Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3569-3573, April 1994 Pharmacology Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase (bone ceil/nitric oxide/aminguanidine) THOMAS P. KASTEN*, PATRICIA COLLIN-OSDOBYt, NiRAJ PATELt, PHILIP OSDOBYt, MARILYN KRUKOWSKIt, THOMAS P. MISKO*, STEVEN L. SETTLE*, MARK G. CURRIE*, AND G. ALLEN NICKOLS*t§ *Department of Molecular Pharmacology, Monsanto Corporate Research, St. Louis, MO 63167; tDepartment of Biology and Division of Bone and Mineral Diseases, Washington University, St. Louis, MO 63130; and tDepartment of Pharmacological and Physiological Sciences, St. Louis University Medical School, St. Louis, MO 63104 Communicated by Philip Needleman, December 27, 1993

ABSTRACT We have examined the effects of modulating histochemical level, Schmidt et al. (11) have demonstrated nitric oxide (NO) levels on osteoclast-mediated bone resorption that nitric oxide synthase (NOS) was present in areas ofbone in vitro and the effects ofnitric oxide synthase (NOS) inhibitors coincident with osteoclast and bone-remodeling activity. The on bone mineral density in vivo. Diaphorase-based histochem- report of MacIntyre et al. (12) indicated that NO-generating ical staining for NOS activity of bone sections or highly agents caused a decrease in isolated rat osteoclast cell spread enriched osteoclast cultures suggested that osteoclasts exhibit area and bone resorption. Also, Howard (13) reported that substantial NOS activity that may account for basal NO NO-generating compounds may increase cGMP levels in production. Chicken osteoclasts were cultured for 36 hr on isolated chicken osteoclasts. Furthermore, sodium nitroprus- bovine bone slices in the presence or absence of the NO- side (SNP) has been shown to inhibit the parathyroid hor- generating agent sodium nitroprusside or the NOS inhibitors mone or 1,25-(OH)2-vitamin D3 stimulation of resorption in N-nitro-L-arginine methyl ester and aminoguanidine. Nitro- the 19-day fetal rat limb resorption assay system, with prusside markedly decreased the number of bone pits and the concomitant increases in cGMP (14). average pit area in comparison with control cultures. On the The current study was designed to investigate the role of other hand, NOS inhibition by N-nitro-L-arginine methyl ester NO in both an isolated in vitro avian osteoclast system and or aminoguanidine dramatically increased the number of bone an in vivo rat osteoporosis model system using a NO- pits and the average resorption area per pit. In a model of generating agent and selective NOS inhibitors. These find- osteoporosis, aminoguanidine potentiated the loss of bone ings demonstrate that NO regulates osteoclast bone-resorp- mineral density in ovariectomized rats. Aminoguanidine also tion activity in vitro and in vivo and that similar effects are caused a loss ofbone mineral density in the sham-operated rats. seen in birds and mammals. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings MATERIALS AND METHODS suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and Animals. Three-month-old female Sprague-Dawley rats NO levels by cells within the bone microenvironment may be a (250-300 g) from Charles River Breeding Laboratories were sensitive mechanism for local control ofosteoclast bone resorp- used in all in vivo experiments. Pathogen-free White Leghorn tion. fertile eggs were hatched at Washington University. Animal protocols were approved by the institutional Animal Care and Bone-remodeling disorders such as osteoporosis, osteoar- Use Committees. thritis, and periodontal disease are frequently associated with Osteoclast Isolation and Culture. Osteoclasts were isolated perturbations in the interplay between local and systemic from White Leghorn chickens maintained on a low calcium bone-remodeling regulatory pathways. Inflammatory cyto- diet for a period of 4 weeks by the method of Oursler et al. kines and arachidonic acid derivatives have been implicated (15). Osteoclasts were cultured in phenol red-free medium as intercellular messengers involved in humoral-mediated 199 with Earle's salts supplemented with 8.3 mM NaHCO3, and local osteopenia (1, 2). Postmenopausal bone loss asso- 100 mM Hepes (pH 6.8), 1% antibiotic/antimycotic solution ciated with diminished estrogen levels is correlated with (GIBCO) with 5% charcoal-stripped fetal calf serum at 370C increased levels of interleukin 1 and stromal cell-derived in an atmosphere of 95% air/5% CO2. For all cGMP studies, interleukin 6, cytokines known to stimulate osteoclast activ- osteoclasts were cultured at 1-2 x 105 osteoclasts per well in ity and development (3, 4). Estrogen also directly inhibits 24-well tissue culture dishes overnight before SNP addition. osteoclast-mediated resorption (5, 6) and affects other bone For bone-resorption studies, 5 x 104 osteoclasts were plated cells (4, 7). The complexity of signals associated with normal directly onto bovine cortical bone slabs in 48-well tissue and pathological bone remodeling underscores the interac- culture dishes and cultured overnight, modulators of NO tive nature of this tissue. were added, and the resorption assay was stopped after an Bone-degrading osteoclasts arise from cells within the additional 36 hr by fixation in 1% formaldehyde. monocyte-macrophage lineage (8) and, although possessing Diaphorase Staining for NOS Activity. NOS-associated a unique ability to resorb bone, share various characteristics NADPH-dependent diaphorase activity was examined on with macrophages. Macrophages release the cytotoxic short- cultured isolated avian osteoclasts and on frozen sections of lived reactive radical nitric oxide (NO) in response to inflam- matory cytokines and agents (9, 10). Osteoclasts probably Abbreviations: NOS, nitric oxide synthase; NAME, N-nitro-L- make NO and also serve as targets for NO action. At the arginine methyl ester; AG, aminoguanidine; TRAP, tartrate- resistant acid phosphatase; BMD, bone mineral density; SNP, sodium nitroprusside. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed at: Monsanto payment. This article must therefore be hereby marked "advertisement" Corporate Research, Building T3P, 800 North Lindbergh Boule- in accordance with 18 U.S.C. §1734 solely to indicate this fact. vard, St. Louis, MO 63167.

3569 Downloaded by guest on September 29, 2021 3570 Pharmacology: Kasten et al. Proc. NatL Acad. Sci. USA 91 (1994) avian tibias obtained from animals maintained on a low calcium diet, which were fixed and processed according to Sainte-Marie (16). Isolated osteoclasts were plated onto bovine cortical bone slices or glass coverslips. Cultured osteoclasts were rinsed in Hanks' balanced salt solution, pH 7.2 (HBSS), fixed in 2.5% glutaraldehyde/HBSS, rinsed, air-dried, and stained for NOS activity using the diaphorase staining protocol of Schmidt et al. (11). Staining was done with and without NADPH to determine NADPH-dependent NOS diaphorase activity. cGMP Measurement. Isolated osteoclasts cultured over- night on tissue culture dishes were treated with isobutyl- methylxanthine (1 mM for 10 min) plus or minus SNP. The medium was removed, cGMP was extracted by addition of ice-cold HCl (0.1 M for 10 min), and cGMP concentrations were determined by RIA. Quantitative Pit Resorption Assay. Osteoclasts cultured on bone slices were rinsed in HBSS, fixed in 1% formalin/ HBSS, rinsed, and stained for tartrate-resistant acid phos- phatase (TRAP) (15). The number of TRAP-positive, multi- nucleated cells for each treatment was obtained by exami- nation of eight random fields per slice (two slices per treatment) using a photomask for size, orientation, and reference for each field. After this determination, the cells were removed with 0.01 M NH40H and rubbing, so that the number and area of resorption pits could be quantitated. Dark-field reflective microscopy permitted excellent visual- ization of pits, and all measurements were performed on Ai video-captured images linked to a Leica Quantimet image ...... analysis program. Total area, mean pit area, pits per cells, and percentage of total area resorbed were determined. Bone Mineral Density (BMD) in Normal and Ovariectomized Rats. Female Sprague-Dawley rats were housed individually B and maintained on a 0.4% calcium diet and deionized water throughout the protocol. On day zero, the rats were divided into groups (n = 6 per group), weighed, and anesthetized with FIG. 1. Diaphorase staining of osteoclasts. (A) Photomicrograph xylaxine at 10 mg/kg/ketamine at 50 mg/kg (i.m.). The left of a frozen section of chicken tibia from an animal maintained on a femur and lumbar vertebrae were scanned for BMD by using low calcium diet for 4 weeks. Section was stained for NOS NADPH- a Hologic (Waltham, MA) model QDR-1000 dual energy dependent diaphorase activity. Large intensely stained osteoclasts x-ray absorptiometer. Animals were ovariectomized or (arrowheads) are observed closely associated with bone trabeculae with (b). Lining cells or osteoblasts also exhibit positive staining for NOS sham-operated by externalization ofthe ovaries removal diaphorase activity (arrows). (x290.) (B) Photomicrograph using or examination and replacement. Aminoguanidine (AG) was bright-field reflective light microscopy of isolated osteoclasts cul- administered by continuous infusion (130 mg/kg per day) tured on cortical bone slices and stained for NOS using the diapho- using s.c. implanted osmotic minipumps fitted to indwelling rase stain. Two osteoclasts (arrowheads) are stained intensely for catheters placed in the jugular vein. 3-Estradiol was admin- NOS and are closely associated with resorption pits, as visualized by istered as an implant that yielded estrogen levels of 60-70 the light refractive circles (arrows). (X219.) pg/ml of serum (17). Every 2 weeks rats were anesthetized and scanned to measure femoral and vertebral BMD. Mean Osteoclasts associated with bone matrix exhibited stronger arterial pressure and heart rate were determined after treat- reactivity when compared to osteoblasts, and only focal ment (18). staining was observed in the marrow cavity in areas of Hitology. At the end of4 weeks of drug treatment, femurs hematopoietic activity. NADPH-dependent diaphorase and lumbar spine were removed from the rats, cleaned ofsoft staining of isolated avian osteoclasts cultured on cortical tissue, and fixed in 10%6 buffered formalin, decalcified, de- bone slices also revealed intense reactivity, and in many hydrated, and processed for either paraffin or plastic embed- cases these cells were associated with multilobulated resorp- ding. Ten-micrometer sections of decalcified samples were tion pits (Fig. 1B). In contrast, freshly isolated osteoclasts or stained with either hematoxylin/eosin or for localization of those plated on tissue culture plastic displayed little diapho- TRAP (15). Sections were viewed and photographed with a rase staining, indicating that such osteoclasts had minimal Leitz Diaplan microscope. NOS activity (data not shown). Data Evaluation. Data are presented as averages ± SEMs. cGMP. Isolated avian osteoclast cultures were exposed to Significance was determined by Student's t test for unpaired SNP to ascertain whether or not osteoclast cGMP levels were data with significance indicated as P < 0.05. BMD data were modified in response to NO. Fig. 2 demonstrates that intra- analyzed by ANOVA with repeated measures. The details of cellular cGMP levels increased in a concentration-dependent group comparisons are described in the text. manner in response to SNP (P < 0.01 each dose vs. basal). Avian osteoclasts were therefore responsive to the NO generator SNP, as indicated by increased intracellular con- RESULTS centrations of cGMP. NOS Activity in Osteoclasts. NADPH-dependent diapho- In Vitro Bone Resorption. Modulators of NOS activity rase staining for NOS activity revealed that osteoclasts and affected isolated avian osteoclast bone-resorptive activity osteoblasts on the long bones ofchickens maintained on a low (Table 1). Bone resorption was inhibited in a dose-dependent calcium diet exhibited intense diaphorase staining (Fig. 1A). manner by SNP. Although fewer osteoclasts were present on Downloaded by guest on September 29, 2021 Pharmacology: Kasten et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3571 so - T

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0 Basal 0.01 0.1 I Nitroprusside (mM) N FIG. 2. SNP-mediated increase in cGMP concentrations in iso- E lated chicken osteoclasts. Chicken osteoclasts were isolated and cm cultured overnight. Isolated osteoclasts demonstrated nondetectable E (ND) basal levels ofcGMP. SNP at the concentrations indicated was added to the cells in the presence ofisobutylmethylxanthine (1 mM). m Incubations were terminated after 10 min. E

the bone slices with increased SNP concentrations, the ._ C number of pits per cell was indistinguishable from control a) samples. The reduced total area of bone resorbed could be, in part, the result of the diminished cell number. However, co 0 when the cell activity was determined, as reflected in the 4 mean area resorbed per pit, there was significantly reduced Time, weeks resorption of bone by SNP-treated osteoclasts as compared with controls. The reduced numbers ofosteoclasts associated FIG. 3. AG-induced bone loss in sham-operated and ovariecto- with bone after treatment with 1 mM SNP may represent a mized rats. Ovariectomized (OVX) or sham-operated (Sham) rats toxic effect or be related to physiological cell retraction and (225 g) were maintained on a 0.4% calcium diet with deionized water release. and treated as indicated. The NOS inhibitor AG was administered by In contrast to the action of SNP, the NOS inhibitors continuous infusion (130 mg/kg per day) via osmotic minipump with N-methyl-L-arginine methyl ester (NAME) and AG each direct delivery into the jugular vein. f-Estradiol (EST) was admin- istered as a silastic implant. BMD was determined by dual energy enhanced osteoclast resorptive activity. Although the lower x-ray absorptiometry. On day zero the average body weight was 278 concentration of NAME did not markedly alter osteoclast ± 3.3 g, the average spinal BMD was 174 ± 1.8 mg/cm2, and the number or activity, the higher concentration (1 mM) elicited average femoral BMD was 200 ± 1.2 mg/cm2 for all groups. a doubling in the number of pits per cell and a 33% increase in the average resorption area per pit, resulting in a nearly increases in the number of pits, number of pits per cell, and 3-fold increase in overall resorptive activity (49,184-143,577 a 54% increase in the average area per pit. pm2). AG substantially increased the number ofpits per cell, In Vivo Bone Resorption. AG, a selective inhibitor of the total area resorbed, and the area per pit resorbed. Unlike inducible NOS, was examined for its effect on BMD in the 0.1 mM NAME, 0.1 mM AG significantly stimulated resorp- ovariectomized rat. Spinal BMD was reduced after ovariec- tive activity in comparison with controls. This stimulation tomy over the 8 weeks studied (-20.0 ± 6.2 mg/cm2) (P < was primarily reflected in the increased number of pits per 0.01) as compared with control sham-operated rats that cell as opposed to the area per pit resorbed. The higher increased BMD (+11.2 ± 4.1 mg/cm2) (P < 0.01) (Fig. 3A). concentration of AG (1 mM) elicited a nearly 3-fold increase The decline in BMD of ovariectomized rats was completely in resorptive activity, similar to that obtained for 1 mM blocked by treatment with estradiol. AG treatment of sham- NAME. The increased resorptive activity was related to operated animals decreased BMD ofthe spine to -10.2 ± 6.8 Table 1. NO modulators affect bone resorption by isolated osteoclasts Total Mean Total area Change in TRAP+, TRAP+/ Pits, area resorption resorbed, resorption, Treatment no. field no. Pits/cell resorbed area/pit Area/field % % Control 100 6.25 ± 0.69 20 0.2 + 0.018 49,184 2459 ± 373 3074 ± 693 14.5 100 SNP (0.1 mM) 60 3.75 ± 0.87 15 0.25 ± 0.043 28,650 1910 ± 533 1910 ± 687 8.2 56.2 SNP (1 mM) 31 1.94 ± 0.85 7 0.23 ± 0.14 8,554 1222 ± 349 535 ± 351 2.5 77.4 NAME (0.1 mM) 77 4.81 ± 0.83 19 0.25 ± 0.06 45,130 2375 ± 370 2821 ± 959 13.1 91.8 NAME (1 mM) 88 5.5 ± 1.08 35 0.4 ± 0.06 143,577 4101 ± 481 8974 ± 2920 42.0 291.9 AG (0.1 mM) 68 4.25 ± 0.93 44 0.65 ± 0.18 104,309 2371 ± 327 6519 ± 2447 30.0 212.1 AG (1 mM) 111 6.93 ± 1.32 47 0.42 ± 0.14 147,671 3142 ± 279 9229 ± 2231 43.0 300.2 Isolated osteoclasts were cultured on devitalized bone slices and treated with SNP or the NOS inhibitors, NAME or AG, as described. Data are presented as totals determined for random fields per bone slice for two bone slices (a total of 16 fields) per treatment. The number of multinucleated, TRAP+ cells in the 16 fields (TRAP+, no.) and per observation field (TRAP+/field), number ofexcavations in the 16 fields (Pits, no.), mean number of excavations per cell (Pits/cell), total area resorbed over the 16 fields (Area), mean area per excavation (Area/pit), and percentage of total area resorbed and the change in resorption compared with control. Units for area measurements are Im2. Data are representative of three separate experiments that yielded comparable results. Results are described as means ± SEMs. Downloaded by guest on September 29, 2021 3572 Pharmacology: Kasten et al. Proc. NatL Acad Sci. USA 91 (1994)

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;~P 6 FIG. 4. Light micrographs of bone sections from spines ot i.: AG-treated ovariectomized rats. (A) Paraffin sections of shanm- operated rat vertebrae stained using hematox'yIn cosin. lMany activ e osteoblasts (arrows) are observed lining the bony (b) trabeculae. The trabeculae are relatively smooth and few. if anr'. osteoclasts can be seen. (B) Paraffin section from an ovariectormized rat vertebrae illustrating moderate resorptive activity. (scalloping of trabeculae) and occasional osteoclasts (arrowhead). Few'er osteoblasts appear to line the trabecular surfaces. Megakarvocytes can also he observed in A~~~~~~~~~~~~~~~ the marrow space. (C) Paraffin section from an ovariectomized ~~~~X AG-treated rat vertebrae, illustrating extensive trabecular scallop- 3 vat ing. indicative of resorptive activity and increased numbers of' osteoclasts (arrowheads) associated with bone. (A-C. 190. mg/cm2 (P < 0.05 compared with vehicle-treated sham- NO-releasing agents SNP and 3-morpholinosydnonimine hy- operated animals), but not to the low level seen in the drochloride. In their system, NO caused cell retraction and ovariectomized rats. Similar to the effects of AG in the inhibited osteoclast-mediated bone resorption in culture. sham-operated animals, AG potentiated the loss of BMD in Stern and Diamond (14) subsequently demonstrated in bone the spine of ovariectomized rats, resulting in an overall organ culture studies that nitroprusside increased cGMP, but decline of -32.0 ± 4.7 mg/cm2 in 8 weeks (P < 0.05 not cAMP levels, and inhibited the resorptive effects of compared with vehicle-treated ovariectomized rats) and a parathyroid hormone and vitamin D. A relationship between difference of 43 mg/cm2 when compared with the control NO and osteoclasts was also indicated by the experiments of sham-operated group (P < 0.01). Femur BMD changes Schmidt et al. (11), suggesting that NOS activity was present resembled those seen in the spine (Fig. 3B). Therefore, in in areas ofbone coincident with osteoclast activity and bone both the spine and femur the BMD was reduced by AG in the remodeling. They suggested that the diaphorase staining sham-operated and ovariectomized rats. Mean arterial pres- represented inducible NOS based on a lack ofcostaining with sures and heart rates at the end of8 weeks were not different a constitutive NOS antibody. From the reported inhibitory for any of the groups (data not shown). Thus, AG treatment effects of NO on osteoclast activity and the proposed pres- had no significant effect upon the constitutive NOS, as ence ofinducible NOS in areas ofbone metabolic activity we assessed by resting blood pressure. hypothesized that NO produced by inducible NOS in bone In Vivo Bone Histology. Longitudinal histological sections may inhibit osteoclast activity and depression of NO may of spine from sham-operated rats showed a smooth bone stimulate resorption. surface adjacent to the marrow cavity (Fig. 4A). Ovariectomy This postulate was evaluated in vitro by using isolated resulted in a more rugged contour of this bone surface with avian osteoclasts cultured on bone slices to examine resorp- an increase in the number of osteoclasts present in any area tive activity, diaphorase staining ofbone sections or isolated and a marked increase in osteoclast resorption activity (Fig. osteoclasts cultured on bone slices, and cGMP responsive- 4B). AG treatment of ovariectomized rats further enhanced ness. The resorption assay demonstrated that SNP addition the scalloped bone surface, suggesting an increase in osteo- inhibited avian osteoclast bone-resorption activity analogous clast activity when compared with either sham-operated or to the effects upon rat osteoclasts in the earlier report of ovariectomized animals (Fig. 4C). MacIntyre et al. (12). In the present studies, the average size of each excavation pit was reduced by SNP treatment. Conversely, inhibition of NOS with the constitutive NOS DISCUSSION selective inhibitor NAME or the inducible NOS selective A relationship between osteoclast function and NO was inhibitor AG dramatically increased the ability ofosteoclasts suggested by MacIntyre et al. (12) in experiments demon- to excavate bone. The number ofpits per cell increased with strating the inhibition of osteoclast activity by NO or the each inhibitor, and the area of the pits was increased, leading Downloaded by guest on September 29, 2021 Pharmacology: Kasten et al. Proc. NatL. Acad. Sci. USA 91 (1994) 3573 to a substantial enhancement of the total area excavated. ectomized rats treated with AG. Thus, NOS inhibition in an Moreover, the observation that the lower dose of AG was in vivo system substantially altered bone metabolic activity more effective in stimulating resorption when compared with by increasing osteoclast-mediated bone resorption, regard- an equivalent molar concentration of NAME supports the less of the estrogen status of the animal. hypothesis that osteoclasts possess the inducible form of In the bone environment a variety of cells other than NOS. In addition, the increased diaphorase staining of os- osteoclasts may produce NO, including osteoblasts, endo- teoclasts on bone compared with those on plastic would thelial cells, osteoclast precursors, and stromal cells. The suggest that osteoclast NOS can be regulated. Osteoclasts in lack of effect of AG upon blood pressure suggests that the vitro responded to exogenously added NO, and the results constitutive form of NOS in endothelial cells was not in- with the NOS inhibitors suggested an endogenous source of volved in the potentiation of bone loss mediated by this NO. Because osteoblasts and other cells in the bone envi- inhibitor. However, the cellular and enzymatic source(s) of ronment also exhibit NOS activity, the observed effect could NO, as well as the regulation of the NOS in bone, deserves be mediated indirectly. However, the strong diaphorase further investigation. Moreover, because NO inhibits osteo- staining of osteoclasts and the fact that the preparation of clastic resorption and because active osteoclasts exhibit high isolated avian osteoclasts represents a highly enriched os- NOS activity, there are likely to be counterbalancing signals teoclast population suggest that one source of NO in bone is for NO modulation ofresorption. These findings suggest that derived from osteoclasts. modulation of NOS and NO levels by osteoclasts may be a Consistent with the osteoclast resorption studies that dem- sensitive mechanism for local control of osteoclast bone onstrated their responsiveness to NO, biochemical respon- resorption and overall bone integrity. siveness was also assessed by measuring cGMP concentra- tions in avian osteoclasts treated with NO. SNP exposure This work was, in part, supported by Monsanto, National Insti- increased cGMP concentrations in osteoclasts in a concen- tutes of Health Grants AR32927 and AR32087, American Heart tration-dependent manner (Fig. 2), supporting the studies of Association Grant-in-Aid 900885, and a Howard Hughes undergrad- Howard (13) using chicken osteoclasts and Stem and Dia- uate research fellowship (to N.P.). mond (14) using rat bone cells. These experiments and those in 2 that 1. Gowen, M. (1992) in Cytokines and Bone Metabolism, ed. Fig. suggest cGMP may mediate the actions of NO Gowen, M. (CRC, Boca Raton, FL), pp. 71-91. on osteoclast function, as it does in smooth muscle cells (19). 2. Mundy, G. (1992) in Biology and Physiology ofthe Osteoclast, However, MacIntyre et al. (12) proposed that cGMP does not eds. Rifkin, B. & Gay, C. (CRC, Boca Raton, FL), pp. 171-186. mediate the osteoclast inhibition caused by NO because 3. Pacifici, R., Rifas, L., Teitelbaum, S., Slatopolsky, E., Mc- cGMP analogues were without effect on osteoclast-mediated Cracken, R., Bergfeld, M., Lee, W., Avioli, L. & Peck, W. pit formation. Thus, despite the observed increase in cGMP (1987) Proc. Natl. Acad. Sci. USA 84, 4616-4620. elicited by NO in our studies, the role of cGMP in the 4. Jilka, R., Hangoc, G., Girasole, G., Passeri, G., Williams, D., inhibition ofosteoclast activity remains unclear and warrants Abrams, J., Boyce, B., Broxmeyer, H. & Manolagas, S. (1992) direct investigation. A nonsignaling role for NO as a modu- Science 257, 88-91. 5. Oursler, M., Osdoby, P., Pyfferoen, J. & Riggs, L. (1991) Proc. lator ofbone metabolism may also be important because NO Natl. Acad. Sci. USA 88, 6613-6617. acts as a cytotoxic agent in the host-defense mechanism. 6. Oursler, M., Pederson, L., Pyfferoen, J., Osdoby, P., Fitz- The diaphorase staining and cGMP experiments suggest patrick, L. & Spelsberg, T. (1993) Endocrinology 132, 1373- that NO may act as an autocrine factor as well as a paracrine 1380. mediator for osteoclasts. NADPH-diaphorase staining, 7. Erricksen, E., Colvard, D., Berg, N., Graham, M., Mann, L., which has been reported to specifically indicate NOS activity Spelsberg, T. & Riggs, L. (1988) Science 241, 84-86. ofisolated osteoclasts cultured on bone or osteoclasts in bone 8. Nijweide, P. & deGrooth, R. (1992) in Biology and Physiology sections (11), established the presence of high NOS activity ofthe Osteoclast, eds. Rifkin, B. & Gay, C. (CRC, Boca Raton, in osteoclasts. These studies were done in the rat, establish- FL), pp. 82-104. 9. Stuehr, D. J. & Nathan, C. F. (1989) J. Exp. Med. 169, 1543- ing the link between NOS and osteoclasts in a mammalian 1545. species. Therefore, avian and rat osteoclasts may both pro- 10. Hibbs, J. B., Jr., Taintor, R. R., Vavrin, Z. & Rachlin, E. M. duce NO and respond to this modulator. Furthermore, be- (1989) Biochem. Biophys. Res. Commun. 157, 87-94. cause osteoclasts maintained on bone appear to have higher 11. Schmidt, H. H. H. W., Gagne, G. D., Nakane, M., Pollock, NOS activity, despite the observation that NO inhibits re- J. S., Miller, M. F. & Murad, F. (1992) J. Histochem. Cy- sorption, this system may act as a rapid mechanism for tochem. 40, 1439-1456. controlling osteoclast resorption. 12. MacIntyre, I., Zaida, M., Alam, A. S. M. T., Datta, H. K., Analogues of L-arginine, the substrate for NOS, have been Moonga, B. S., Lidbury, P. S., Hecker, M. & Vane, J. R. developed as competitive inhibitors of NOS. Infusion of the (1991) Proc. Natl. Acad. Sci. USA 88, 2936-2940. 40-fold selective 13. Howard, G. A. (1985) in Normal and Abnormal Bone Growth: constitutive NOS inhibitor NAME into Basic and Clinical Research, eds. Dixon, A. D. & Sarnat, B. G. normotensive animals and humans markedly increases blood (Liss, New York), pp. 67-76. pressure (19, 20). In contrast, AG has been demonstrated as 14. Stem, P. H. & Diamond, J. (1992) Res. Commun. Chem. a 20- to 30-fold selective inhibitor of the inducible isoform of Pathol. Pharmacol. 75, 19-28. NOS by direct enzyme assays (21). 15. Oursler, M. J., Collin-Osdoby, P., Anderson, F., Li, L., Web- AG produced bone loss in the spine and femur of sham- ber, D. & Osdoby, P. (1991) J. Bone Miner. Res. 6, 375-385. operated rats and potentiated bone loss in the lumbar spine 16. Sainte-Maire, G. (1%2) J. Histochem. Cytochem. 10, 250-256. and femur of ovariectomized rats. The estrogen-deficient 17. Bridges, R. S. (1984) Endocrinology 114, 930-940. state of the ovariectomized rats induced substantial loss of 18. Wilkins, M. R., Settle, S. L. & Needleman, P. (1990) J. Clin. bone in Invest. 85, 1274-1279. mineral, particularly the first few weeks after sur- 19. Moncada, S., Palmer, R. M. J. & Higgs, E. A. (1991) Phar- gery. But the effect of AG on BMD was a loss that was macol. Rev. 43, 109-142. additive to that of estrogen deficiency. This bone-wasting 20. Vallance, P., Collier, J. & Moncada, S. (1989) Lancet 2, 997. effect of AG was also seen in the sham-operated animals. 21. Misko, T. P., Moore, W. M., Kasten, T. P., Nickols, G. A., Histological examination confirmed the potentiation of os- Corbett, J. A., Tilton, R. G., McDaniel, M. L., Williamson, teoclast activity and perhaps numbers in the spine of ovari- J. R. & Currie, M. G. (1993) Eur. J. Pharmacol. 233, 119-125. Downloaded by guest on September 29, 2021