation on bone, incubation on plastic, glass, or vitronectin-coated plastic substrates did not induce secretion of TRAP. This suggests that the induction of resorptive behavior in osteoclasts depends upon stimulation by bone matrix of a putative osteoclastic "mineral receptor." Release of TRAP by osteoclasts thus represents not only a productive approach to the analysis of the mechanisms that modulate the rate of resorptive activity, but also a system whereby the mechanism through which bone substrates induce resorptive behavior can be identified.     

Influence of metal ion solutions on rabbit osteoclast activities in vitro

The purpose of the present study was to compare the effects of various metal ions (aluminium, chromium, cobalt, gold, iron, strontium, titanium and vanadium) on rabbit osteoclast activities, with respect to their number, size, resorptive capacity and their capacity to release proteinases. Marked heterogeneous osteoclastic behaviour was observed early in culture with metal ions (24 h) in term of resorption parameters. In contrast, protease activities (cysteine-proteinase and metalloproteinase activities) were not modulated in our culture conditions. Aluminium, iron, gold and titanium reduced the number of osteoclasts significantly. Aluminium and gold had no effect on osteoclast-mediated resorption on dentin-slices, although aluminium induced a greater number of very small lacunae. Titanium reduced only the mean surface area per lacunae, cobalt reduced the mean surface area of lacunae and increased their number, and iron reduced both parameters. Strontium had no effect on osteoclast formation and on total dentin slice surface resorbed. However, strontium increased the number of small lacunae formed on dentin-slices by osteoclasts. Chromium had no effect on osteoclast activities. These findings indicate that metal ions induce very early effects on osteoclasts, which can contribute to periprosthetic pathologies via different cellular mechanisms.     

A new superoxide-generating oxidase in murine osteoclasts

Superoxide production contributes to osteoclastic bone resorption. Evidence strongly indicates that NADPH oxidase is an enzyme system responsible for superoxide generation in osteoclasts. A membrane-bound subunit, p91, is the catalytic domain of NADPH oxidase. However, osteoclasts from p91 knockout mice still produce superoxide at a rate similar to that observed in wild type mice. This unexpected phenomenon prompted us to examine the osteoclasts for an alternative to the p91-containing oxidase. In this study, the cloning of a NADPH oxidase subunit (Nox 4) with 578 amino acids is reported. Nox 4 has 58% similarity in amino acids with the known p91 subunit of NADPH oxidase. Nox 4 is present and active in osteoclasts. Antisense oligonucleotides of Nox 4 reduced osteoclastic superoxide generation as well as resorption pit formation by osteoclasts. This new oxidase complex was present and functional in osteoclasts from p91 knockout mice, explaining the normal resorptive activity seen in the osteoclasts where no p91 is present.     

Bone resorption and bone remodelling in juvenile carp, Cyprinus carpio L.

The present study considers the important role of bone resorption for bone growth in general, and aims to clarify if and how bone resorption contributes to the skeletal development of carp, Cyprinus carpio L., a teleost species with ‘normal’ osteocyte‐containing (cellular) bone. To ensure the identification of osteoclasts and sites of bone resorption independently from the morphology of the bony cells, bones were studied by histological procedures, and by demonstration of the enzymes which serve as osteoclast markers, viz. tartrate resistant acid phosphatase (TRAP), ATPase and a vacuolar proton pump. Two types of bone‐resorbing cells were observed in juvenile carp: (1) multinucleated giant cells displaying morphological and biochemical attributes which are known from mammalian osteoclasts; and (b) flat cells which lack a visible ruffled border and for which identification requires the performance of enzyme histochemical procedures. Bone resorption performed by osteoclasts mainly occurs at endosteal bone surfaces. To a lesser extent, bone resorption also takes place at periosteal bone surfaces, but without an apparent connection to bone growth. The latter observation, and the occurrence of bone remodelling, suggest that the endoskeleton of juvenile carp might be involved in mineral metabolism. Morphological differences and biochemical similarities to bone resorption in teleosts with acellular bone are discussed.     

Parathyroid hormone (PTH) and PTH-like protein (PLP) stimulate interleukin-6 production by osteogenic cells: a possible role of interleukin-6 in osteoclastogenesis

Osteogenic cells mediate PTH-stimulated osteoclastic bone resorption by a yet unidentified mechanism. We show that primairy rat osteoblast-like cells and the clonal osteogenic sarcoma cell line UMR-106 produce interleukin-6 (IL-6) and that bPTH(1-84) and synthetic hPLP(1-34) stimulate this production dose-dependently. With both peptides a close relation between IL-6 and cyclic-AMP production was found, though for PTH concentrations higher than 2.10(-8) M a clear dissociation was observed. Significant IL-6 activity was also detected in media of cultures of 17-day-old fetal mouse radii and metacarpals which was clearly stimulated by PTH. The source of IL-6 in these bone explants seems to be the osteogenic (cartilage) cells. Treatment of bone explants with IL-6 induced osteoclastic resorption which, however, depended on the bone resorption system used. This bone resorbing action of IL-6 is exerted probably through an effect on the formation of osteoclasts (osteoclastogenesis) rather than on the activation of already existing mature osteoclasts. We suggest that IL-6 produced by osteogenic cells may be a mediator in PTH-stimulated osteoclastic bone resorption.     

Rac-GTPase, osteoclast cytoskeleton and bone resorption.

The members of the Rho-GTPase subfamily, Rac1 and Rac2, are intimately involved in the organization of the cytoskeleton, and the p21-activated kinases or PAKs are targets of these proteins. Rac1 and Rac2 are also essential components of NADPH oxidase, the enzyme responsible for generating free radicals. The cytoskeleton modulates the adhesion of osteoclasts to bone and its subsequent resorption. These cells contain NADPH diaphorase activity, and free radicals influence bone resorption. The influence of Rac1, Rac2 and PAK1 on the cytoskeleton, resorbing activity and NADPH diaphorase activity of disaggregated rat osteoclasts was investigated by permeabilisation with saponin and introducing specific anti-Rac1, anti-Rac2 or anti-PAK1 antibodies. Rhodamine-phalloidin stain was used to identify actin in osteoclasts cultured on plastic slides, and the bone-slice method was used to measure resorption. Saponin permeabilisation did not affect the cytoskeletal organization or bone resorption. Anti-Rac antibodies caused dose- and time-dependent cytoskeletal changes. The osteoclasts rounded up and developed retraction fibers; actin rings were disrupted and large actin dots were seen at the periphery of the cells. Osteoclast resorptive activity was depressed after incubation with the antibodies. The total area resorbed by treated cells and the mean pit area were smaller than those of controls. Anti-PAK1 antibody caused similar changes. None of the antibodies altered the NADPH diaphorase activity. Thus, Rac-GTPases are present in rat osteoclasts and are involved in the organization of the actin cytoskeleton and in resorptive activity. These effects may be mediated by PAK1 kinase, but do not influence osteoclast NADPH diaphorase activity.     

Immunocytochemical localization of cathepsin D in the rat osteoclast.

We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 micron) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsin-negative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

The in vitro effect of pH on osteoclasts and bone resorption in the cat: implications for the pathogenesis of FORL

Dental disease due to osteoclast over-activity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline osteoclastic resorptive lesions (FORL) involve extensive resorption of the tooth leaving it liable to root fracture and subsequent tooth loss. The aetio-pathogenesis of FORL is not known. Recent work has shown that systemic acidosis causes increased osteoclast activation and that loci of infection or inflammation in cat mouth are likely to be acidotic. To investigate this, we generated osteoclasts from cat blood and found that they formed in large numbers (approximately 400) in cultures on bovine cortical bone slices. Acidosis caused an increase in the size of cells-in cultures maintained up to 14 days at basal pH 7.25, mean osteoclast area was 0.01 +/- 0.003 mm(2), whereas an 8.6-fold increase was observed in cells cultured between 11 and 14 days at pH 7.15 (0.086 +/- 0.004 mm(2)). Acidosis caused a modest increase in the number of osteoclasts. Exposure to pH 6.92 exhibited a 5-fold increase in the area of bone slices covered by resorption lacunae ( approximately 70% bone slice resorbed). In line with this finding, significant increases were observed in the expression of cathepsin K and proton pump enzymes (both approximately 3-fold) that are key enzymes reflective of resorptive activity in osteoclasts. These results demonstrate that acidosis is a major regulator of osteoclast formation and functional activation in the cat, and suggest that local pH changes may play a significant role in the pathogenesis of FORL.     

Na+/H(+)-antiporter activity is essential for the induction, but not the maintenance of osteoclastic bone resorption and cytoplasmic spreading.

We have examined the kinetics of the effects of inhibitors of the Na+/H(+)-antiporter (dimethylamiloride) and the vacuolar H(+)-ATPase (bafilomycin A1) on bone resorption by disaggregated rat osteoclasts in the bone slice assay. Bafilomycin A1 (100 nM) inhibited resorption by approximately 95%, 75%, 80% and 60% respectively, when added at t = 0, 1, 3 or 6 hr after osteoclast adherence to bone slices, during a 24 hr culture period. The incomplete inhibition by bafilomycin A1 when added after the start of incubation was presumably accounted for by resorption that had occurred prior to addition of the compound. Dimethylamiloride (100 microM) inhibited bone resorption by 80% and 65% when added at t = 0 or 1 hr after osteoclast adherence, but was without effect when added at t = 3 or 6 hr. In addition, dimethylamiloride but not bafilomycin A1 strongly inhibited osteoclast cytoplasmic spreading. The results indicate that Na+/H(+)-antiporter activity is essential for controlling intracellular pH during early activation events stimulated by the adherence of osteoclasts to mineralized bone surfaces, which lead to cytoskeletal activation, cell spreading and bone resorption.     

Osteoclast biology in the osteopetrotic (op) rat

Osteopetrosis is a metabolic bone disease characterized by reduced bone resorption. From experimental studies of various osteopetrotic mutations has emerged the hypothesis that each is unique with respect to mechanisms whereby osteoclast development and/or function are reduced. The osteopetrotic (op) mutation in the rat was discovered in Fatty/ORL stock over a decade ago. The paucity of data about osteoclast biology in this mutation prompted this study of cytological, cytochemical, and ultrastructural features of osteoclasts. In op rats, osteoclasts are significantly reduced in number, but are larger and more vacuolated than in normal littermates. Mutant osteoclasts can form ruffled borders and clear zones, but their ability to fragment and excavate bone surfaces is greatly impaired. Cytoplasmic vacuoles in op osteoclasts are randomly distributed and greatly enlarged, and they stain weakly for two cytochemical characteristics of osteoclasts, tartrate-resistant acid phosphatase and acid ATPase. These findings suggest that an abnormality in the lysosomal/vacuolar system, an important component of the resorptive mechanism, may be involved in the interception of osteoclast function in this mutation.