Expression of endothelial nitric oxide synthase protein is not necessary for mechanical strain-induced nitric oxide production by cultured osteoblasts

Summary

Regulation of nitric oxide (NO) production is considered essential in mechanical load-related osteogenesis. We examined whether osteoblast endothelial NO synthase (eNOS)-derived NO production was regulated by HSP90. We found that HSP90 is essential for strain-related NO release but appears to be independent of eNOS in cultured osteoblasts.

Introduction

NO is a key regulator of bone mass, and its production by bone cells is regarded as essential in mechanical strain-related osteogenesis. We sought to identify whether bone cell NO production relied upon eNOS, considered to be the predominant NOS isoform in bone, and whether this was regulated by an HSP90-dependent mechanism.

Methods

Using primary rat long bone-derived osteoblasts, the ROS 17/2.8 cell line and primary mouse osteoblasts, derived from wild-type and eNOS-deficient (eNOS^?/?) mice, we examined by immunoblotting the expression of eNOS using a range of well-characterised antibodies and extraction methods, measured NOS activity by monitoring the conversion of radiolabelled l-arginine to citrulline and examined the production of NO by bone cells subjected to mechanical strain application under various conditions.

Results

Our studies have revealed that eNOS protein and activity were both undetectable in osteoblast-like cells, that mechanical strain-induced NO production was retained in bone cells from eNOS-deficient mice, but that this strain-related induction of NO production was, however, dependent upon HSP90.

Conclusions

Together, our studies indicate that HSP90 activity is essential for strain-related NO release by cultured osteoblasts and that this is highly likely to be achieved by an eNOS-independent mechanism.     

The role of nitric oxide in the mechanical repression of RANKL in bone stromal cells

Both mechanical loading and nitric oxide (NO) have positive influences on bone mass. NO production is induced by mechanical strain via upregulation of eNOS mRNA and protein, the predominant NOS in adult bone. At the same time, strain causes decreased expression of RANKL, a factor critical for osteoclastogenesis. In this study, we harvested primary stromal cells from wild-type (WT) and eNOS(-/-) mice to test whether induction of NO by mechanical strain was necessary for transducing mechanical inhibition of RANKL. We found that strain inhibition of RANKL expression was prevented by NOS inhibitors (L-NAME and L-NMMA) in WT stromal cells. Surprisingly, stromal cells from eNOS(-/-) mice showed significant mechanical repression of RANKL expression (p<0.05). Mechanical strain still increased NO production in the absence of eNOS, and was abolished by SMTC, a specific nNOS inhibitor. nNOS mRNA and protein expression were increased by strain in eNOS(-/-) but not in WT cells, revealing that nNOS was mechanically sensitive. When NO synthesis was blocked with either SMTC or siRNA targeting nNOS in eNOS(-/-) cells however, strain still was able to suppress RANKL expression by 34%. This indicated that strain suppression of RANKL can also occur through non-NO dependent pathways. While our results confirm the importance of NO in the mechanical control of skeletal remodeling, they also suggest alternative signaling pathways by which mechanical force can produce anti-catabolic effects on the skeleton.     

Nitric oxide in fracture repair. Differential localisation, expression and activity of nitric oxide synthases

Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, responsible for the generation of NO, are expressed during the healing of fractures. To localise the sites of expression compared with those in normal bone we made standardised, stabilised, unilateral tibial fractures in male Wistar rats. Immunostaining was used to determine the precise tissue localisation of the different NOS isoforms. Western blotting was used to assess expression of NOS isoform protein and L-citrulline assays for studies on NOS activity. Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the early phase of fracture repair. Western blot and image analysis confirmed this initial increase. Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture healing. Western blotting showed a reduction in iNOS during the early healing period. Significantly reduced calcium-independent NOS activity was also seen. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blood flow recognised during fracture healing. eNOS expression in osteocytes may occur in response to changes in either mechanical or local fluid shear stress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair.     

Involvement of different ion channels in osteoblasts'' and osteocytes'' early responses to mechanical strain

The involvement of functional ion channels in previously documented early responses of osteocytes and osteoblasts to mechanical strain in bone tissue was investigated in explants of rat ulnae by the use of ion channel blockers. Gadolinium chloride (a blocker of stretch/shear-sensitive cation channels) elevated basal prostaglandin (PG) E₂ and prostacyclin (PGI₂) release and osteocyte glucose-6-phosphate dehydrogenase (G6PD) activity, but was associated with a reduction in basal nitric oxide (NO) production. Gadolinium abolished loading-related increases in the release of PGI₂ and NO and osteocyte G6PD activity. Gadolinium also reduced the loading-related release of PGE₂ assumed to originate from osteoblasts and the magnitude of loading-related increases in G6PD activity in these cells. Nifedipine (a blocker of L-type voltage-dependent calcium channels) had no effect on basal levels of prostanoid or NO release, or G6PD activity in osteocytes or osteoblasts, and did not affect loading-related release of PGI₂ or increase in osteocyte G6PD. However, nifedipine prevented loading-related increases in PGE₂ and NO release and osteoblast G6PD activity. These results are consistent with osteocytes' response to bone loading requiring activatable ion channels sensitive to gadolinium, but not those sensitive to nifedipine. In osteoblasts, the early responses to bone loading appear to be associated with ion channels sensitive to gadolinium and nifedipine; however, the nifedipine-sensitive channels seem to have the dominant effect.     

Expression and functional role of nitric oxide synthase isoforms in human osteoblast-like cells

Previous studies have shown evidence of constitutive and cytokine-inducible nitric oxide (NO) synthase activity in cultured osteoblast-like cells from various species. Although cytokine-induced NO production has been found to inhibit osteoblast growth, the role of constitutive NO production in regulating osteoblast function is less clear and the isoforms of nitric oxide synthase (NOS) that are expressed by human osteoblasts have not been determined. Here, we investigated NOS expression in cultured human osteoblast-like cells and studied the effects of constitutive and cytokine-induced NO on osteoblast growth and differentiation. Low levels of NO were produced constitutively by osteoblast-like cells as reflected by analysis of medium nitrite concentrations, and evidence of ecNOS mRNA, protein, and bioactivity was found in primary osteoblasts (hOBs), TE85, and MG63 osteosarcoma cells. None of the osteoblast-like cells expressed nNOS, however, and iNOS was produced only by hOB cells after stimulation with the cytokines IL-1beta, TNF-alpha, and IFN-gamma. The NOS inhibitor, L-NMMA, did not affect growth or alkaline phosphatase activity in unstimulated osteoblasts. Incubation of hOB cells with cytokines inhibited growth and stimulated alkaline phosphatase activity and these effects were abrogated by L-NMMA. Cytokines also inhibited growth of TE85 cells and MG63 cells, but these effects appeared to be NO independent because they were not influenced by L-NMMA. Our experiments show that human osteoblasts constitutively produce NO through the ecNOS pathway, but demonstrate that this does not appear to exert an appreciable effect on osteoblast growth or differentiation under basal conditions. In contrast, IL-1beta, TNF-alpha, and IFN-gamma exerted growth-inhibiting and differentiation-inducing effects on osteoblasts that were partly NO dependent, indicating that NO may act predominantly as a modulator of cytokine-induced effects on osteoblast function.     

Endothelial Nitric Oxide Synthase is Not Essential for Nitric Oxide Production by Osteoblasts Subjected to Fluid Shear Stress In Vitro

Endothelial nitric oxide synthase (eNOS) has long been held responsible for NO production by mechanically stimulated osteoblasts, but this has recently been disputed. We investigated whether one of the three known NOS isoforms is essential for NO production by mechanically stimulated osteoblasts in vitro and revisited the bone phenotype of the eNOS^?/? mouse. Osteoblasts, obtained as outgrowths from mouse calvaria or long bones of wild-type (WT), eNOS^?/?, inducible NOS^?/? (iNOS^?/?), or neuronal NOS^?/? (nNOS^?/?) mice, were subjected to mechanical stimulation by means of pulsating fluid flow (PFF); and NO production was determined. Tibiae and femora from 8-week-old mice were subjected to μCT and three-point bending tests. Deletion of single NOS isoforms did not lead to significant upregulation of alternate isoforms in cultured osteoblasts from WT, eNOS^?/?, iNOS^?/?, or nNOS^?/? mice. Expression of eNOS mRNA in osteoblasts was below our detection limit, and no differences in growth between WT and eNOS^?/? osteoblasts were found. PFF increased NO production by approximately fourfold in WT and eNOS^?/? osteoblasts and significantly stimulated NO production in iNOS^?/? and nNOS^?/? osteoblasts. Tibiae and femora from WT and eNOS^?/? mice showed no difference in bone volume and architecture or in mechanical parameters. Our data suggest that mechanical stimuli can enhance NO production by cultured osteoblasts singly deficient for each known NOS isoform and that lack of eNOS does not significantly affect bone mass and strength at 8 weeks of age. Our data challenge the notion that eNOS is a key effector of mechanically induced bone maintenance.     

Nitric oxide signaling in mechanical adaptation of bone

One of the most serious healthcare problems in the world is bone loss and fractures due to a lack of physical activity in elderly people as well as in bedridden patients or otherwise inactive youth. Crucial here are the osteocytes. Buried within our bones, these cells are believed to be the mechanosensors that stimulate bone formation in the presence of mechanical stimuli and bone resorption in the absence of such stimuli. Intercellular signaling is an important physiological phenomenon involved in maintaining homeostasis in all tissues. In bone, intercellular communication via chemical signals like NO plays a critical role in the dynamic process of bone remodeling. If bones are mechanically loaded, fluid flows through minute channels in the bone matrix, resulting in shear stress on the cell membrane that activates the osteocyte. Activated osteocytes produce signaling molecules like NO, which modulate the activity of the bone-forming osteoblasts and the bone-resorbing osteoclasts, thereby orchestrating bone adaptation to mechanical loading. In this review, we highlight current insights in the role of NO in the mechanical adaptation of bone mass and structure, with emphasis on its role in local bone gain and loss as well as in remodeling supervised by osteocytes. Since mechanical stimuli and NO production enhance bone strength and fracture resistance, these new insights may facilitate the development of novel osteoporosis treatments.     

Role of inducible nitric oxide synthase in skeletal adaptation to acute increases in mechanical loading.

To clarify the role of nitric oxide (NO) in regulation of bone metabolism in response to skeletal loading, we examined inducible NO synthase (iNOS) gene knockout mice in the tail-suspension model. Histomorphometric analyses of proximal tibias revealed that 7 days of tail suspension decreased the bone volume (BV/TV) and bone formation rate (BFR/BS) and increased the osteoclast surface (Oc.S/BS) in mice with all iNOS genotypes. Both iNOS+/+ and iNOS+/- mice responded to subsequent 14-day reloading, with increases in BV/TV and BFR/BS and a decrease in Oc.S/BS, whereas these responses were abolished in iNOS-/- mice. The osteoblasts flattened after tail suspension appeared cuboidal during subsequent reloading. Immunoreactivity for iNOS was detected in these osteoblasts and osteocytes by immunohistochemistry. These defective responses after reloading were rescued in iNOS-/- mice by treatment with an NO donor nitroglycerine (NG). Conversely, the responses in iNOS+/+ mice were inhibited by treatment with an NOS inhibitor aminoguanidine (AG). In bone marrow cell cultures, mineralized nodules derived from iNOS-/- mice after reloading were significantly reduced. Taken together, our results suggest that NO generated by iNOS in osteoblasts plays a critical role in adjusting bone turnover and increasing osteogenic activity in response to the acute increase in mechanical loading after tail suspension.     

Osteocytes use estrogen receptor alpha to respond to strain but their ERalpha content is regulated by estrogen.

The role of mechanical strain and estrogen status in regulating ERalpha levels in bone cells was studied in female rats. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas habitual strain and artificial loading has only a small but positive effect, except on the ulna's medial surface, where artificial loading stimulates reversal of resorption to formation. INTRODUCTION: Osteoporosis is the most widespread failure of bones' ability to match their architectural strength to their habitual load bearing. In men and women, the severity of bone loss is associated with bioavailability of estrogen. This association could result from the estrogen receptor (ER) involvement in bone cells' adaptive response to loading. MATERIALS AND METHODS: In vivo semiquantitative analysis of the amount of ERalpha protein per osteocyte was performed in immuno-cytochemically stained sections from control and loaded rat ulna, as well as tibias of ovariectomy (OVX) and sham-operated female rats. In vitro, the effect of exogenous estrogen (10(-8) M) and mechanical strain (3400 microepsilon, 1 Hz, 600 cycles) on the expression of ERalpha mRNA levels was assessed in ROS 17/2.8 cells in monolayers using real-time PCR and ER promoter activity. ERalpha translocation in response to exogenous estrogen and mechanical strain was assessed in both ROS 17/2.8 and MLO-Y4 cells. RESULTS: More than 90 percent of tibial osteocytes express ERalpha, the level/osteocyte being higher in cortical than cancellous bone. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas in the ulna habitual strain and that caused by artificial loading had only a small but positive effect, except on the medial surface, where loading stimulates reversal of resorption to formation. In unstimulated osteocytes and osteoblasts in situ, and osteocyte-like and osteoblast-like cells in vitro, ERalpha is predominantly cytoplasmic. In vitro, both strain and estrogen stimulate transient ERalpha translocation to the nucleus and transient changes in ERalpha mRNA. Strain but not estrogen also induces discrete membrane localization of ERalpha. CONCLUSIONS: Bone cells' responses to both strain and estrogen involve ERalpha, but only estrogen regulates its cellular concentration. This is consistent with the hypothesis that bone loss associated with estrogen deficiency is a consequence of reduction in ERalpha number/activity associated with lower estrogen concentration reducing the effectiveness of bone cells' anabolic response to strain.     

The role of prostaglandins and nitric oxide in the response of bone to mechanical forces.

We have developed an experimental model whereby bone is exposed to a brief episode of mechanical stimulation, which is followed by bone formation. The earliest response is in osteocytes, which express c-fos and insulin-like growth factor (IGF-1) within 30-60min. Thirty-six to 72h after loading bone matrix gene expression occurs on bone surfaces. The osteogenic response can be suppressed by a single dose of nitric oxide synthase (NOS) or prostaglandin (PG) synthase inhibitors, if these are administered just before mechanical stimulation: similar doses after stimulation have no effect. There is a later phase of indomethacin-sensitivity associated with COX-2 expression in bone at 6h. Thus, mechanically induced osteogenesis involves early expression of c-fos and IGF-1 by osteocytes, which are believed to be the strain-sensitive cells in bone. Both NOS and PG synthase, either in parallel or in sequence, are crucial to the initial transduction of the mechanical stimulus into an osteogenic response.     

Effects of hind limb unloading and reloading on nitric oxide synthase expression and apoptosis of osteocytes and chondrocytes

In rat bone, the absence of mechanical load results in a reduction in bone formation, inhibition of longitudinal growth, and a decrease in the number of osteoblasts and osteoprogenitors in cancellous bone. Unloading has also been linked to an increase in apoptosis of osteocytes and chondrocytes through production of nitric oxide (NO) and increased expression of NO synthases (NOS). Reloading results in recovery of bone volume within 14 days, although osteoblast and osteoclast numbers remain below control values, suggesting decreased bone turnover. This study was designed to evaluate the effects of hind limb unloading and subsequent reloading on apoptosis, NOS expression, and histomorphometric parameters in trabecular and cortical bone, articular cartilage, and growth plate cartilage of the proximal tibia of the hind limbs. Compared to ambulatory controls, 2 weeks of unloading resulted in a 66% increase in the percentage of apoptotic osteocytes in the trabecular metaphysis, a 14% increase in osteoclast number and a 48% decrease in bone volume. The percentage of eNOS- or iNOS-positive osteocytes was unchanged. Upon reloading, the percentage of apoptotic osteocytes and bone volume returned to baseline whereas the percentage of iNOS-positive osteocytes increased by 50% and osteoclast number decreased by 30% compared to ambulatory controls. More striking changes were observed in articular and growth plate cartilage. Unloading resulted in a 230% increase in apoptotic chondrocytes, a 400% increase in iNOS-positive chondrocytes, and a 17% reduction in width in articular cartilage. Reloading for 2 weeks resulted in partial recovery. Chondrocytes in the proliferative and hypertrophic zones of the growth plate responded similarly to those in the articular cartilage. In summary, we observed that 14 days of unloading increased apoptosis of osteocytes and chondrocytes. This was associated with an increase in the proportion of iNOS-positive chondrocytes whereas the proportion of iNOS-positive osteocytes remained unchanged. Reloading for 14 days restored osteocyte apoptosis to control levels but the percentage of iNOS- and eNOS-positive osteocytes increased in reloaded bone compared to controls. This was associated with a decrease in osteoclast number. In cartilage, reloading for 2 weeks did not result in a return to baseline in any of the parameters measured, suggesting that the effects of unloading on articular cartilage and the growth plate last longer than those in bone and may have prolonged effects on joint biomechanics and longitudinal bone growth.     

Effects of nitric oxide synthase inhibitors on bone formation in rats

NO is synthesized from -arginine by at least three isoforms of nitric oxide synthase (NOS) and is known to function as a vasodilator and neurotransmitter. NO is produced by bone cells but its function in bone biology is, as yet, unclear. We hypothesized that NOS mediates bone formation in remodeling regions of the skeleton. We studied the effects of two NOS inhibitors: N^G-nitro- -arginine methyl ester ( -NAME), which is a general inhibitor of NOS activity and is known to inhibit the vasodilatory effects of the endothelial NOS (eNOS) isoform; and aminoguanidine, which is a selective inhibitor of the inducible NOS (iNOS) isoform. Our hypothesis was tested by treating rats with NOS inhibitors and measuring bone formation rates in the tibial epiphysis and diaphysis. Bone formation indices were measured using standard bone histomorphometry. -NAME treatment significantly raised mean arterial blood pressure (MAP). This effect was partially reversed by addition of -arginine. Aminoguanidine had no significant effect on MAP, indicating that it did not block eNOS. The treatments also had substantial effects on bone formation in remodeling trabecular bone. -NAME did not significantly change trabecular bone formation rate, whereas aminoguanidine reduced bone formation rate in the tibial epiphysis by 79% compared with control. This reduction was completely reversed by -arginine, suggesting that bone formation during remodeling is, in part, mediated through -arginine metabolism. No effect of aminoguanidine on bone formation was seen in the tibial diaphysis, a site that undergoes minimal bone remodeling. This finding suggests that the -arginine-NO pathway is important in bone remodeling.     

<Emphasis Type="Italic">In Vivo</Emphasis> Mechanical Loading Modulates Insulin-Like Growth Factor Binding Protein-2 Gene Expression in Rat Osteocytes

Mechanical stimulation is essential for maintaining skeletal integrity. Mechanosensitive osteocytes are important during the osteogenic response. The growth hormone-insulin-like growth factor (GH-IGF) axis plays a key role during regulation of bone formation and remodeling. Insulin-like growth factor binding proteins (IGFBPs) are able to modulate IGF activity. The aim of this study was to characterize the role of IGFBP-2 in the translation of mechanical stimuli into bone formation locally in rat tibiae. Female Wistar rats were assigned to three groups (n = 5): load, sham, and control. The four-point bending model was used to induce a single period of mechanical loading on the tibial shaft. The effect on IGFBP-2 mRNA expression 6 hours after stimulation was determined with nonradioactive in situ hybridization on decalcified tibial sections. Endogenous IGFBP-2 mRNA was expressed in trabecular and cortical osteoblasts, some trabecular and subendocortical osteocytes, intracortical endothelial cells of blood vessels, and periosteum. Megakaryocytes, macrophages, and myeloid cells also expressed IGFBP-2 mRNA. Loading and sham loading did not affect IGFBP-2 mRNA expression in osteoblasts, bone marrow cells, and chondrocytes. An increase of IGFBP-2 mRNA-positive osteocytes was shown in loaded (1.68-fold) and sham-loaded (1.35-fold) endocortical tibial shaft. In conclusion, 6 hours after a single loading session, the number of IGFBP-2 mRNA-expressing osteocytes at the endosteal side of the shaft and inner lamellae was increased in squeezed and bended tibiae. Mechanical stimulation modulates IGFBP-2 mRNA expression in endocortical osteocytes. We suggest that IGFBP-2 plays a role in the lamellar bone formation process.     

Patterns of osteocytic endothelial nitric oxide synthase expression in the femoral neck cortex: differences between cases of intracapsular hip fracture and controls

Evidence indicates that extensive amalgamation of adjacent resorbing osteons is responsible for destroying the microstructural integrity of the femoral neck’s inferior cortex in osteoporotic hip fracture. Such osteonal amalgamation is likely to involve a failure to limit excessive resorption, but its mechanistic basis remains enigmatic. Nitric oxide (NO) inhibits osteoclastic bone destruction, and in normal bone cells its generation by endothelial nitric oxide synthase (eNOS, the predominant bone isoform) is enhanced by mechanical stimuli and estrogen, which both protect against fracture. To determine whether eNOS expression in osteocytes reflects their proposed role in regulating remodeling, we have examined patterns of osteocyte eNOS immunolabeling in the femoral neck cortex of seven cases of hip fracture and seven controls (females aged 68–96 years). The density of eNOS⁺ cells (mm⁻²) was 53% lower in the inferior cortex of the fracture cases (p < 0.0004), but was similar in the superior cortex. eNOS⁺ osteocytes were, on average, 22% further from their nearest blood supply, than osteocytes in general (p < 0.0001) and the nearest eNOS⁺ osteocyte was 57% further from its nearest canal surface (p < 0.0001). This differential distribution of eNOS⁺ osteocytes was significantly more pronounced in the cortices of fracture cases (p < 0.0001). We conclude that the normal regional and osteonal pattern of eNOS expression by osteocytes is disrupted in hip fracture, particularly at sites that are loaded most by physical activity. These results suggest that eNOS⁺ osteocytes may normally act as sentinels confining resorption within single osteons. A reduction in their number, coupled to an increase in their remoteness from canal surfaces, may thus permit the irreversible merging of resorbing osteons, and thus contribute to the marked increase in the fragility of osteoporotic bone.     

骨质疏松发生一氧化氮机制及雌激素调控作用研究

目的　研究骨质疏松发生的一氧化氮机制及雌激素防治骨质疏松作用的一氧化氮通路 ,深入理解骨质疏松症病理过程中一氧化氮作用环节。方法　 45只SD雌性大鼠分为①假手术组(Sham组 ) ;②卵巢切除组 (OVX组 ) ;③OVX +倍美力治疗 (Premarin组 )。进行iNOS原位杂交及iNOS、eNOS免疫组化研究 ,测定其平均灰度值及平均积分光密度 (ODI)作统计指标。结果　iNOS原位杂交实验结果显示 :正常大鼠去卵巢后 ,骨髓腔内及骨小梁表面iNOSmRNA有强阳性表达 ,差异有非常显著性 (P <0 0 1) ,iNOS免疫组化实验结果也显示OVX组iNOS阳性表达明显较Sham组增加 ;而Pre marin组iNOS表达强度较OVX组明显降低 (P <0 0 5 )。OVX组与Sham组eNOS表达差异无显著性 ,Premarin组较OVX组eNOS表达强度则明显增加 (P <0 0 5 )。结论　NO是调节OB、OC功能活动的一种重要效应因子 ,NO导致细胞因子诱导性骨吸收增强与绝经后雌激素缺乏OP密切相关。雌激素可下调iNOSmRNA表达 ,抑制iNOS蛋白合成 ,从而减轻功能亢进的OC性骨吸收 ,而对骨组织正常生理活动中的eNOS合成 ,有适度促进作用 ,有利于骨组织的重建及骨量的恢复 ,从而重建骨形成 吸收新平衡偶联     

Osteocytic expression of constitutive NO synthase isoforms in the femoral neck cortex: a case-control study of intracapsular hip fracture.

NO is an osteocytic signaling molecule that can inhibit osteoclasts. The NO synthases eNOS and nNOS were expressed by >50% of osteonal osteocytes in controls. Hip fracture cases showed +NOS osteocytes only in deep osteonal bone, and 25-35% reduced expression overall. These data are consistent with increased osteonal vulnerability to deep osteoclastic attack. INTRODUCTION: Osteocytes may regulate the response to mechanical stimuli in bone through the production of local signaling molecules such as NO derived from the NO synthase eNOS. Because NO is inhibitory to osteoclastic resorption, it has been suggested that osteocytes expressing eNOS act as sentinels, confining resorption within single osteons. Recently, nNOS has been shown to be present in osteocytes of adult human bone. MATERIALS AND METHODS: Cross-sections of the femoral neck (eight female cases of intracapsular hip fracture and seven postmortem controls; age, 68-91 years) were analyzed by immunohistochemistry. The percentages of osteocytes expressing each of these two isoforms were calculated, and their distances to the nearest canal surface were measured. RESULTS: The percentage of +nNOS osteocytes was lower in the fracture cases than in the controls (cases: 43.12 +/- 1.49, controls: 56.68 +/- 1.45; p < 0.0001). Compared with nNOS, eNOS expression was further reduced (p = 0.009) in the cases but was not different in the controls (cases: 36.41 +/- 1.53, controls: 56.47 +/- 2.41; p < 0.0001). The minimum distance of +eNOS or +nNOS osteocytes to a canal surface was higher in the cases compared with controls (eNOS: controls; 44.4 +/- 2.2 microm, cases: 61.7 +/- 2.0 microm; p < 0.0001; nNOS: controls: 52.4 +/- 1.7 microm, cases: 60.2 +/- 2.1 microm; p = 0.0039). +eNOS osteocytes were closer to the canal surfaces than +nNOS osteocytes in the controls by 8.00 +/- 4.0 microm (p = 0.0012). CONCLUSION: The proportions of osteocytes expressing nNOS and eNOS were both reduced in the fracture cases, suggesting that the capacity to generate NO might be reduced. Furthermore, the reduction in NOS expression occurs in those osteocytes closest to the canal surface, suggesting that the ability of NO to minimize resorption depth might be impaired. Further studies are needed on the regulation of the expression and activity of these distinct NOS isoforms.     

Evidence for the involvement of endothelial nitric oxide synthase from smooth muscle cells in the erectile function of the human corpus cavernosum

Nitric oxide (NO) is an important mediator in the relaxation of cavernosal smooth muscle. The present study examines the existence and location of the constitutive isoform eNOS (endothelial NO synthase) accompanying the already substantiated neurogenic NOS (nNOS) in the human corpus cavernosum of men with and without erectile dysfunction. Activities of NOS enzymes were examined in specimens of 11 potent and nine long-term impotent patients by means of light and electron microscopy using NADPH-diaphorase staining and immunohistochemical eNOS-specific, smooth muscle actin-specific and nNOS-specific markers. Cavernosal smooth muscle shows a distinct expression of eNOS. In contrast to the weaker expression of eNOS and nitrinergic innervation found in larger veins, the small intracavernosal helicine arteries express large quantities of eNOS and possess a dense nitrinergic innervation. Long-term impotent patients display a broad heterogeneity in eNOS expression and nitrinergic innervation while no overall correlation between NOS expression and erectile function was observed. The expression of eNOS indicates eNOS as a main source of NO alongside nNOS. The differentiated localization of eNOS supports at least a role of this isoform in vascular regulation. Received: 21 April 1997 / Accepted 11 November 1997