Enhancement of the inducible NO synthase activation by retinoic acid is mimicked by RARalpha agonist in vivo

We have previously shown that all-trans retinoic acid (atRA), the active metabolite of vitamin A, enhances the activation of the inducible nitric oxide synthase (NOS II) pathway, a component of innate immunity, in rats in vivo. We investigated the relative contribution of retinoic acid receptor-alpha (RARalpha) and retinoid X receptors (RXRs) to NOS II activation triggered by LPS. Five-day supplementation with 10 mg/kg of either atRA or the RARalpha selective agonist Ro-40-6055, but not with 10 mg/kg of the pan-RXR agonist Ro-25-7386, enhanced the LPS-induced NOS II mRNA, protein expression in liver, and plasma nitrite/nitrate concentration. Both atRA and the RARalpha agonist (but not the RXR agonist) increased the number of peripheral T helper lymphocytes and plasma interferon-gamma concentration. Synergism between retinoids and LPS on NOS II activation within an organ coincided with synergism on interferon regulatory factor-1 mRNA expression but not with the level of expression of the RARalpha protein. These results suggest that, in vivo, atRA activates NOS II through RARalpha and contributes to characterizing the complex effect of retinoids on the host inflammatory/immune response.     

Antihypertensive effects of onion on NO synthase inhibitor-induced hypertensive rats and spontaneously hypertensive rats

This study was designed to show the effects of onion on blood pressure in N(G)-nitro-L-arginine methyl ester (L-NAME) induced-hypertensive rats and stroke prone spontaneously hypertensive rats (SHRSP) using dried onion at 5% in their diets. For the experiment with L-NAME induced-hypertensive rats, male 6-weeks-old Sprague-Dawley rats were given tap water containing L-NAME to deliver 50 mg/kg BW/day. In this experiment, we found distinct antihypertensive effects of onion on the L-NAME induced-hypertensive rats and the SHRSP. Dietary onion decreased the thiobarbituric acid reactive substances (TBARS) in plasma in these hypertensive rats. Also, onion increased the nitrate/nitrite (products of nitric oxide (NO)) excreted in urine and the NO synthase (NOS) activity in the kidneys in SHRSP. These results suggested that the increased NO caused by the greater NOS activity, and additionally by the increased saving of NO by the antioxidative activity of onion, was one of the cause of the antihypertensive effect of onion in SHRSP. In the L-NAME induced hypertensive rats, onion did not significantly block the inhibition of NOS activity by L-NAME, and decreased nitrate/nitrite excretion in urine was not restored. The mechanism of the antihypertensive effect of onion probably involves increased saving of NO by antioxidative activity of onion in L-NAME induced-hypertensive rats.     

GH-releasing peptide-2 administration prevents liver inflammatory response in endotoxemia

It has been reported that growth hormone (GH)-releasing peptide-2 (GHRP-2), a ghrelin receptor agonist, has an anti-inflammatory effect. We investigated whether this GH secretagogue attenuates liver injury in LPS-treated rats. Wistar rats were simultaneously injected (ip) with LPS (1 mg/kg) and/or GHRP-2 (100 microg/kg). Serum levels of aspartate and alanine transaminases were measured as an index of liver damage. Circulating nitrites/nitrates and hepatic IGF-I and TNF-alpha were evaluated as possible mediators of GHRP-2 actions. LPS increased serum levels of transaminases and nitrites/nitrates. Moreover, LPS increased hepatic TNF-alpha and decreased hepatic IGF-I mRNAs. GHRP-2 administration attenuated the effects of LPS on transaminases, nitrites/nitrates, TNF-alpha, and IGF-I in vivo. This GHRP-2 effect does not seem to be due to modifications in food intake, since fasting did not modify serum levels of transaminases, serum nitrites/nitrates, and hepatic TNF-alpha mRNA both in vehicle rats and in LPS-injected rats. To elucidate whether GHRP-2 is acting directly on the liver, cocultures of hepatocytes and nonparenchymal cells and monocultures of isolated hepatocytes were incubated with LPS and GHRP-2. The ghrelin receptor agonist prevented an endotoxin-induced increase in transaminases and nitrite/nitrate release as well as in TNF-alpha mRNA and increased IGF-I mRNA from cocultures of hepatocytes and nonparenchymal cells, but not from monocultures. In summary, these data indicate that GHRP-2 has a protective effect on the liver in LPS-injected rats that seems to be mediated by IGF-I, TNF-alpha, and nitric oxide. Our data also suggest that the anti-inflammatory effect of GHRP-2 in the liver is exerted on nonparenchymal cells.     

Streptozotocin may provide protection against subsequent oxidative stress of endotoxin or streptozotocin in rats

Endotoxin lipopolysaccharide (LPS) and streptozotocin-induced diabetes are known to cause oxidative stress in vivo. There is some evidence that a sublethal dose of LPS provides protection against subsequent oxidative stress. Because of its wide use as a diabetogenic agent, this study was undertaken to determine if streptozotocin can likewise provide a protective effect against further oxidative stress in rats. Female Sprague–Dawley rats were given streptozotocin (50 mg/kg intraperitoneally once) prior to exposure to either bacterial endotoxin from Salmonella abortus equii (5 mg/kg intraperitoneally) or three additional daily doses of streptozotocin (50 mg/kg intraperitoneally). One week after LPS or streptozotocin treatments, oxidative stress was determined by measuring changes in antioxidant activity (glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, glutathione S-transferase, and γ-glutamyltranspeptidase) and in concentrations of glutathione, nitrite, and thiobarbituric acid reactants in liver, kidney, intestine, and spleen. High levels of some antioxidants in the LPS-control and streptozotocin-control rats, in contrast to normal levels found in diabetes + LPS and multidose-streptozotocin rats, suggest that streptozotocin, like LPS, may confer a protective effect against subsequent oxidative stress. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 143–149, 1998     

Modulation of cerebellar and hepatic nitric oxide synthase by exogenous arginine and endotoxin.

We have studied in mice the effect of treatment with exogenous arginine and/or LPS by monitoring serum nitrite/nitrate levels and by investigating the response of cerebellar and liver nitric oxide synthase (NOS). We measured NOS activity in cerebellar extracts while changes in iNOS mRNA were followed in the liver since direct assay of NOS activity proved unreliable with this tissue. In fact, liver and cerebellum extracts were both very active in converting arginine into a citrulline-like metabolite, but only cerebellum conversion was dependent on addition of NADPH and inhibitable by N(G)-methyl-l-arginine. Treatment with LPS, on its own, increased serum nitrite/nitrate levels at 5 and 20 h after injection, while treatment with LPS and arginine produced nitrite/nitrate levels in the serum even greater at 5 h, but significantly lower at 20 h. Liver iNOS mRNA levels were markedly increased by LPS, and this effect was significantly decreased when mice were also given exogenous arginine. A stimulatory effect of LPS was also found on NOS activity in the cerebellum, where a very small stimulation may have also been caused by arginine feeding. These findings indicate that LPS stimulates NOS expression/activity both in the cerebellum and in the liver and suggest a complex pattern of modulation of iNOS by arginine, with NO being first produced in excess and then downregulating iNOS expression.     

Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats

Free radicals have been implicated in the etiology of cardiac dysfunction during sepsis, but the actual species responsible remains unclear. We studied the alterations in myocardial nitric oxide (NO), superoxide, and peroxynitrite generation along with cardiac mechanical function and efficiency in hearts from lipopolysaccharide (LPS)-treated rats. Six hours after LPS (4 mg/kg ip) or saline (control) treatment, hearts were isolated and perfused for 1 h with recirculating Krebs-Henseleit buffer and paced at 300 beats/min. Cardiac work, O(2) consumption, and cardiac efficiency were markedly depressed in LPS hearts compared with controls. Plasma nitrate/nitrite level was elevated in LPS rats, and ventricular NO production was enhanced as measured by electron spin resonance spectroscopy, Ca(2+)-independent NO synthase (NOS) activity, and inducible NOS immunohistochemistry. Ventricular superoxide production was also enhanced in LPS-treated hearts as seen by lucigenin chemiluminescence and xanthine oxidase activity. Increased nitrotyrosine staining (immunohistochemistry) and higher lipid hydroperoxides levels were also detected in LPS-treated hearts, indicating oxygen radical-induced stress. Enhanced generation of both NO and superoxide, and thus peroxynitrite, occur in dysfunctional hearts from endotoxemic rats.     

Troglitazone inhibits the expression of inducible nitric oxide synthase in adipocytes in vitro and in vivo study in 3T3-L1 cells and Otsuka Long-Evans Tokushima Fatty rats

The aim of this study was to determine the mechanism of troglitazone action on nitric oxide (NO) production via inducible NO synthase (iNOS) in adipocytes in vitro and in vivo. The treatment of 3T3-L1 adipocytes with the combination of lipopolysaccharide (LPS), tumor necrosis factor-alpha and interferon-gamma synergistically induced de novo iNOS expression leading to enhanced NO production. The NO production was inhibited by co-treatment with aminoguanidine or N-nitro-L-arginine methylester hydrochloride. Troglitazone inhibited the NO production in a dose dependent manner by the suppression of iNOS expression. In the 24 week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, the mean weight and the blood glucose were 21% and 30%, respectively, higher than in their lean counterparts. The serum nitrite concentration was increased after injection of LPS (4 mg/kg, i.p.), more markedly in OLETF rats than in the lean rats. The epididymal fats from LPS-injected groups, but not the ones from the non-injected groups, expressed mRNA and protein of iNOS. Troglitazone pre-treatment blocked the LPS-induced expression of iNOS in adipose tissue and the increase in serum nitrite concentration. These results suggest that troglitazone inhibits the cytokine-induced NO production in adipocytes by blocking iNOS expression both in vitro and in vivo.     

Retinoic acid-induced differentiation in a human neuroblastoma cell line is associated with an increase in nitric oxide synthesis

The human neuroblastoma cell line SK-N-BE, after incubation with 10 μM retinoic acid (RA) or 20 nM phorbol 12-myristate 13-acetate (PMA), underwent biochemical and morphological signs of differentiation within 10–14 days. In parallel, SK-N-BE cells produced significantly higher amounts of nitric oxide (NO) in comparison with controls, as assessed by the measurement of nitrite and nitrate in the culture supernatant and of NO synthase (NOS) activity in the cell lysates (measured as ability to convert [³H]arginine into [³H]citrulline and as NADPH diaphorase activity). Nitrite/nitrate production was abolished by adding the NO scavenger hemoglobin in the culture medium and was inhibited by aminoguanidine (AG, a selective inhibitor of the inducible NOS isoform) but not by the less selective inhibitor N^G-nitro-L -arginine methylester (NAME). Western blotting experiments with monoclonal antibodies against the ncNOS and iNOS isoforms suggest that RA-elicited NOS activation is not attributable to an increased expression of the protein. NAME and AG were not able to revert inhibition of proliferation induced by RA, and the NO donor sodium nitroprusside did not mimic the effect of RA and PMA. These data indicate that increased NO synthesis does not mediate RA- or PMA-induced differentiation but may be an additional marker of differentiation into sympathetic-like neuronal cells. J. Cell. Physiol. 174:99–106, 1998. © 1998 Wiley-Liss, Inc.     

Reduced inotropic heart response in selenium-deficient mice relates with inducible nitric oxide synthase

Atria from mice fed a selenium-deficient (Se(-)) diet have a diminished beta-adrenoceptor-inotropic cardiac response to isoproterenol or norepinephrine compared with atria from mice fed the same diet supplemented with 0.2 mg/kg Se as sodium selenite (Se(+)). This diminished response could be reversed by feeding Se(-) mice the Se(+) diet for 1 wk or by pretreatment with nitric oxide synthase (NOS) inhibitors such as N(G)-monomethyl-l-arginine or aminopyridine. Elevated serum concentrations of nitrite/nitrate as well as a threefold increase in the atrial NOS activity were seen in the Se(-) versus Se(+) mice. Western blotting and indirect immunofluorescence indicated an enhanced expression of inducible NOS in hearts from Se(-) mice. Increased expression and activity of NOS and increased nitrite/nitrate levels from Se(-) mice correlated with an impaired response to beta-adrenoceptor inotropic cardiac stimulation. Elevated nitric oxide levels may account for some of the pathophysiological effects of Se deficiency on the heart.     

Blood plasma response and urinary excretion of nitrite and nitrate in milk-fed calves after oral nitrite and nitrate administration

There is marked endogenous production of nitrate in young calves. Here we have studied the contribution of exogenous nitrate and nitrite to plasma concentrations and urinary excretion of nitrite and nitrate in milk-fed calves. In experiment 1, calves were fed 0 or 200 &mgr;mol nitrate or nitrite/kg(0.75) or 100 &mgr;mol nitrite plus 100 &mgr;mol nitrate/kg(0.75) with milk for 3 d. In experiment 2, calves were fed 400 &mgr;mol nitrate or nitrite/kg(0.75) with milk for 1 d. Plasma nitrate rapidly and comparably increased after feeding nitrite, nitrate or nitrite plus nitrate. The rise of plasma nitrate was greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. Plasma nitrate decreased slowly after the 3-d administration of 200 &mgr;mol nitrate or nitrite/kg(0.75) and reached pre-experimental concentrations 4 d later. Urinary nitrate excretions nearly identically increased if nitrate, nitrite or nitrite plus nitrate were administered and excreted amounts were greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. After nitrite ingestion plasma nitrite only transiently increased after 2 and 4 h and urinary excretion rates remained unchanged. Plasma nitrate concentration remained unchanged if milk was not supplemented with nitrite or nitrate. Nitrate concentrations were stable for 24 h after addition of nitrite to full blood in vitro, whereas nitrite concentrations decreased within 2 h. In conclusion, plasma nitrate concentrations and urinary nitrate excretions are enhanced dose-dependently by feeding low amounts of nitrate and nitrite, whereas after ingested nitrite only a transient and small rise of plasma nitrite is observed because of rapid conversion to nitrate.     

N-acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase.

This in vivo study evaluates the effect of N-acetylcysteine (NAC) administration on nitric oxide (NO) production by the inducible form of nitric oxide synthase (iNOS). NO production was induced in the rat by the ip administration of 2 mg/100 g lipopolysaccharide (LPS). This treatment caused: (1) a decrease in body temperature within 90 min, followed by a slow return to normal levels; (2) an increase in plasma levels of urea, nitrite/nitrate, and citrulline; (3) the appearance in blood of nitrosyl-hemoglobin (NO-Hb) and in liver of dinitrosyl-iron-dithiolate complexes (DNIC); and (4) increased expression of iNOS mRNA in peripheral blood mononuclear cells (PBMC). Rat treatment with 15 mg/100 g NAC ip, 30 min before LPS, resulted in a significant decrease in blood NO-Hb levels, plasma nitrite/nitrate and citrulline concentrations, and liver DNIC complexes. PBMC also showed a decreased expression of iNOS mRNA. NAC pretreatment did not modify the increased levels of plasma urea or the hypothermic effect induced by the endotoxin. The administration of NAC following LPS intoxication (15 min prior to sacrifice) did not affect NO-Hb levels. These results demonstrate that NAC administration can modulate the massive NO production induced by LPS. This can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression. This hypothesis is also supported by the lack of effect of late NAC administration.     

Vasopressin and nitric oxide synthesis after three days of water or food deprivation

Nitric oxide has been suggested to be involved in the regulation of fluid and nutrient homeostasis. In the present investigation, vasopressin and nitric oxide metabolite (nitrite and nitrate) levels were determined in plasma of male Wistar rats submitted to water or food deprivation for three days. Hematocrit and plasma sodium showed marked increase in dehydrated and starved rats. Potassium levels and plasma volume decreased in both treated groups. Plasma osmolality and vasopressin levels were significantly elevated in water deprived (362.8 +/- 7.1 mOsm/kg H2O, 17.3 +/- 2.7 pg/ml, respectively, p < 0.001) rats, but not in food deprived (339.9 +/- 5.0, 1.34 +/- 0.28) rats, compared to the controls (326.1 +/- 4.1, 1.47 +/- 0.32). The alterations observed in plasma vasopressin levels were related to plasma osmolality rather than plasma volume. Plasma levels of nitrite and nitrate were markedly increased in both water and food deprived rats (respectively, 2.19 +/- 0.29 mg/l and 2.22 +/- 0.17 mg/l versus 1.33 +/- 0.19 mg/l, both p < 0.01). There was a significant negative correlation between plasma nitrite and nitrate concentration and plasma volume. These results suggest that both dehydration and starvation increase plasma nitric oxide, probably by activation of nitric oxide synthases. The release of nitric oxide may participate in the regulation of the alteration in blood flow, fluid and nutrient metabolism caused by water deprivation or starvation.     

Nitric oxide- and oxygen-derived free radical generation from control and lipopolysaccharide-treated rat polymorphonuclear leukocyte.

Previous studies from this lab have shown NO-mediated modulation of free radical generation from polymorphonuclear leukocytes (PMNs), following hypoxic-reoxygenation as well as in the normoxic cells. The present study is an attempt to investigate further the regulation of NO and free radical generation in the lipopolysaccharide (LPS)-treated PMNs. PMNs were isolated from the rat blood and peritoneal cavity, 4 h after LPS (1 mg/kg, i.p.) treatment. Nitric oxide synthase (NOS) activity and nitrite content were increased in the peripheral and peritoneal PMNs following LPS treatment. An increase in the apparent V(max) for l-arginine uptake was also observed in the LPS-treated peripheral PMNs, while peritoneal PMNs exhibited increase in both apparent V(max) and affinity for l-arginine. Synthesis of nitrite did not augment after increasing the availability of substrate to control PMNs, however, peripheral and peritoneal PMNs from LPS-treated rats utilized l-arginine more efficiently for nitrite synthesis. NOS activity, l-arginine uptake, and its utilization were maximal in the peritoneal PMNs. Arachidonic acid (AA, 1 x 10(-6) M)-induced free radical generation from PMNs was also enhanced significantly after LPS treatment. Preincubation of PMNs with nitrite elevated the free radical generation and myeloperoxidase (MPO) release. MPO and antioxidant enzyme activity in the PMNs was significantly augmented after LPS treatment. NOS inhibitors, aminoguanidine and 7-nitroindazole, inhibited arachidonic acid-induced free radical generation from LPS treated PMNs. The results obtained thus indicate that augmentation of free radical generation from rat PMNs following LPS treatment appears to be regulated by NO and MPO.     

The acute phase response alters cationic amino acid transporter expression in growing chickens (Gallus gallus domesticus)

The effect of an acute phase response (APR) on cationic amino acid transporter (CAT1-3) mRNA expression in liver, muscle, bursa and thymus was determined in broiler strain chickens. The APR was initiated by injecting Salmonella typhimurium lipopolysaccharide subcutaneously (LPS; 1 mg/kg bw). In Experiment 1, CAT1-3 mRNA expression was determined at multiple time points following LPS administration. LPS increased bursa and liver total and high affinity CAT mRNA expression (P<0.05) and transiently increased pectoralis total CAT mRNA expression (P<0.05). Total CAT mRNA expression in the thymus decreased 7.7-fold from 0 to 8 h after LPS injection (P<0.05). In Experiment 2, fasted chicks were uninjected or LPS-injected. LPS increased total and high affinity CAT mRNA 2-fold in both the bursa and liver (P<0.05) and did not change thymus total and high affinity CAT mRNA expression (P>0.05). LPS increased liver weight only (P<0.05) and did not alter the plasma lysine and arginine concentration (P>0.05). In Experiments 3 and 4, thymocyte proliferation and total protein content were dependent upon the media lysine concentration (P<0.001). The inability of the thymus to compete for lysine and arginine during the APR may limit the ability of thymocytes to develop during infections.     

Inhibition by nitric oxide of cytochrome P450 4A activity contributes to endotoxin-induced hypotension in rats.

Nitric oxide (NO) production during endotoxemia is associated with decreased total CYP content, CYP 1A1/2, 2B1/2, 2C6, 2C11, 3A1, and 3A2 mRNA, protein expression or activity which is prevented by NO synthase (NOS) inhibitors in rats. This study was conducted to determine if endotoxin-induced hypotension caused by NO production is mediated by inhibition of renal CYP 4A protein expression and activity. In conscious male Sprague-Dawley rats, endotoxin (10 mg/kg, ip) reduced mean arterial pressure (MAP), increased serum and renal nitrite levels, and inducible NOS (iNOS), and decreased renal CYP 4A1/A3 protein and CYP 4A activity. The selective iNOS inhibitor 1,3-PBIT (10 mg/kg, ip; 1h after endotoxin) prevented endotoxin-induced decrease in MAP, renal CYP 4A1/A3 protein level and CYP 4A activity and increase in systemic and renal nitrite production. The selective constitutive NOS (cNOS) inhibitor N(G)-nitro-L-arginine (L-NNA; 20 mg/kg, ip; 1 h after endotoxin) partially attenuated endotoxin-induced decrease in MAP. The selective CYP 4A inhibitor, aminobenzotriazole (50 mg/kg, ip; 1 h after endotoxin) diminished CYP 4A1/A3 protein level and CYP 4A activity. Aminobenzotriazole did not alter the endotoxin-induced decrease in MAP, but it reversed the effect of 1,3-PBIT in preventing endotoxin-induced fall in MAP and CYP 4A activity. These data suggest that the endotoxemia-induced increase in NO production primarily via iNOS suppresses renal CYP 4A expression and activity, and inhibition of iNOS with 1,3-PBIT restores renal CYP 4A protein and activity and MAP presumably due to increased production of arachidonic acid metabolites derived from CYP 4A.     

In vivo and in vitro effects of lysine clonixinate on nitric oxide synthase in LPS-treated and untreated rat lung preparations.

Recent studies have shown that some nonsteroidal antiinflammatory drugs (NSAIDS) inhibited the inducible NO synthase (iNOS) without direct effect on the catalytic activity of this enzyme. This study was conducted to investigate the in vitro and in vivo effects of lysine clonixinate (LC) and indomethacin (INDO) on NOS activity in rat lung preparation. LC is a drug with antiinflammatory, antipyretic, and analgesic action. In the in vitro experiments, rats were injected with saline or lipopolysaccharide (LPS) and killed 6 h after treatment. Lung preparations were incubated with LC at 2.3 x 10(-5) M or 3.8 x 10(-5) M. The minimum concentration did not modify NOS activity in control or LPS-treated rats but the maximum dose inhibited increased NO production induced by LPS. Furthermore, INDO at 10(-6) M had no effect on enzymatic activity in control or LPS-treated rats. In the in vivo experiments, 40 mg/kg of LC were injected ip. Such a dose did not affect basal production of NO. When LC and LPS were injected simultaneously 6 h before sacrifice, a significant decrease in LPS-induced NOS activity was observed. INDO 10 mg/kg injected in control animals had no effect on NOS activity and did not block LPS induced stimulation of NO production when injected simultaneously. Finally, when LC (40 mg/kg) was injected 3 h after LPS, the enzymatic activity remained unchanged. Expression of iNOS was detected by Western blotting in rats treated with LPS plus 4, 10, 20, and 40 mg/kg of LC. The lowest dose was the only one showing no effect on LPS-induced increase of iNOS. In short, LC is a NSAID with inhibitory action on the expression of LPS-induced NOS, effect that was not seen with INDO in our experimental conditions.     

Asymmetric dimethylarginine is not involved in ovariectomy-induced osteopenia in rats

Previous studies have indicated that the plasma concentration of nitric oxide synthase inhibitor, asymmetric dimethylarginine (ADMA), was increased in postmenopausal women. In the study reported here, we tested the relationship between the decrease of bone mineral density (BMD) and ADMA concentration in ovariectomized (OVX) rats. Ovariectomized rats at 8 months of age were treated with 17beta-estradiol (10 or 30 microg/kg of body weight/day, s.c.) or L-arginine (300 mg/kg/day, i.p.) for 12 weeks (n = 10 for each group). Pre- and posttreatment total BMD, posttreatment plasma nitrite/nitrate and ADMA concentrations, and posttreatment BMD in the lumbar part of the spine (L4-L6), femurs, and tibias were examined. Ovariectomy caused a significant decrease in several BMD indexes, which was reversed by estrogen treatment (P < 0.05). Plasma nitrite/nitrate concentration was significantly decreased in OVX rats, but was restored by estrogen treatment (P < 0.05). There were no differences in the plasma concentration of ADMA in OVX or estrogen-treated rats. L-Arginine had no effect on plasma nitrite/nitrate concentration and BMD in OVX rats. These results suggest that ovariectomy does not influence the plasma concentration of ADMA, and that ADMA is not involved in ovariectomy-induced osteopenia in rats.