Carbonic anhydrase XIV: luminal expression suggests key role in renal acidification

BACKGROUND: Carbonic anhydrase (CA) plays a fundamental role in regulation of systemic acid-base homeostasis by facilitating urinary acidification. Four CA isozymes (CA II, IV, XII, XIV) have been identified in kidney. Until now, luminal CA IV, a GPI-anchored isozyme, was thought to mediate most bicarbonate absorption. Although CA XIV mRNA has been demonstrated in mouse and human kidney, the localization of this newly discovered CA has not been established. METHODS: RT-PCR and Western blot analyses were used to demonstrate CA XIV mRNA and protein in extracts of cortex and medulla of mouse kidney. Polyclonal antibodies against mouse CA XIV were utilized for immunofluorescence to examine the pattern of expression of CA XIV in the nephron of both rat and mouse kidney. RESULTS: Immunofluorescence staining showed abundant expression of CA XIV in apical plasma membranes of the S1 and S2 segments of proximal tubules, and weaker staining in the basolateral membranes. Also, strong staining was seen in the initial portion of the thin descending limb of Henle. These results show that luminal CA XIV is strongly expressed in regions of the rodent nephron that have been thought to be important in urinary acidification. Staining for CA XIV and CA IV in the same sections showed some areas of co-expression, but also some areas where each was expressed without the other. CONCLUSIONS: Luminal CA XIV may account for a substantial fraction of the bicarbonate reabsorption previously attributed to CA IV. If so, CA XIV and CA IV may be functionally redundant.     

Inhibition of the classical NF-κB pathway prevents osteoclast bone-resorbing activity

The classical NF-κB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-κB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-κB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-κB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p < 0.001) and depths (p < 0.05) of pits. The reduced resorption by the peptide was due to reduced osteoclast bone-resorbing activity, but not reduced differentiation as the number of osteoclasts was similar in all groups. The peptide inhibited bone resorption by reducing TRAP activity, disrupting actin rings and preventing osteoclast migration. Gene expressions of a panel of bone resorption markers were significantly reduced. The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption. Therefore, these data suggest that the classical NF-κB pathway plays a pivotal role in osteoclast bone-resorbing activity.     

Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts

Osteoclasts are multinucleated cells that carry out bone resorption. Analysis of the direct effect of hormones on the bone-resorbing activity of human osteoclasts has been limited by difficulties in isolating these cells from the human skeleton. In this study, human osteoclasts formed from cultures of peripheral blood mononuclear precursors (PBMCs) on a Type-I collagen gel were isolated by collagenase treatment for investigating their resorptive activity. PBMCs were cultured in the presence of M-CSF, soluble RANKL, dexamethasone, and 1,25(OH)2D3. The isolated multinucleated cells expressed the osteoclast markers, TRAP, VNR, cathepsin K, calcitonin receptors and were capable of extensive lacunar resorption. Calcitonin inhibited the motility and resorptive activity of osteoclasts. RANKL significantly stimulated osteoclast resorption, but 1,25(OH)2D3, PTH, and OPG did not. These findings indicate that calcitonin and RANKL act directly on human osteoclasts to inhibit and stimulate osteoclast bone-resorbing activity, respectively, and that PTH, 1,25(OH)2D3, and OPG are more likely to influence osteoclast activity indirectly. This technique of human osteoclast isolation should permit the effects of cellular and hormonal/humoral factors on the bone-resorbing activity of mature human osteoclasts to be assessed independently of any effect such factors have on osteoclast formation. It should also make it possible to examine directly the resorptive activity and other characteristics of osteoclasts in specific bone disorders such as Paget's disease.     

An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors.

The present study provides a novel assay system to examine the differentiation of osteoclast progenitors on devitalized bone slices. We used the population of bone cells liberated enzymatically from 14-day-old mouse embryonal calvariae as a source of osteoclast progenitors. The analysis of differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts was assessed in terms of the formation of TRAP-positive cells and pits or resorption lacunae, respectively, on devitalized bone slices. Osteoclasts having bone-resorbing activity appeared when the calvarial cell population was cultured in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. The resorbing activity increased in a 1 alpha,25-(OH)2D3 dose-related manner. However, calcitonin, a potent inhibitor of differentiation and activation of osteoclast lineage cells, reduced the area of the resorption lacunae in a dose-dependent fashion. The bone-resorbing cells on the bone slices expressed an obvious ruffled border and clear zone, structures specific to mature osteoclasts. These results suggest that osteoclast progenitors in the mouse calvarial population examined differentiated into mature osteoclasts in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. Further, using this assay system we assessed the effect of some other osteotropic factors on the differentiation of osteoclast progenitors to mature osteoclasts. IL-1, IL-6, and PTH increased the formation of TRAP-positive cells and pits and the area of resorption lacunae in a dose-dependent fashion. However, prostaglandin E2 was unable to induce the formation of resorption lacunae, although a significant appearance of TRAP-positive cells was observed at a concentration of 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of adjuvant-induced arthritic bone destruction by cyclooxygenase-2 selective agents with and without inhibitory potency against carbonic anhydrase II.

In vitro assays revealed that COX-2 inhibitors with CA II inhibitory potency suppressed both differentiation and activity of osteoclasts, whereas that without the potency reduced only osteoclast differentiation. However, all COX-2 inhibitors similarly suppressed bone destruction in adjuvant-induced arthritic rats, indicating that suppression of osteoclast differentiation is more effective than that of osteoclast activity for the treatment. INTRODUCTION: Cyclooxygenase (COX)-2 and carbonic anhydrase II (CA II) are known to play important roles in the differentiation of osteoclasts and the activity of mature osteoclasts, respectively. Because several COX-2 selective agents were recently found to possess an inhibitory potency against CA II, this study compared the bone sparing effects of COX-2 selective agents with and without the CA II inhibitory potency. MATERIALS AND METHODS: Osteoclast differentiation was determined by the mouse co-culture system of osteoblasts and bone marrow cells, and mature osteoclast activity was measured by the pit area on a dentine slice resorbed by osteoclasts generated and isolated from bone marrow cells. In vivo effects on arthritic bone destruction were determined by radiological and histological analyses of hind-paws of adjuvant-induced arthritic (AIA) rats. RESULTS: CA II was expressed predominantly in mature osteoclasts, but not in the precursors. CA II activity was inhibited by sulfonamide-type COX-2 selective agents celecoxib and JTE-522 similarly to a CA II inhibitor acetazolamide, but not by a methylsulfone-type COX-2 inhibitor rofecoxib. In vitro assays clearly revealed that celecoxib and JTE-522 suppressed both differentiation and activity of osteoclasts, whereas rofecoxib and acetazolamide suppressed only osteoclast differentiation and activation, respectively. However, bone destruction in AIA rats was potently and similarly suppressed by all COX-2 selective agents whether with or without CA II inhibitory potency, although only moderately by acetazolamide. CONCLUSIONS: Suppression of osteoclast differentiation by COX-2 inhibition is more effective than suppression of mature osteoclast activity by CA II inhibition for the treatment of arthritic bone destruction.     

Acid pH Increases Carbonic Anhydrase II and Calcitonin Receptor mRNA Expression in Mature Osteoclasts

Numerous resorptive stimuli have been shown to enhance osteoclast differentiation, increasing osteoclast numbers and accelerating bone resorption. Currently, there is much less understanding of regulation of mature osteoclast activity. Indeed, there is presently only minimal evidence of changes in gene expression as a mechanism for altering bone resorption. We investigate here, in the mature osteoclast, regulation of 2 genes—carbonic anhydrase II (CAII) and calcitonin receptor (CTR) in response to acidosis, which is known to increase bone resorption. We studied the effect of acid pH on CAII and CTR mRNA expression in mature osteoclasts raised in coculture of ST-2 and primary marrow cells. On day 6 of culture, stromal cells were removed with collagenase, the remaining osteoclasts were incubated overnight, and then exposed to varying pH. RT-PCR was performed on total RNA using primers for CAII, CTR, or glyceraldehyde dehydrogenase phosphate (GAP). Expression of CTR mRNA was increased 2.14 ± 0.41 and 2.56 ± 0.45 (P < 0.05)-fold by a 4-hour exposure to pH 6.75 and 6.5, respectively. CAII mRNA was similarly increased 2.18 ± 0.42 and 2.63 ± 0.48 (P < 0.05)-fold by pH 6.75 and 6.5, respectively. Increased expression of CAII and CTR mRNA was seen by 2 hours and maximally by 4 hours. Increased expression of CTR and CAII mRNA was not explained by increases in osteoclast numbers: pH 7.4–100 ± 3.7, 6.75–133 ± 8.3, 6.5–124 ± 7.8. These results demonstrate upregulation of two osteoclast genes in response to acidosis, illustrating the ability of the mature osteoclast to respond to resorptive signals with increased functional gene expression. Received: 28 June 1999 / Accepted: 4 February 2000     

Cellular biology and biochemical mechanism of bone resorption. A review of recent developments on the formation, activation, and mode of action of osteoclasts

The newest knowledge on the osteoclast allows us to consider bone resorption in a global perspective, as the resultant of three successive steps that may each be individually regulated by physiopathologic or pharmacologic agents. The first involves the formation of osteoclast progenitors in hematopoietic tissues followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone. The second consists in the activation of osteoclasts at the contact of mineralized bone. Osteoblasts appear to control this step by exposing the mineral to osteoclasts and preosteoclasts and/or by releasing a soluble factor that activates these cells. In a third step, activated osteoclasts resorb both the mineral and the organic of mineralized bone through the action of agents that they secrete in the segregated zone underlying their ruffled border. The mineral appears to be solubilized by hydrogen ions secreted by an ATP-driven proton pump located at that border and fed by protons generated from CO2 by carbonic anhydrase. The removal of organic matrix, which could be prepared by osteoblast collagenase at the level of nonmineralized bone surfaces, appears dependent on acid proteinases, particularly cysteine-proteinases, secreted, together with other lysosomal enzymes, in the acid microenvironment of the resorption zone.     

Osteoclastic acidification pathways during bone resorption

Osteoclasts resorb bone by attaching to the surface and then secreting protons into an extracellular compartment formed between osteoclast and bone surface. This secretion is necessary for bone mineral solubilization and the digestion of organic bone matrix by acid proteases. This study summarizes the characterization and role of each type of ion transport and defines the main biochemical mechanisms involved in the dissolution of bone mineral during bone resorption. The primary mechanism responsible for acidification of the osteoclast-bone interface is vacuolar H+-adenosine triphosphatase (ATPase) coupled with Cl- conductance localized to the ruffled membrane. Carbonic anhydrase II (CAII) provides the proton source for extracellular acidification by H+-ATPase and the HCO3- source for the HCO3-/Cl- exchanger. Whereas some transporters are responsible for the bone resorption process, others are essential for pH regulation in the osteoclast. The HCO3-/Cl- exchanger, in association with CAII, is the major transporter for maintenance of normal intracellular pH. An Na+/H+ antiporter may also contribute to the recovery of intracellular pH during early osteoclast activation. Once this mechanism has been rendered inoperative, another conductive pathway translocates the protons and modulates cytoplasmic pH. Inward-rectifying K+ channels may also be involved by compensating for the external acidification due to H+ transport. These different effects of transport processes, either on bone resorption or pH homeostasis, increase the number of possible sites for pharmacological intervention in the treatment of metabolic bone diseases.     

Indapamide, a thiazide-like diuretic, decreases bone resorption in vitro.

We recently showed that indapamide (IDP), a thiazide-related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10(-4) M IDP and 10(-4) M AZ, as well as 10(-5) M pamidronate (APD), decreased bone resorption in organ cultures and in cocultures of osteoblast-like cells and bone marrow cells in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We investigated the mechanism of this antiresorptive effect of IDP; IDP decreased osteoclast differentiation as the number of osteoclasts developing in coculture of marrow and osteoblast-like cells was decreased markedly. We then investigated whether IDP affected osteoblast-like cells because these cells are involved in the osteoclast differentiation. Indeed, IDP increased osteoblast-like cell proliferation and alkaline phosphatase (ALP) expression. Nevertheless, it did not modify the colony-stimulating factor 1 (CSF-1) production by these cells. In addition, osteoblast-like cells expressed the Na+/Cl- cotransporter that is necessary for the renal action of thiazide diuretics, but IDP inhibited bone resorption in mice lacking this cotransporter, so the inhibition of bone resorption and osteoclast differentiation did not involve this pathway. Thus, we hypothesized that IDP may act directly on cells of the osteoclast lineage. We observed that resorption pits produced by spleen cells cultured in the presence of soluble osteoclast differentiation factor (sODF) and CSF-1 were decreased by 10(-4) M IDP as well as 10(-5) M APD. In conclusion, in vitro IDP increased osteoblast proliferation and decreased bone resorption at least in part by decreasing osteoclast differentiation via a direct effect on hematopoietic precursors.     

A band of F-actin containing podosomes is involved in bone resorption by osteoclasts

Isolated rabbit osteoclasts cultured on devitalized thin bone slices excavate resorption lacunae that can be visualized with brightfield or phase-contrast microscopy. Superimposition of the brightfield images of such resorption lacunae and the fluorescence images of the corresponding osteoclasts after fixation and staining with rhodamine-conjugated phalloidin revealed that a bright fluorescent band of F-actin-containing podosomes precisely outlined the resorption lacunae in stationary osteoclasts. When the resorption lacunae were being extended laterally, the clearly delineated band of podosomes corresponded to the advancing edge of the resorbing osteoclast and the most recently excavated part of the lacunae. Reshaping and reorganization of the bright bands preceded development of the lateral boundary of the lacunae. Podosomes forming these bands were highly dynamic, changed in size and location, and appeared and disappeared continuously. Their lifespan varied between 2 and 12 min. Similar bands were also seen in vivo in bone-resorbing osteoclasts on the endocranial surface of growing calvariae. Podosomes disappeared in osteoclasts treated with calcitonin, resulting in the disruption of the fluorescent bands. Our results suggest that podosomes are an essential part of the resorption apparatus of osteoclasts.     

Prostaglandin E2 Directly Inhibits Bone-Resorbing Activity of Isolated Mature Osteoclasts Mainly Through the EP4 Receptor

Prostaglandins (PGs) are well known to be important local factors in regulating bone formation and resorption. PGE₂ is a potent stimulator of bone resorption because of enhancing osteoclast formation by its indirect action through stromal cells. However, the direct action of PGE₂ on functionally mature osteoclasts is still controversial. In this study using highly purified rabbit mature osteoclasts, we examined the direct effect of PGE₂ on osteoclastic bone-resorbing activity and its mechanism. PGE₂ inhibited resorption pit formation on a dentine slice by the purified osteoclasts in a dose- and time-dependent manner. The inhibitory effect appeared as early as 4 hours after the PGE₂ addition. Forskolin and 12-0-tetradecanoyl phorbol-13-acetate (TPA), respective activators of adenylate cyclase and protein kinase C, also decreased the osteoclastic bone-resorbing activity. PGE₂ increased the content of intracellular cAMP in a dose range effective for the inhibition of bone resorption, whereas the prostanoid did not alter the intracellular level of inositol triphosphate. The inhibition of osteoclastic bone resorption by PGE₂ was amplified and diminished by a cAMP phosphodiesterase inhibitor (isobutyl methylxanthine) and a protein kinase A inhibitor (Rp-cAMP), respectively. Of four different subtypes of PGE₂ receptors (EPs), EP4 mRNA was predominantly expressed in isolated osteoclasts, whereas the other types of EP mRNA were detected in only small amounts. These results suggest that the PGE₂ inhibitory effect was mediated by an adenylate cyclase system coupled with EP4. This possible association of PGE₂ with EP4 in mature osteoclasts was supported by the finding that a specific agonist of EP4 (AE-604) inhibited the bone-resorbing activity and elevated the intracellular cAMP content. However, butaprost, a selective EP2 agonist, also mimicked the PGE₂ effects on isolated osteoclasts although EP2 mRNA expression was minimal. In conclusion, PGE₂ directly inhibits bone-resorbing activity of functionally mature osteoclasts by activation of the adenylate cyclase system, perhaps mainly through EP4. Received: 21 July 1999 / Accepted: 31 January 2000     

Vasoactive intestinal peptide regulates osteoclast activity via specific binding sites on both osteoclasts and osteoblasts

Clinical and experimental observations, together with immunohistochemical findings, suggest that neuro-osteogenic interactions may occur in the skeleton. In this study, we have examined the effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in bone, on the activity of the bone-resorbing osteoclast. Effects on bone resorption were assessed by counting the number of pits formed by rat osteoclasts incubated on devitalized slices of bovine cortical bone. Under conditions with an initially sparse density of stromal cells/osteoblasts, VIP caused a rapid cytoplasmic contraction and decreased motility of osteoclasts. This was coupled with a decrease in the number of resorption lacunae and a decrease in the total area resorbed by the osteoclasts in 48-h cultures. Time-course experiments revealed that the inhibitory effects on contraction and motility were transient and that the cells gradually regained their activity, such that, when culture time was prolonged to 120 h, a stimulatory effect by VIP on bone resorption was observed. When osteoclasts were incubated on bone slices, in the presence of an initially large number of stromal cells/osteoblasts, VIP treatment increased the number of resorption pits and total bone area resorbed in 48-h cultures. Using atomic force microscopy, we provide direct evidence that both osteoclasts and stromal cells/osteoblasts bind VIP. Also, VIP was shown to cause a rapid rise of intracellular calcium in osteoclasts and in a proportion (20%) of stromal cells/osteoblasts. Taken together, these data suggest that differentiated osteoclasts are equipped with receptors for VIP that are linked to a transient inhibition of osteoclast activity and, in addition, that stromal cells/osteoblasts have VIP receptors coupled to a delayed stimulation of osteoclastic resorption.     

Evaluation of the osteoclastic population in iliac crest biopsies from 36 normal subjects: a histoenzymologic and histomorphometric study

After histochemical staining of tartrate-resistant acid phosphatase (TRAP) activity, the total and active trabecular resorption surfaces and the number of osteoclasts were determined by histomorphometry on iliac crest biopsies from 36 healthy volunteers. The subjects were separated into three groups according to age and sex. Total trabecular resorption surface showed no significant variation in any group, but the fraction of active resorption surface was significantly higher in the older population. The number of TRAP cells per mm2 of section area, related to trabecular bone volume or surface, showed a significant increase in elderly subjects. The mean osteoclast interface was similar in all the groups. We found a significant decrease in resorption depth between young and old populations. These results are consistent with a reduced activity of bone-resorbing cells in advancing age. These normal values, established after histochemical identification of osteoclasts, may be applied for evaluating abnormal bone-resorbing cell activity in metabolic bone diseases.     

Alendronate Disturbs Vesicular Trafficking in Osteoclasts

The nitrogen-containing bisphosphonate alendronate inhibits osteoclast-mediated bone resorption through inhibition of the mevalonate pathway. This results in impaired protein prenylation and may affect the function of small GTPases in osteoclasts. Since these proteins are important regulators of vesicle transport in cells, we investigated the possible interference of alendronate with these processes in isolated rat osteoclasts. We show here that alendronate-induced inhibition of bone resorption coincides with accumulation of tartrate-resistant acid phosphatase- and electron dense material-containing tubular vesicles in osteoclasts. Alendronate-induced changes in osteoclasts also included widening of the sealing zone areas and incomplete organization of tight attachments and ruffled borders. Osteoclasts also appeared partially detached from the bone surface, and organic matrix was typically dissolved only at the edges of the resorption pits on alendronate-coated bone slices. In contrast, resorption pits on the control and clodronate-coated bone slices were thoroughly resorbed. Inhibition of bone resorption by alendronate was not, however, related to a decrease in osteoclast number. In conclusion, our findings suggest that alendronate inactivates osteoclasts by mechanisms that impair their intracellular vesicle transport, apoptosis being only a secondary phenomenon to this.     

The dynamin inhibitor dynasore inhibits bone resorption by rapidly disrupting actin rings of osteoclasts

The cytoskeletal organization of osteoclasts is required for bone resorption. Binding of dynamin with guanosine triphosphate (GTP) was previously suggested to be required for the organization of the actin cytoskeleton. However, the role of the GTPase activity of dynamin in the organization of the actin cytoskeleton as well as in the bone-resorbing activity of osteoclasts remains unclear. This study investigated the effects of dynasore, an inhibitor of the GTPase activity of dynamin, on the bone-resorbing activity of and actin ring formation in mouse osteoclasts in vitro and in vivo. Dynasore inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation and rapidly disrupting actin rings in osteoclasts. A time-lapse image analysis showed that dynasore shrank actin rings in osteoclasts within 30 min. The intraperitoneal administration of dynasore inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced trabecular bone loss in mouse femurs. These in vitro and in vivo results suggest that the GTPase activity of dynamin is critical for the bone-resorbing activity of osteoclasts and that dynasore is a seed for the development of novel anti-resorbing agents.     

Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts

Bone morphogenetic proteins (BMPs) play an important role in various kinds of pattern formation and organogenesis during vertebrate development. In the skeleton, BMPs induce the differentiation of cells of chondrocytic and osteoblastic cell lineage and enhance their function. However, the action of BMPs on osteoclastic bone resorption, a process essential for pathophysiological bone development and regeneration, is still controversial. In this study, we examine the direct effect of BMPs on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. BMP-2 caused a dose- and time-dependent increase in bone resorption pits excavated by the isolated osteoclasts. BMP-4 also stimulated osteoclastic bone resorption. The increase in osteoclastic bone resorption induced by BMP-2 was abolished by the simultaneous addition of follistatin, a BMP/activin binding protein that negates their biological activity. Just as it increased bone resorption, BMP-2 also elevated the messenger RNA expressions of cathepsin K and carbonic anhydrase II, which are key enzymes for the degradation of organic and inorganic bone matrices, respectively. Type IA and II BMP receptors (BMPRs), and their downstream signal transduction molecules, Smad1 and Smad5, were expressed in isolated osteoclasts as well as in osteoblastic cells, whereas type IB BMPR was undetectable. BMPs directly stimulate mature osteoclast function probably mediated by BMPR-IA and BMPR-II and their downstream molecules expressed in osteoclasts. The results presented here expand our understanding of the multifunctional roles of BMPs in bone development.     

In vitro resorptive activity of isolated chick osteoclasts: effects of carbonic anhydrase inhibition

A potent inhibitor of carbonic anhydrase, 5-[3-hydroxybenzoyl]thiophene-2-sulfonamide (HTS), was shown to cause a 37% reduction in the area of resorption pits formed by isolated chick osteoclasts when used at a dose of 10(-7) M. HTS at doses of 10(-9) and 10(-7) M was also effective in reducing acid formation by the osteoclasts (14 and 36%, respectively). Additionally, the effect of HTS was found to be readily reversed by removing the agent, showing that it does not exert a toxic effect on the cells. This study indicates that the inhibitory effect of HTS on bone resorption is at the level of the acid-forming mechanism in osteoclasts and supports the view that carbonic anhydrase has a central role in the process.     

A comparison of the effects of inhibitors of carbonic anhydrase on osteoclastic bone resorption and purified carbonic anhydrase isozyme II

Summary We have assessed the effects of five sulfonamides with widely varying inhibitory activity for carbonic anhydrase (CA) in the bone slice assay using disaggregated rat osteoclasts (OCs), and in the Maren assay where the catalytic activity of purified CA isozyme II (CA II) was measured. There was an excellent correlation between the relative potencies of the compounds in the two assays: ethoxzolamide (ETH)>acetazolamide (AZ)>M&B 21659>M&B 9811>M&B 7973. In the bone slice assay, ETH and AZ were found to be the most potent inhibitors of OC bone resorption, with IC₅₀ values of 0.09 and 0.8 μM, respectively (from plan surface area of bone resorbed). These results support previous observations showing that OCs use CA II to generate protons during bone resorption and that CA II activity is essential for OCs to be able to resorb bone.     

Effect of chronic carbonic anhydrase inhibitor therapy on bone mineral density in white women

A limited, dual-photon absorptiometry, single-center study of bone mineral density (BMD) was conducted on white female glaucoma subjects who were chronic users of the carbonic anhydrase (CA) inhibitors acetazolamide (Az) or methazolamide (Mz). In postmenopausal subjects long-term (greater than 4 years) CA inhibitor use was associated with a bone-sparing effect as judged by spinal BMD in comparison to controls matched for age, sex, weight, and ethnic group or in comparison to a national normative data base. Short-term (0-2 years) postmenopausal CA inhibitor users and premenopausal subjects using CA inhibitors showed no sparing of spinal BMD. Femoral neck BMD was not affected by CA inhibitor therapy in any of the groups. This study supports a proposed role for carbonic anhydrase in human bone resorption and suggests a possible future utility for carbonic anhydrase inhibitors in prophylaxis or management of primary involutional osteoporosis. Future studies are necessary to verify and expand these findings, assess the effects of CA inhibitors on bone mechanical competence, and further develop CA inhibitors with some specificity for bone.