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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast-like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non–load-bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast-like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

It is well established from both human(1,2) and animal(3–6) studies that mechanical loading is an important regulator of skeletal mass and architecture. However, the mechanisms by which these adaptive responses are initiated and subsequently coordinated are unclear. It is evident that the osteogenic response to load bearing must involve several stages(7) including: initial transduction of mechanical events into cellular signals; communication of local mechanically derived “information” between cells in order to assess strain distribution throughout the structure; and stimulation and coordination of any subsequent changes in modeling and remodeling activity which act either to alter the bone architecture or to maintain the status quo.

Transduction of the physical consequences of load bearing into cellular signals appears to involve changes in cytosolic free calcium and is followed within minutes of loading by prostaglandin (PG) production.(8,9) The involvement of PG production has also been supported by studies indicating an association between PG production and strain application(8,10); PGE2-induced osteogenesis in vivo(11,12) and indomethacin-mediated modulation of loading-induced increases in new bone formation.(13,14) Most of these studies have focused on the role of PGE2, but it is evident that prostacyclin (PGI2) is also involved.(15–17)

By analogy with observations made in endothelial cells, indicating that pulsatile shear stresses rapidly enhance the release of PGE2(18) and both PGI2(19) and nitric oxide (NO),(20) it has been established that bone cells maintained in vitro show both strain-related and fluid flow–related increases in both PGI2(21) and NO production.(22,23) Furthermore, the obligatory involvement of NO in bone's adaptive response to load bearing is suggested by two in vivo studies which have shown that nonselective inhibitors of NO synthase (NOS) activity L-NMMA and L-NAME, abrogated the osteogenic response to a short period of loading in rat tail vertebrae(24) and rat tibiae,(25) respectively.

NO can be produced by three distinct isoforms of NOS: an inducible form (iNOS, type II) which is transcriptionally up-regulated over a period of hours by specific cytokine combinations; and two constitutively expressed isoforms, eNOS (type III) and nNOS (type I), which were originally identified in endothelial and neuroneal tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies suggest that cultured osteoblast-like cells can express all three isoforms of NOS: iNOS,(22,26–28) eNOS,(26) and nNOS.(22) However, the rapidity with which mechanical strain induces increases in NO release from bone cells suggests that this response involves the activation of one of the constitutively expressed isoforms, eNOS or nNOS.

To establish the mechanism by which loading-induced NO production may be regulated within the bone cell network, we have: i) using Northern blot hybridization, in situ hybridization, and immunohistochemistry, examined the prevalence and distribution of the different NOS isoforms in load-bearing rat ulnae and non–load-bearing calvariae, which differ in their sensitivity to mechanical loading–induced stimuli; ii) determined the effect of single and multiple sessions of mechanical loading in vivo on NOS mRNA expression in extracted total ulnar RNA; and iii) examined the effect of mechanical strain on NO production by primary cultures of osteoblast-like cells derived from rat long bones and calvariae, and osteoblast-like cells and osteocytes isolated and purified from embryonic long bones. Since some of our studies, and those of many others, are conducted in organ culture, we have also examined how mRNA expression of NOS isoforms is influenced by isolation and maintenance of rat ulnae and calvariae explants for 5 h in vitro.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In vivo loading and subsequent treatment of rat ulnae

The ulnae of 16 male 100 g Sprague-Dawley rats (Charles River, Margate, Kent, U.K.) were axially loaded nonsurgically as described previously.(29) Briefly, anesthesia was induced by an intraperitoneal injection of xylazine (11 mg/kg, Rompun; Bayer, Bury St. Edmunds, U.K.), and ketamine hydrochloride (55 mg/kg, Vetalar; Parke Davis and Co., Pontypool, U.K.). In each case the left ulna was loaded and the right ulna served as a control. Dynamic axial loads were applied for 10 minutes at 2 Hz as a ramped square wave, producing a longitudinal peak compressive strain of 2000 μϵ at the medial surface. The waveform was generated using Snapmaster for Windows (HEM Data Corp., Southfield, MI, U.S.A.) and the loads applied by an Instron servohydraulic material testing machine. After the loading period, animals were allowed to recover and were killed 6 h later by an overdose of sodium pentobarbitone PhEur (Euthsate; Willows Francis Veterinary, Crawley, U.K.).

The left and right ulnae were carefully dissected and all their surrounding muscle removed leaving the periosteum intact. The diaphyseal region was dissected and the marrow cavity flushed thoroughly with phosphate-buffered saline (PBS). Thereafter, both control and loaded bones were further subdivided into four groups, each containing four bones. These were snap-frozen in liquid nitrogen for Northern blot hybridization studies.

In a second experiment, the left ulna of 12 250 g Sprague-Dawley male rats were loaded once a day for 5 days with 1200 cycles, at 2 Hz, with peak compressive strains of 4000 μϵ at a strain rate of 30,000 μϵ/s at the medial surface. Animals were killed 6 h after the last loading session, and both left and right (control) ulnae were removed and processed as described above. Control and loaded bones were subdivided into four groups, each containing three bones, and these were snap-frozen in liquid nitrogen for total RNA extraction and subsequent Northern blot hybridization analyses.

In vitro loading and subsequent treatment of rat ulnae and calvariae

Rat ulnae were excised and maintained in culture as described by Cheng et al.(30) Briefly, male 110 g Sprague-Dawley rats (Charles River) were killed by barbiturate overdose, the ulnae dissected aseptically, and adherent muscle removed leaving the periosteum intact. A central 12 mm section of the shaft (cartilaginous ends having been removed and marrow aspirated by flushing with medium) was cultured at the air/medium interface in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% dextran-charcoal purified FCS, 2 mM L-glutamine, penicillin/streptomycin (100 IU/ml:100 mg/ml; Life Technologies, Paisley, Scotland, U.K.) in a humidified 5% CO2 incubator at 37°C.

Following a preincubation period of 5 h, pairs of ulnae were placed in a loading apparatus,(31) and axial compressive loads were applied to generate physiological levels of mechanical strain (−1200 μϵ, for 600 cycles at 1 Hz) to one of each pair of ulnae by a pneumatically operated actuator. Contralateral bones were treated in a similar manner, except the dynamic loads were not applied. Fresh medium was added just prior to treatment and collected at the time(s) specified.

In complementary studies, calvariae were removed from 110 g Sprague-Dawley rats and cut to produce two equal sized rectangular portions of contralateral parietal bones (free of suture and other attendant soft tissue) and cultured as described above. After a preincubation period of 5 h, one of each pair was loaded to produce strains of either −100 or −1000 μϵ (600 cycles at 1 Hz)(31) using apparatus described above. Contralateral bones were held in the loading device for 10 minutes without loading. As above, fresh medium was added prior to treatment and collected at the time(s) specified.

Both calvariae and ulnae, prepared as above, were snap-frozen in liquid nitrogen either immediately post mortem or following the 5 h preincubation for the Northern blot hybridization or chilled in n-hexane (Merck, Lutterworth, U.K.) at −70°C for immunocytochemistry.

Isolation and subsequent treatment of primary osteoblast-like cells

Bone cells were derived by outgrowth from cortical explants of male rat femora (long bone–derived osteoblast-like cells(17,32,33) and calvariae (calvarial-derived osteoblast-like cells). To harvest sufficient cells, an incubation period of 21 days was allowed. Long bone–derived and calvarial-derived osteoblast-like cells were washed in PBS and released with 0.05% trypsin and 0.02% EDTA (Sigma, Ltd., Dorset, U.K.). Cells were seeded onto presterilized plastic strips and maintained for 24 h in DMEM (5% FCS, 2 mM L-glutamine, 50 mg/ml gentamycin, 50 mg/ml ascorbic acid, and 1 mg/ml glucose). Subsequently, cells were deprived of FCS for 24 h prior to any treatment. On several occasions, both long bone–derived and calvarial-derived osteoblast-like cells were tested to confirm their phenotype as measured by alkaline phosphatase activity and osteocalcin expression, collagen mRNA expression, and potential for mineralization as described previously.(17) These cells were subsequently used for in vitro strain application after which an assessment of medium nitrite accumulation was made and their total RNA isolated for Northern blot hybridization.

Isolation and subsequent treatment of chick osteocytes and osteoblasts

Chick osteoblast-like cells were derived by collagenase digestion from cortical explants of 18 day embryonic chick tibiotarsi. Since there are no antibodies available to enable isolation of osteocytes other than chick, it is the only species that can be used for osteocyte purification. Osteocytes were extracted from 18 day embryonic chick tibiotarsi using the chick osteocyte selective monoclonal antibody 7.3(34) (a gift from Prof. P.J. Nijweide), in the immunomagnetic isolation/purification procedure described by van der Plas and Nijweide.(35) Isolated cells were seeded onto plastic strips and maintained as described above.

Strain application to cells in culture

Cultured cells were seeded onto plastic strips which were subjected to controlled mechanical strains by using a four-point bending apparatus.(17) The cells were maintained in serum-free medium, in a humidified atmosphere of 95% air/5%CO2, for 24 h and then tensile mechanical strain applied (peak strain 3400 μϵ, for 600 cycles at 1 Hz). Similar strips with cells attached, subjected to cyclic perturbation of the medium without applied load, served as controls. In all other respects test and control groups were subjected to the same conditions. Medium added just prior to the application of mechanical strain was sampled or collected at the time(s) specified.

RNA isolation and Northern blot hybridization

Snap-frozen bone segments were ground to a fine powder in liquid nitrogen using a baked pestle and mortar. Total RNA was extracted from these preparations and from cultured osteoblasts by using the Ultraspec (Biotex Laboratories, Gaithersburg, MD, U.S.A.) adaptation to methods described by Chomczymski and Saachi.(36) Total RNA was quantitated spectrophotometrically, and equal samples (15 μg) were fractionated onto a 1% agarose gel containing 2.2 M formaldehyde (Merck). The gel was stained with ethidium bromide to visualize ribosomal RNA (28 s and 18 s). The intensity of the ribosomal bands was checked to confirm equal loading of RNA across the gel lanes. The RNA was transferred and fixed onto Hybond-N nylon membrane (Amersham, Amersham, U.K.) using the manufacturer's recommendations.

Rat NOS isoforms and β-actin cDNA-specific sequences were amplified by RT-PCR as described earlier(17) (eNOS primers were a gift from Dr. S. Ralston, U.K.). The amplicons were gel purified and labeled to a high specific activity with [α-32P]deoxycytosine triphosphate (3000 Ci/mmol; Amersham) by random hexanucleotide-primed second-strand synthesis.(37)

The blots were hybridized in Quickhyb hybridization buffer (Stratagene, Cambridge, U.K.) at 68°C and washed under conditions of progressively increasing stringency. The final wash was with 0.1× SSC (20× SSC contains 3 M sodium chloride and 0.3 M sodium citrate, pH 7.0) and 0.1% SDS for 60 minutes at 65°C. Autoradiography was carried out using intensifying screens and Hyperfilm MP (Amersham). The Hyperfilm was preflashed with Sensitise (Amersham) and exposed at −70°C in order to improve the linearity of the response. The intensity of the autoradiogram bands was estimated by scanning densitometry (Bio-Rad Imaging Densitometer with Molecular Analyst Software, Richmond, CA, U.S.A.). The levels of mRNA for NOS isoforms were normalized against β-actin mRNA levels in the same samples.

In situ hybridization for NOS isoforms

Neonatal rats were used for in situ hybridization studies because this technique involves high temperature and stringency washes that usually completely destroy the structure of mineralized bone from older animals. Tibiae were excised from 1-day-old neonatal Wistar rats, fixed in 4% paraformaldehyde, mounted, and quick-frozen. Serial sections (7 μm) were cut and captured onto Vectabond-treated slides (Vector Laboratories, Ltd., Peterborough, U.K.). A 324 bp cDNA complementary to the rat eNOS coding region inserted in a bluescript vector was transcribed to RNA and labeled with digoxigenin (Riboprobe System-T7; Promega, Southampton, U.K.). Sections were treated with proteinase K (10 μg/ml) then incubated overnight with hybridization solution (50% formamide, 5× SSC, 5× Denhard's solution, 100 μg/ml denatured herring sperm DNA, 10% dextran sulfate, and digoxigenin-labeled probe). Negative controls were treated with 400 μg/ml RNAse for 30 minutes. Sections were washed (2× SSC, RT), treated with 10 μg/ml RNAse, washed again (2× SSC, 1× SSC at 50°C), and finally developed with ALP-conjugated antidigoxigenin using a Dig Nucleic Acid detection kit (Boehringer Mannheim UK Ltd., Lewes, U.K.).

Immunocytochemistry for NOS isoforms

Monoclonal antibodies to eNOS were raised against a 20.4 kDa protein fragment corresponding to amino acids 1034–1029 of human NOS and purified from mouse ascites (Transduction Laboratories, Lexington, KY, U.S.A.). Polyclonal antibodies for both iNOS and nNOS were raised against corresponding synthetic peptides and were kindly donated by Drs. S. Moncada and V. Riveros-Moreno (London, U.K.).

Unfixed (10 μm) transverse sections of calcified rat calvariae or ulnae (from 90 g or 125 g male Sprague-Dawley rats) were cut in a Bright's cryostat (Bright's Instruments, Huntingdon, U.K.) with the cabinet temperature at or below −25°C. The sections were fixed for 10 minutes in an ethanol/water mixture (3:1), their endogenous peroxidase activity inhibited by incubation in 0.3% hydrogen peroxide in methanol for 20 minutes and subsequently exposed to either normal goat or horse sera for 30 minutes. Thereafter, sections were transferred to a humidified chamber and incubated overnight at 4°C with either eNOS (diluted 1:2000), iNOS (diluted 1:1500), or nNOS (diluted 1:1000) antibodies. Following a brief wash in 0.1 M PBS at pH 7.4, sections were incubated with biotinylated goat anti-rabbit IgG or horse anti-mouse IgG (Vector Laboratories) at 1:100 dilution in PBS containing 0.1% bovine serum albumin (w/v) for 30 minutes, washed, and incubated for 60 minutes with 1:200 avidin biotinylated horseradish peroxidase complex (ABC complex; Vector Laboratories) in PBS at pH 7.4. Immunoreactivity (activity of immunologically bound enzyme conjugate) was visualized by reaction in a chromogenic solution containing glucose oxidase-3.3′diaminobenzidine-nickel ammonium sulfate. Omission of primary anti-NOS antibodies was used as a negative control. Histology of these sections was compared with serial sections stained with either conventional hematoxylin and eosin or with 0.1% toluidine blue (in 0.1 M acetate buffer, pH 6.1), washed, dehydrated, and mounted in DPX (Merck).

Measurement of nitrite concentration

The supernatant of each sample of medium was removed and stored at −20°C until the nitrite concentration (a stable metabolite of NO) was assessed by chemiluminescence.(38,39) NOS activity was confirmed by inhibition using either L-NAME (10–100 mM; Sigma) or L-N5-(1-Iminoethyl) ornithine (L-NIO; 10–100 μM, Calbiochem, La Jolla, CA, U.S.A.) added to culture medium 30 minutes prior to treatment, and during subsequent treatment (strain application of physiological magnitude).

Statistics

Data are presented as mean values ± SEM. Quantitative data were analyzed by unpaired t-test and considered significantly different at p < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

NOS mRNA expression in bones and primary bone cell cultures

Northern blot hybridization analysis of total RNA extracted immediately post mortem from rat ulnae and calvariae and from long bone–derived primary osteoblast-like cells showed the presence of eNOS mRNA transcripts (Fig. 1). In contrast, there was little or no evidence of expression of either nNOS or iNOS mRNA transcripts. Total RNA extracted from rat brain and cultured rat bones served as positive controls for nNOS and iNOS mRNA expression, respectively.

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Figure FIG. 1.. Expression, by Northern analysis, of the mRNA for NOS isoforms in total RNA extracted from: lane 1, rat cerebellum; lane 2, fresh ulnar biopsies; lane 3, ulnae cultured for 5 h in DMEM in the presence of 10% FCS; lane 4, ulnae cultured for 5 h in DMEM in the absence of FCS; lanes 5–8, long bone–derived osteoblasts; lane 9, calvariae fresh; lane 10, calvariae cultured for 5 h in DMEM in the presence of 10% FCS; and lane 11, calvariae cultured for 5 h in the absence of FCS. The lower panel shows ethidium bromide–stained total RNA with discrete 28S and 18S ribosomal bands.

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NOS localization by immunocytochemistry and in situ hybridization

NOS isoforms identified by immunostaining showed distinct patterns of expression which were consistent with the observations from the Northern blot hybridizations. In ulnae, eNOS was present in both osteoblasts and osteocytes, being particularly prominent in osteocytes of the midcortical region (Fig. 2A). Little if any iNOS immunoreactivity was detected in either of these cell types in fresh rat ulnae (Fig. 2B). In calvaria, weak immunoreactivity for eNOS was seen in osteoblasts but was absent from osteocytes (Fig. 2C). As in the rat ulnae, neither calvarial osteoblasts nor osteocytes showed significant immunoreactivity for iNOS (Fig. 2D). Weak, if any, nNOS immunoreactivity was seen in some calvarial and ulnar osteoblasts, some calvarial osteocytes, and no significant nNOS labeling was evident in ulnar osteocytes (data not shown).

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Figure FIG. 2.. Endothelial NOS expression in rat ulna, calvaria, and tibia. Immunocytochemistry revealed: (A) eNOS-immunoreactive osteocytes (arrows) in the cortical midregion of adult rat ulna; (B) an absence of iNOS-immunoreactivity in osteocytes from similar tissue (arrows); (C) osteoblast-like cells at the periosteal surface of rat calvaria expressing eNOS; and (D) the absence of iNOS immunoreactivity in serial section. In situ hybridization revealed: (E) eNOS mRNA in osteocytes in neonatal tibia; (F) absence of eNOS mRNA in a serial section treated with RNAse. (A–F) Magnification ×440, bar = 25 μm.

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In situ hybridization analysis of rat long-bone sections showed prominent eNOS mRNA expression in tibial osteocytes and osteoblasts (Figs. 2E and 2F). These findings are consistent with the pattern of mRNA and protein expression in fresh ulnae as shown by Northern blot hybridization and immunocytochemistry.

Effect of organ culture on NOS mRNA levels

Because many studies are undertaken in organ culture the aim of these studies was to establish whether in vivo NOS isoform mRNA expression profiles were affected by isolation and subsequent organ culture. Five hour culture of explants of ulnae and calvariae, with or without FCS (10%, charcoal-dextran stripped), produced a marked stimulation in iNOS mRNA levels but no change in the expression of either eNOS or nNOS (Fig. 1).

Effect of in vivo mechanical-loading on NOS isoform mRNA levels

Microdensitometric assessment of Northern blot hybridization analysis of total RNA extracted from ulnae 6 h after the final loading session showed that neither a single period of mechanical loading in vivo (−2000 μϵ, for 1200 cycles at 2 Hz; data not shown) nor five consecutive in vivo loading sessions (−4000 μϵ, for 1200 cycles at 2 Hz; Fig. 3) resulted in any statistically significant changes in eNOS mRNA transcript levels. There was similarly no change in the low levels of nNOS or iNOS mRNA transcripts evident in these bones (data not shown).

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Figure FIG. 3.. Expression of the mRNA for eNOS in control and in vivo loaded rat ulnae (–4000 μϵ for 1200 cycles at 2 Hz, once a day for 5 days). Total RNA (20 μg) was subjected to Northern analysis. The lower panel shows ethidium bromide–stained total RNA with discete 28S and 18S ribosomal bands.

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Effect of mechanical loading on NO release in organ culture

Dynamic axial compressive loading of ulna explants at 1 Hz for 10 minutes, producing peak strains of −1200 μϵ resulted in a rapid and statistically significant increase in medium nitrite concentration (54.8 ± 9.4% increase, p = 0.004) (Fig. 4A). In contrast, loading explants of rat calvariae (1 Hz for 10 minutes), at peak strain levels similar to those measured physiologically at this location (−100 μϵ) and also an order of magnitude higher (−1000 μϵ, data not shown), did not significantly increase nitrite concentration in the medium (10 ± 8% increase; p = NS; Fig. 4A).

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Figure FIG. 4.. Effect of mechanical strain on nitrite release (percentage of respective control, mean ± SEM) in medium conditioned by: (A) rat long bones (ulnae, six experiments, n is equal to at least four pairs of bones in each, control = 0.50 ± 0.08, load = 0.77 ± 0.1 [unnormalized data]) and calvarial bones (parietal, three experiments, n is equal to at least four pairs of bones in each, control = 0.72 ± 0.07, load = 0.76 ± 0.13); (B) Rat long bone–derived primary osteoblasts (femoral, LOBS, n = 5 experiments, control = 0.76 ± 0.16, load = 1.32 ± 0.27 [unnormalized data]) and calvarial-derived primary osteoblasts (COBS, n = 6, control = 1.07 ± 0.047, load = 3.28 ± 1.41). *Significance at level of p < 0.05. In all instances, the medium was collected within 5 minutes after the cessation of mechanical strain application (3400 μϵ for 600 cycles at 1 Hz).

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Comparison of NO production by calvarial and long bone–derived rat osteoblast-like cells in culture

In contrast to the situation in explants, cultured osteoblasts derived from both ulnae and calvaria responded to a single 10-minute period of dynamic mechanical strain by significant increases in medium nitrite (Fig. 4B). Calvarial osteoblasts produced more marked increases in the rate of nitrite accumulation than those from the ulnae (ulnar osteoblasts showed an increase of 81 ± 32% and calvarial osteoblasts 194 ± 44%, p < 0.05; Fig. 4B).

Comparison of strain-induced NO production by osteoblasts and osteocytes both derived from embryonic chick tibiotarsi

In a direct comparison of the response to similar levels of tensile mechanical strain (3400 μϵ, 2 Hz for 10 minutes), cultures of purified embryonic osteocytes derived from chick tibiae produced significantly greater strain-related nitrite accumulation in the medium (8.32 ± 2.40 pM NO/cell/h, p < 0.05) than osteoblast-like cells derived from the same bones (2.40 ± 0.10 pM NO/cell/h, p < 0.05; Fig. 5).

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Figure FIG. 5.. (A) Alterations in nitrite (μM) release by isolated, purified, embryonic chick long bone–derived osteocytes (O.cytes) and primary osteoblasts (O.blasts) induced by mechanical strain (3400 μϵ for 600 cycles at 1 Hz). (B) Rate of strain-induced nitrite accumulation in medium conditioned by embryonic chick O.cytes (n = 3 experiments) and O.blasts (n = 5 experiments). *Significance at level of p < 0.05.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The results presented here show that eNOS is the predominant NOS isoform expressed by resident bone cells of the osteoblast/osteocyte lineage. Further, because isolated osteocytes produce greater increases in strain-induced NO release than isolated osteoblasts and are also more numerous, it is likely that the NO produced as a result of mechanical loading in intact long bones is predominantly derived from eNOS in resident osteocytes. Since it has been shown that NO produced at the time of loading is obligatory for the subsequent osteogenic response,(24,25) our findings imply that such osteocyte-derived NO may play an important regulatory role in the adaptive response. Our results also show that exposure to mechanical loading in vivo has no effect on the levels of eNOS mRNA expression or the other NOS isoforms. Furthermore, calvaria, which normally experience only low strains, and which previously have been reported to be unresponsive to loading,(31) fail to show any loading-related increases in NO production when maintained in explant culture. This contrasts with the situation in ulnae.

Our data on the predominance of eNOS expression from Northern blot analysis contrasts with those from previous RT-PCR studies,(22,26,40) which collectively suggest that all three NOS isoforms are expressed in a variety of osteoblast-like cells, cell lines, and fresh bone biopsies. Many studies have observed a similar discrepancy, where RT-PCR detectable transcripts, presumably with very low abundance, were undetectable by either Northern blot analysis or immunocytochemistry.(41–45) This difference may be ascribed to the RT-PCR technique which is useful for detecting particularly rare transcripts but less useful for comparing relative abundance. Furthermore, it is possible that the expression profiles of the different NOS isoforms detectable by RT-PCR may be skewed by different efficiencies with which the distinct primers amplify their respective mRNAs. Our findings using the less sensitive, yet quantitatively more precise approach of Northern blot hybridization analysis(17) suggest that eNOS is the predominant isoform expressed in cells of the osteoblastic lineage in long bones in situ. This interpretation is also supported by our findings from in situ hybridization and immunocytochemistry.

A number of recent studies(21,46–48) show that the rapid increases in NO production stimulated by loading do not persist after the removal of mechanical stimulation. Using rat tail vertebrae loaded in vitro, we have shown that loading-induced increases in NO release are only evident during each (of three) distinct loading episode, and that during intervening periods (24 h) NO release returned to basal levels.(22) Our results support the notion that rapid loading-related increases in NO release are mediated by eNOS activation and that no transcriptional up-regulation in mRNA levels for any of the NOS isoforms is induced. That strain should stimulate the release of NO but not upregulate expression of the isoform responsible for producing it is consistent with NO being associated with strain measurement. In a “measuring” system it is not desirable that the sensitivity of the system should be influenced by previous exposure to the parameter being measured. This conclusion appears to be consistent with the magnitude- dependent increases in NO shown by cultured ulnae exposed to increasing strains.(22) This interpretation is also supported by both our findings from in situ and by previous immunocytochemistry.(49–51)

By subjecting osteocytes and osteoblasts derived from the same embryonic chick long bones to similar strains, it is evident that osteocytes are stimulated to release more NO per cell per hour than osteoblasts. This is consistent with the intense labeling of mid-cortical osteocytes for eNOS. Since osteocytes are also the most numerous cell type in bone,(52) these findings suggest that the predominant source of NO, in normal and mechanically loaded intact long bones, is likely to be derived from rapid, post-transcriptional activation of eNOS in osteocytes. This contrasts with osteocytes generally lower level of activity than osteoblasts(53) and suggests that NO production is an important activity of these cells.

The situation in long bones appears to be substantially different from that in calvaria. Immunostaining did not show any eNOS positive osteocytes in rat calvaria, and mechanical loading of rat calvaria did not produce any significant short-term increases in nitrite accumulation in the medium. However, eNOS mRNA is detectable in total RNA extracted from calvariae, suggesting limited translation of eNOS mRNA and NO production. These observations are compatible with the different roles of calvariae and long bones.(31)

In conclusion, our present results are consistent with rapid loading-related increases in NO production being an integral part of bone cells' early response to mechanical loading. This response may be controlled by osteocyte-derived NO released as a result of rapid activation of eNOS but does not appear to involve the transcriptional up-regulation of any of the NOS isoforms. These results support the hypothesis that in long bones, osteocyte eNOS-derived NO release may constitute an integral part of the adaptive response of long bones to mechanical loading.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by grants from The Wellcome Trust, The BBSRC, The MRC, and The Dunhill Medical Trust.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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