TNF-alpha induces apoptosis in rat fetal brown adipocytes in primary culture

The balding (bal) mutation of the mouse is an autosomal recessive mutation that causes alopecia and immunologic anomalies. A new allele was identified by allelism testing after using an interspecific backcross to localize the mutation to the centromeric end of mouse chromosome 18. We investigated the skin and hair histologic lesions of two alleles (bal(J) and bal(Pas)) at this locus and analyzed the expression of several keratinocyte markers and the production of autoantibodies by immunofluorescence on frozen skin sections. The lesions observed included separation of the inner and outer root sheath in anagen follicles resulting in the hair fiber being very easily plucked from the follicle. Vesicles on the ventral tongue, mucocutaneous junction of the eyelid, foot pads, and rarely in skin were also evident. Separation occurred between the basal and suprabasilar cells forming an empty cleft, resembling that observed in human pemphigus vulgaris. Immunofluorescence studies did not reveal the presence of tissue-bound or circulating autoantibodies. Expression of keratinocyte markers in hair follicles was normal. Keratin 6-positive cells were found on either side of the follicular separation suggesting a molecular defect in adhesion molecules between the inner layer of the outer root sheath cells to layers on either sides. This hypothesis has been confirmed by another group who demonstrated that the bal(J) mutation is due to the insertion of a thymidine in the desmoglein 3 gene, resulting in a premature stop codon.     

Macrophage-induced inhibition of nitric oxide production in primary rat hepatocyte cultures via prostaglandin E2 release

Kupffer cells and other macrophages play an important role in pathogenesis of toxicants in the liver. The aim of this study was to evaluate the effect of macrophages on hepatocyte production of nitric oxide (NO), which has been previously reported to be protective toward oxidative stress induced in primary rat hepatocytes. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocytes at various ratios between macrophages and hepatocytes. These cocultures were supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) for 23 hours to induce NO synthase and trigger NO production. NO production was followed by quantification of nitrites in culture medium and dinitrosyl iron complexes (DNIC) in intact hepatocytes after separation from macrophages. In cocultured hepatocytes incubated with LPS and IFN-gamma, DNIC and nitrite levels decreased compared with those observed in hepatocytes cultured without macrophages in the same conditions. Moreover, inhibition of NO production in hepatocyte cocultures was macrophage-number-dependent. Macrophage-conditioned medium also inhibited NO production in hepatocytes, suggesting that the effect of macrophages was mediated by soluble factors. Among the soluble factors known to decrease NO levels are some cytokines, growth factors, reactive oxygen species, and prostaglandins. Ultrafiltration of macrophage-conditioned medium through a 500-d membrane to rule out higher-molecular-weight molecules, such as anti-inflammatory cytokines and growth factors, failed to restore NO production. In the same way, the use of superoxide dismutase (SOD) and catalase (CAT) to eliminate reactive oxygen species produced by macrophages did not lead to recovery of NO levels in hepatocytes. However, when NO synthesis was inhibited in macrophages by NG-monomethyl-L-arginine (L-NMMA), hepatocytes recovered the capacity to produce NO. A net decrease of prostaglandin E2 (PGE2) release by macrophages was concomitantly observed. Moreover, inhibition of PGE2 production in macrophages by indomethacin led to restoration of NO levels. Taken together, our observations suggest that NO synthesized by macrophages can decrease NO production in hepatocytes via PGE2 release. Because of the protective role of NO toward many liver injuries, it may be postulated that macrophages contribute through this mechanism to liver damage.     

The role of nitric oxide in mercury chloride-induced vasculitis in the brown Norway rat

The molecular epidemiology of Pseudomonas aeruginosa infection in cystic fibrosis (CF) siblings was analysed by DNA fingerprinting using arbitrary primed polymerase chain reaction. A total of 306 strains collected from six pairs of siblings over a period of 20-126 months (median 64) was studied. Fifty-four different P. aeruginosa genotypes were recognized. Two out of six pairs of siblings were ultimately colonized by identical strains, and it was shown that a single P. aeruginosa clone can persist in an individual patient for over ten years. No overlap in P. aeruginosa genotypes was encountered between families, whereas in all families at least transient cross-colonization with the same genotype was observed. This finding demonstrates that P. aeruginosa cross-infection or acquisition of the same strain from an identical environmental source exists within the family situation, but does not always result in a long-term colonization by identical genotypes in all family members suffering from CF.     

Effects of ether lipid 1-O-octadecyl-2-methoxy-rac-glycero- 3-phosphocholine and its analogs PAF and CPAF on the release of nitric oxide in primary cultures of rat astrocytes

Ether lipid 1-O-octadecyl-2-O-methoxy-rac-glicero-3-phosphocholine (ET-18-OCH3) is an immunomodulator with antineoplastic activity. Its analog compounds PAF and CPAF share some of its biological effects. In our experiments, even very small amounts of ET-18-OCH3 released a remarkable quantity of nitric oxide (NO) from rat astrocytes cultured in vitro. The NO biosynthesis was inhibited by pretreatment with the antagonist BN 50730. The effect of ET-18-OCH3 was greater than that of the LPS inducer. PAF did not produce NO, even at high doses, while the nonmetabolizable CPAF only induced a significant release of NO from 12 micrograms/ml onwards. These results demonstrate that ET-18-OCH3 is functionally active also in astrocyte cultures. Stimulation of NO biosynthesis is of a great value on account of its the known effect as a neurotransmitter, potentiator of immune defences and possible modulator of cerebral circulation.     

Effects of insulin-like growth factor I and II on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

We compared the effects of insulin-like growth factor I (IGF-I) and II (IGF-II) on DNA synthesis and proliferation and investigated various signal transduction mechanisms involved in insulin-like growth factor-induced mitogenesis in primary cultures of adult rat hepatocytes. IGF-I stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 75 ng/ml within 4 h of culture. These effects were sensitive to the IGF-I concentration and cell density. Hepatocyte proliferation induced by IGF-I was potentiated by metaproterenol (10(-6) M) as well as by 8-bromo-cAMP, phorbol 12-myristate 13-acetate (PMA; 10(-8) M) and was inhibited by U-73122 (1-(-[[17beta-3-methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-+ ++1Hpyrrol-2,5-dione)), genistein, wortmannin, PD98059 (2'-amino-3'-methoxyflavone) and rapamycin. The IGF-I effect was independent of pertussis toxin (100 ng/ml). IGF-II also dose dependently stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 0.75 ng/ml within 4 h of culture. However, these effects were not dependent on the initial plating density. The stimulatory effects of IGF-II were potentiated by UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (10(-5) M) and inhibited by phenylephrine, PMA, metaproterenol, 8-bromo-cAMP, PD98059, rapamycin, and pertussis toxin. The IGF-II effects were not affected by genistein, U-73122, and wortmannin. These results suggest that IGF-I and IGF-II rapidly stimulate the DNA synthesis and proliferation of adult rat hepatocytes by separate mechanisms.     

Effects of curcumin on P-glycoprotein in primary cultures of rat hepatocytes

Curcumin is a natural phenolic compound found in the rhizomes of Curcuma longa and endowed with beneficial biological activities including antioxidant, anticarcinogenic and hepatoprotective effects. In this study curcumin was tested for its potential ability to interact in vitro with hepatic P-glycoprotein (Pgp), in a model system represented by primary cultures of rat hepatocytes, in which spontaneous overexpression of multidrug resistance (mdr) genes occurs. In both freshly-plated hepatocytes, containing low levels of Pgp, and 72 hour-cultured hepatocytes, containing high levels of Pgp, the Rhodamine-123 (R-123) efflux, which represents a specific functional test for Pgp-mediated transport, was inhibited by curcumin in a dose-dependent manner. Western blot analysis showed that 25microM curcumin, when included in the culture medium throughout the experimental observation (72 hours), was able to significantly lower the increase of mAb C219-immunoreactive protein spontaneously occurring in the cells during culture. Curcumin, at doses ranging from 50 to 150microM was cytotoxic for freshly-plated hepatocytes, as shown by the strong decrease in the cell ability to exclude trypan blue 24 hours later, but it was significantly less cytotoxic when added to 24 or 48 hour-cultured cells. The resistance to curcumin, progressively acquired by cells during culture, was significantly reduced by high concentrations of dexamethasone (DEX) or dimethyl-sulfoxide (DMSO), culture conditions known to inhibit the spontaneous overexpression of Pgp. In addition, in a concentration-dependent manner, verapamil reverted curcumin resistance in Pgp overexpressing hepatocytes. In photoaffinity labeling studies, curcumin competed with azidopine for binding to Pgp, suggesting a direct interaction with glycoprotein. These results suggest that curcumin is able to modulate in vitro both expression and function of hepatic Pgp and support the hypothesis that curcumin, a chemopreventive phytochemical, could reveal itself also as a compound endowed with chemosensitizing properties on mdr phenotype.     

Effects of thermal injury on production of nitric oxide in rat heart

To elucidate the relationship between thermal injury and production of nitric oxide (NO), we measured the levels of heart No2-/NO3- (the stable and products of NO) and GMP in rats with full thickness burn of 30% TBSA. A higher level of heart tissue NO2-/NO3- was observed from 3 to 24 hours after burn injury. Parallel to the changes of NO2-/NO3-, there were increases of tissue cGMP level and heart tissue water content in burned rats. L-NMMA, a specific inhibitor of NO synthetase (NOS), can block the rise of heart NO2-/NO3- and the enhancement of heart water content. The results indicate that the heart injury caused by burn injury may be associated with induction of NOS and formation of NO.     

Effects of dithiocarbamates on toxicity of cadmium in rat primary hepatocyte cultures

The protective effects of N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD) on the toxicity of Cd in the rat primary hepatocyte cultures were studied. Cytotoxicity was assessed by measuring cell viability, extra cellular lactic dehydrogenase (LDH) activity, and intracellular lipid peroxidation and active oxygen species. Primary hepatocyte cultures were treated with 109CdCl2 (5, 10 or 50 microM Cd and 1.7 KBq of 109Cd/well) for 30 min or 4 h. BGD or HBGD was added to the culture medium to make the final concentration of 100 microM and incubated for 4.5 h in 30 min Cd exposure or 1 h in 4 h Cd exposure. Decreases in the hepatocyte viability caused by all Cd exposure concentrations were significantly prevented by treatment with BGD or HBGD. The treatment with the chelating agents for 4.5 h after Cd exposure for 30 min significantly prevented increases in extracellular LDH activity. Increases in the lipid peroxidation in hepatocytes exposed to Cd for 30 min or 4 h were prevented significantly by treatment with BGD or HBGD for 4.5 h or 1 h, respectively. Moreover, the increases in the level of active oxygen species caused by Cd exposure for 30 min were significantly prevented by treatment with the chelating agents for 1.5 h. These findings suggest that BGD and HBGD protect against the cytotoxicity of Cd in rat primary hepatocyte cultures and that the protective effects of chelating agents presumably result from a decrease in the Cd level, the effective sequestration of the reactive Cd ion, and the direct preventive effect on the active oxygen species in the hepatocytes.     

Direct action of nitric oxide on osteoblastic differentiation

The effect of nitric oxide (NO) on osteoblastic differentiation was examined in cultured mouse osteoblasts. Interleukin-1beta and tumor necrosis factor-alpha expressed inducible NO synthase gene with little effect on constitutive NO synthase gene. These cytokines increased NO production, which was inhibited by L-NMMA pretreatment, and decreased alkaline phosphatase (AIPase) activity, which was not restored by L-NMMA. Furthermore, NO donors, sodium nitroprusside and NONOate dose-dependently elevated AIPase activity and expression of osteocalcin gene. These results suggest that NO directly facilitates osteoblastic differentiation and the cytokine-induced inhibition of AIPase activity is mediated via mechanism other than NO.     

Effects of a nitric oxide synthase inhibitor on NMDA receptor function in organotypic hippocampal cultures

Nitric oxide (NO) has been proposed to trigger long-term potentiation (LTP) at CA3 to CA1 synapses. We previously reported that NO synthesis inhibitors and blockers reduce an electrophysiological index of NMDA receptor activation in acute hippocampal slices. We now show that the NOS inhibitor, NG-methyl-L-arginine (MLA), also reversibly prevents LTP induction in organotypic hippocampal slices and significantly reduces a biochemical index of NMDA receptor function. These results results further indicate that MLA inhibits LTP induction by interfering with NMDA receptor functions.     

Effects of progesterone on proliferation and differentiation of fetal rat calvarial osteoblasts in vitro

OBJECTIVES: To investigate the influence of progesterone on proliferation and differentiation of osteoblast at the levels of gene expression and cell functions. METHODS: Fetal rat calvarial osteoblasts were cultured in vitro in the presence of (10(-9) mol/L-10(-6) mol/L) progesterone. Cell proliferation, alkaline phosphalase (ALP) activity, osteocalcin mRNA expression and osteocalcin secretion in the medium and bone nodule formation were analyzed. RESULTS: Progesterone did not influence cell proliferation; Progesterone enhanced the ALP activity in rat osteoblasts; Progesterone stimulated osteocalcin mRNA expression in a dose-dependent manner and increased the amount of osteocalcin in the culture medium; Progesterone increased both number and area of bone nodule formation. CONCLUSION: Progesterone has a multi-stimulating effect on the differentiation of fetal rat calvarial osteoblast, hut no effect on cell proliferation.     

Effects of gadolinium on proliferation, differentiation and calcification of primary mouse osteoblasts in vitro

To evaluate the effects of Gd on proliferation, differentiation and mineralization function of primary osteoblasts (OBs) in vitro, we tested cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cell differentiation by alkaline phosphatase (ALP) activity assay, synthesis of type ? collagen, and oil red O and alizarin red S (ARS) stain assays. The results indicated that effects of Gd on the proliferation, osteogenic differentiation, mineralization function and adipocytic transdifferentiation of primary OBs de-pended on concentration and incubation time, but were not dose-dependent. It was suggested that the effect of Gd on bone metabolism was complicated, and concentration and culture time were key factors for switching the biological effects of Gd from damage to protection.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

Inhibitory effects of nitric oxide donors on nitric oxide synthesis in rat gastric myenteric plexus

We investigated whether nitric oxide (NO) exerts an inhibition on its own synthesis in the gastric myenteric plexus in rats. Nonadrenergic, noncholinergic relaxations in response to transmural electrical stimulation (TS) were markedly antagonized by NG-nitro-L-arginine methyl ester, (10(-4) M) and abolished by tetrodotoxin (10(-6) M). Pretreatment with various NO donors (3-morpholino-sydnonymide [SIN-1 (3 x 10(-7) to 3 x 10(-6) M)], S-nitroso-N-acetylpenicillamine (10(-6) to 10(-5) M), sodium nitroprusside (10(-8) to 3 x 10(-8) M) and 8-bromoquanosine 3', 5'-cyclic monophosphate [8-bromo-cGMP (10(-6) to 3 x 10(-6) M)]) significantly inhibited TS-evoked nonadrenergic, noncholinergic relaxations in a dose-dependent manner. In contrast, vasoactive intestinal polypeptide (10(-8) M)-induced relaxations were not affected by SIN-1 or 8-bromo-cGMP. TS evoked a significant increase in 3H-citrulline formation, which was completely abolished by calcium-free medium, NG-nitro-L-arginine methyl ester, (10(-4) M) and tetrodotoxin (10(-6) M). 3H-citrulline formation evoked by TS was significantly inhibited by SIN-1 (10(-7) to 10(-5) M) and 8-bromo-cGMP (10(-7) to 10(-5) M) in a dose-dependent manner. The inhibitory effect of SIN-1 was partially prevented by 1H-[1,2, 4]oxadiazolo[3,4-a]quinoxalin-1-one (10(-5) M), a guanylate cyclase inhibitor. We conclude that NO synthesis in the gastric myenteric plexus is negatively regulated by NO and cGMP. This suggests an autoregulatory feedback mechanism of NO synthesis in the gastric myenteric plexus.     

Effects of nitric oxide from exogenous nitric oxide donors on osteoblastic metabolism

We examined the effects of nitric oxide (NO) on the differentiation and mineralization of newborn rat calvarial osteoblastic cells (ROB cells) using exogenous NO donors, sodium nitroprusside, 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e (NOC-7) and 2,2'-(hydroxynitrosoydrazino)bis-ethanamine (NOC-18). Sodium nitroprusside and NOC-7 dose-dependently enhanced the rate of production of intracellular cGMP in ROB cells and the rat clonal osteogenic cell line ROB-C26. We used NOC (NOC is the trade name for NO complex manufactured by Dojindo, Kumamoto, Japan) as an NO donor in our experiments because sodium nitroprusside exhibited a marked cytotoxicity. Northern blot analysis revealed that the level of mRNA for osteocalcin, one of the osteoblastic differentiation markers, was enhanced in the ROB cells, which was continuously treated by NOC-18. NOC-18, however, did not affect the level of mRNA for alkaline phosphatase and the activity of alkaline phosphatase. Both the number and the total area of mineralized nodules that are a model of in vitro bone formation were shown to be increased by 10(-5) M NOC-18. Our data suggest that NO might act as a local regulator of the metabolism of osteoblastic cells.     

Effects of nitric oxide synthesis inhibition on methamphetamine-induced dopaminergic and serotonergic neurotoxicity in the rat brain

We examined effects of nitric oxide (NO.) synthesis inhibition on methamphetamine (MA)-induced dopaminergic and serotonergic neurotoxicity. The toxic dose of MA (5 mg/kg, sc, x4) significantly decreased contents of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum (ST), and significantly decreased contents of serotonin (5-HT) in the ST, nucleus accumbens (NA) and medial frontal contex (MFC). Coadministration with a NO. synthase inhibitor, N omega-nitro-L-arginine methyl ester (LNAME) (30 mg/kg, i.p., x2), reduced the MA-induced decreases in contents of DA, DOPAC and HVA in the ST, but not reduced the MA-induced decreases in contents of 5-HT in the ST, NA and MFC. These findings suggest that the MA-induced dopaminergic, but not serotonergic neurotoxicity, may be related to the neural process such as NO. formation caused by the activation of postsynaptic DA receptor.     

Effects of tumor necrosis factor-alpha on glucose and albumin production in primary cultures of rat hepatocytes

Tumor necrosis factor-alpha (TNF) is known to alter significantly in vivo hepatic glucose and albumin metabolism. However, it remains unclear whether the observed effects represent direct actions of this factor or secondary responses due to the recruitment of other mediator systems. The present study was designed to investigate direct actions of TNF on glucose and albumin production in primary cultures of rat hepatocytes. Addition of TNF to the culture medium resulted in a 45% to 50% reduction in glucose production from a control level of 239 +/- 15 nmol/plate.h. This effect was reversed by addition of anti-TNF monoclonal antibody. In glycogen-depleted cells, short-term (5-hour) incubation with TNF did not affect hepatocyte albumin secretion, which was 8.13 +/- 0.29 microgram/plate.h. However, in cells exposed to insulin or in non-glycogen-depleted cells, addition of TNF resulted in a 10% to 25% reduction in albumin production. These findings indicate that TNF exerts direct inhibitory effects on hepatocyte glucose and albumin production, but the effects on the latter process are modest. A notable aspect of the findings is that the albumin effects are insulin or glucose substrate-dependent, which may have implications regarding liver function during nutritional support in critical illness.     

Effects of various nitric oxide synthase inhibitors on NMDA-induced neuronal injury in rat cortical neurons

Two questions were investigated: (1) whether cancer patients (n = 72) and staff (n = 63) have different cognitive representations of the concepts 'caring' (in Swedish: omv?rdnad) and 'clinical care' (in Swedish: v?rd), and (2) whether two different wordings of the response categories used by patients and staff to rank the perceived importance of 50 specific caring behaviors ('Old' response format: 'of importance'--of no importance vs 'New' format: more or less important') in relation to these concepts would produce different results. A Swedish questionnaire version of the CARE-Q instrument was used. Participants were randomized to one of four research conditions: (1) Caring/Old, (2) Caring/New, (3) Clinical care/Old, and (4) Clinical care/New, and asked to rank the importance of the 50 CARE-Q behaviors for that specific concept/response format combination. Results demonstrated that response formats did not affect patient or staff answers. Neither group did to any great extent value CARE-Q behaviors differently when regarded as examples of 'caring' vs 'clinical care'. The assumption that different cognitive representations of the concepts or that a specific wording of response categories had affected previous CARE-Q results was not substantiated.     

Expression of nitric oxide synthase isoforms in bone and bone cell cultures

Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine-NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.