Involvement of inducible isoforms of COX and NOS in streptozotocin-pancreatic damage in the rat: interactions between nitridergic and prostanoid pathway

Streptozotocin-induced pancreatic damage involves nitric oxide (NO) and prostaglandins (PGs) overproduction. In this work we aim to evaluate a putative relationship between the elevated NO levels and the altered prostanoid production in pancreatic tissue from streptozotocin-diabetic rats. Total NOS activity and nitrate/nitrite pancreatic levels in tissues from diabetic rats are decreased when the cyclooxygenase (COX) inhibitor indomethacin (INDO) is added to the incubating medium, while the addition of PGE(2)increases nitrate/nitrite production and NOS levels. INDO and PGE(2)selectively affect Ca(2+)-dependent NOS (iNOS) activity in diabetic tissues, and they have not been able to modify nitrate/nitrite levels, iNOS or Ca(2+)-dependent (cNOS) in control tissues. When the NOS inhibitor L-NMMA was present in the incubating medium, control pancreatic [(14)C]-Arachidonic Acid ([(14)C]-AA) conversion to 6-keto PGF(1 alpha)and to TXB(2)was lower, and PGF(2 alpha), PGE(2)and TXB(2)production from diabetic tissues diminished. The NO donors, spermine nonoate (SN) and SIN-1, enhanced TXB(2)levels in control tissues, while PGF(2 alpha), PGE(2)and TXB(2)levels from diabetic tissues were increased. PGE(2)production from control and diabetic tissues was assessed in the presence of the NO donor SN plus INDO or NS398, a specific PG synthase 2 inhibitor. When SN combined with INDO or NS398 was added, the increment of PGE(2)production was abolished by both inhibitors in equal amounts, indicating that the activating effect of nitric oxide is exerted on the inducible isoform of cyclooxygenase. In the diabetic rat, prostaglandins and NO seem to stimulate the generation of each other, suggesting a lack of regulatory mechanisms that control the levels of vasoactive substances in acute phase of beta-cell destruction.     

Oxalomalate affects the inducible nitric oxide synthase expression and activity

Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.     

Blood cell NO synthesis in response to exercise.

Nitric oxide (NO) is important for the maintenance of cardiovascular homeostasis and is also involved in immunity and inflammation. The aim of our work was to determine the effects of intense exercise on plasma and blood cell NO handling. Nine voluntary male professional cyclists participated in the study. Blood samples were taken in basal conditions and 3h after finishing a mountain cycling stage. Exercise-induced neutrophilia, lymphopenia, and hemolysis. Plasma and erythrocytes maintained basal nitrite levels, whereas neutrophils and lymphocytes decreased nitrite concentration after intense exercise. Basal iNOS levels and SOD activity were similar in neutrophils and lymphocytes. iNOS levels and SOD activity dropped in neutrophils and rose in lymphocytes after exercise. Arginase activity rose only in lymphocytes. Neutrophil nitrite was correlated with SOD activity and iNOS levels, but not in lymphocytes. iNOS levels were correlated with SOD in both neutrophils and lymphocytes. Intense exercise maintained plasma basal arginine and ornithine concentration, and decreased citrulline concentration. Intense exercise induced important changes in NO handling in neutrophils and lymphocytes, yet the basal picture was maintained in erythrocytes.     

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.     

Interleukin-1 beta induces nitric oxide production and inhibits the activity of aconitase without decreasing glucose oxidation rates in isolated mouse pancreatic islets.

The aim of this investigation was to further characterize the process of interleukin-1 beta (IL-1 beta) induced nitric oxide production in isolated pancreatic islets. It was found that both IL-1 beta and nitroprusside increased islet nitrite production. This effect was paralleled by inhibition of islet aconitase activity and glucose oxidation rates. Neither trifluoroperazinen or aminopterin could prevent the IL-1 beta induced increase in nitrite production, aconitase inhibition and decrease in glucose oxidation rates. In a second series of experiments, isolated mouse pancreatic islets were exposed to IL-1 beta for 24 h and subsequently used for nitrite production, aconitase activity and glucose oxidation determinations. The islets responded to IL-1 beta with an increased nitrite production and a decreased activity of aconitase, whereas the islet glucose oxidation rates were not decreased. It is concluded that IL-1 beta in both rat and mouse islets induces nitric oxide formation and that this induction leads to the inhibition of the Krebs cycle enzyme aconitase. In rat islets this probably leads to an inhibited insulin secretion, whereas IL-1 beta in mouse islets suppresses insulin secretion by a non-mitochondrial mechanism.     

Cytokines and lipopolysaccharides induce inducible nitric oxide synthase but not enzyme activity in adult rat cardiomyocytes

There is evidence that nitric oxide (NO) formation in adult cardiomyocytes stimulated with lipopolysaccharide (LPS) is not commensurate with iNOS levels. Tetrahydrobiopterin (BH(4)) is a key factor in the stabilization and NO production by iNOS homodimer. Thus we hypothesized that BH(4) is a limiting factor for NO production in adult cardiomyocytes in response to LPS and cytokines (TNF-alpha, IL-1, IFN-gamma alone, or mixed). It was verified that LPS and cytokines induced iNOS expression which did not translate into increased nitrite or [(14)C]citrulline production. This response coincided with defective BH(4) synthesis and low GTP cyclohydrolase activity. Furthermore, supplementation with BH(4) and ascorbate failed to increase iNOS activity. This effect was related to preferential accumulation of BH(2) rather than BH(4) in these cells. Uncoupled iNOS activity in stimulated cells was examined using mitochondrial aconitase activity as an endogenous marker of superoxide anion radical (O(2)(-)) formation, and found not to be significantly inhibited. 2-Hydroxyethidium also was not significantly increased. We conclude that adult cardiomyocytes are an unlikely source of NO and O(2)(-) in inflammatory conditions. This finding adds a new and unexpected layer of complexity to our understanding of the responses of the adult heart to inflammation.     

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.     

Inhibition of aconitase by nitric oxide leads to induction of the alternative oxidase and to a shift of metabolism towards biosynthesis of amino acids

Nitric oxide (NO) is a free radical molecule involved in signalling and in hypoxic metabolism. This work used the nitrate reductase double mutant of Arabidopsis thaliana (nia) and studied metabolic profiles, aconitase activity, and alternative oxidase (AOX) capacity and expression under normoxia and hypoxia (1% oxygen) in wild-type and nia plants. The roots of nia plants accumulated very little NO as compared to wild-type plants which exhibited ～20-fold increase in NO emission under low oxygen conditions. These data suggest that nitrate reductase is involved in NO production either directly or by supplying nitrite to other sites of NO production (e.g. mitochondria). Various studies revealed that NO can induce AOX in mitochondria, but the mechanism has not been established yet. This study demonstrates that the NO produced in roots of wild-type plants inhibits aconitase which in turn leads to a marked increase in citrate levels. The accumulating citrate enhances AOX capacity, expression, and protein abundance. In contrast to wild-type plants, the nia double mutant failed to show AOX induction. The overall induction of AOX in wild-type roots correlated with accumulation of glycine, serine, leucine, lysine, and other amino acids. The findings show that NO inhibits aconitase under hypoxia which results in accumulation of citrate, the latter in turn inducing AOX and causing a shift of metabolism towards amino acid biosynthesis.     

The relationship between nitric oxide and leptin in the lung of rat with streptozotocin-induced diabetes

Lung structural changes and immunoreactivity of endothelial (eNOS)- and inducible nitric oxide synthase (iNOS) were investigated by light microscopy in lungs of treated and untreated diabetic rats. Diabetes was induced by a single intraperitoneal (i.p.) injection of 65 mg kg(-1) streptozotocin (STZ) in Wistar albino male rats. Diabetic rats received daily i.p. doses of dexamethasone (2 mg kg(-1)), leptin (0.5 microg kg(-1)) and intramuscular insulin (20 U kg(-1)) or a combination of these drugs for 1 week starting 4 weeks after the STZ injections. After treatment, the blood levels of glucose, leptin, insulin and nitrate/nitrite (NO(3) (-)/NO(2) (-)) were measured. Dilatation of alveoli and alveolar ducts, partial alveolar wall thickening and increased eNOS- and iNOS characterized the diabetic rat lungs. High blood glucose and nitrate/nitrite levels as well as low insulin and leptin levels were also present. Treatment with insulin, dexamethasone and a combination of these drugs resulted in improvement of the structural and immunohistochemical abnormalities. The most effective treatment was insulin therapy. Leptin administration resulted in increased relative amounts of extracellular material, which led to noticeable respiratory efficiency in the diabetic rat lungs. All treatments except leptin lowered blood glucose levels. The combination of insulin and dexamethasone increased blood leptin and insulin, while the remaining diabetic rats had blood with low leptin and insulin concentrations. These results suggest that therapy with insulin plus dexamethasone but not therapy with leptin is beneficial for diabetics.     

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.     

The role of inducible nitric oxide synthase inhibitor on the arteriolar hyporesponsiveness in hemorrhagic-shocked rats

Hemorrhagic shock (HS) has been implicated in the induction of inducible nitric oxide synthase (iNOS) that leads to increase production of nitric oxide (NO). Recently, NO has been implicated to cause hyporesponsiveness of blood vessel in vitro towards vasoconstrictors in refractory (decompensated) HS. In our in vivo model, we examined the effects of aminoguanidine (AG), a known iNOS inhibitor, with angiotensin II (ANG II), a vasoconstrictor, following hemorrhagic shock decompensatory phase (HSDP) on percentage survival, vascular responsiveness, mean arterial blood pressure (MABP), heart rate and mean nitrate/nitrite levels in anaesthetized rats. HSDP (3 h) was achieved via constant pressure method (40-45 mmHg). MABP and heart rate was measured via the left carotid artery. Plasma collected from HSDP rats was used to measure nitrate/nitrite levels. Vascular hyporeactivity to ANG II was carried out using HSDP aortic strips, precontracted with KCl and noradrenaline. Sham-operated rats served as controls. HSDP rats decreased percentage survival, vascular contractility to ANG II and noradrenaline, MABP, heart rate while showing increased levels of nitrate/nitrite. Infusion of AG with ANG II, increased percentage survival and had reversed these cardiovascular effects of HSDP rats. This study indicates that excessive NO formation from iNOS activity induces vascular hyporeactivity and decompensation in HSDP. This might suggest that selective NOS inhibitor, AG, when coupled with ANG II, show reduction in NO's effect in HSDP.     

Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats

We hypothesized that aging is characterized by a reduced release of nitric oxide (NO) in response to shear stress in resistance vessels. Mesenteric arterioles and arteries of young (6 mo) and aged (24 mo) male Fischer 344 rats were isolated and cannulated. Shear stress (15 dyn/cm(2))-induced dilation was significantly reduced and shear stress (1, 5, 10, and 15 dyn/cm(2))-induced increases in perfusate nitrite were significantly smaller at all shear stress levels in vessels of aged rats. Inhibition of NO synthesis abolished shear stress-induced release of nitrite. Furthermore, shear stress (15 dyn/cm(2))-induced release of nitrate was significantly higher and total nitrite (nitrite plus nitrate) was significantly lower in vessels of aged rats. Tiron or SOD significantly increased nitrite released from vessels of aged rats, but this was still significantly less than that in young rats. Superoxide production was increased and the activity of SOD was decreased in vessels of aged rats. There were no differences in endothelial NO synthase (eNOS) protein and basal activity or in Cu/Zn-SOD and Mn-SOD proteins in vessels of the two groups, but extracellular SOD was significantly reduced in vessels of aged rats. Maximal release of NO induced by shear stress plus ACh (10(-5) M) was comparable in the two groups, but phospho-eNOS in response to shear stress (15 dyn/cm(2)) was significantly reduced in vessels of aged rats. These data suggest that an increased production of superoxide, a reduced activity of SOD, and an impaired shear stress-induced activation of eNOS are the causes of the decreased shear stress-induced release of NO in vessels of aged rats.     

The oral histone deacetylase inhibitor ITF2357 reduces cytokines and protects islet β cells in vivo and in vitro

In type 1 diabetes, inflammatory and immunocompetent cells enter the islet and produce proinflammatory cytokines such as interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); each contribute to β-cell destruction, mediated in part by nitric oxide. Inhibitors of histone deacetylases (HDAC) are used commonly in humans but also possess antiinflammatory and cytokine-suppressing properties. Here we show that oral administration of the HDAC inhibitor ITF2357 to mice normalized streptozotocin (STZ)-induced hyperglycemia at the clinically relevant doses of 1.25-2.5 mg/kg. Serum nitrite levels returned to nondiabetic values, islet function improved and glucose clearance increased from 14% (STZ) to 50% (STZ + ITF2357). In vitro, at 25 and 250 nmol/L, ITF2357 increased islet cell viability, enhanced insulin secretion, inhibited MIP-1α and MIP-2 release, reduced nitric oxide production and decreased apoptosis rates from 14.3% (vehicle) to 2.6% (ITF2357). Inducible nitric oxide synthase (iNOS) levels decreased in association with reduced islet-derived nitrite levels. In peritoneal macrophages and splenocytes, ITF2357 inhibited the production of nitrite, as well as that of TNFα and IFNγ at an IC(50) of 25-50 nmol/L. In the insulin-producing INS cells challenged with the combination of IL-1β plus IFNγ, apoptosis was reduced by 50% (P < 0.01). Thus at clinically relevant doses, the orally active HDAC inhibitor ITF2357 favors β-cell survival during inflammatory conditions.     

Saccharated colloidal iron enhances lipopolysaccharide-induced nitric oxide production in vivo

We investigated the effects of iron on the production of nitric oxide (NO), inducible NO synthase (iNOS), and plasma cytokines induced by lipopolysaccharide (LPS) in vivo. Male Wistar rats were preloaded with a single intravenous injection of saccharated colloidal iron (Fesin, 70 mg iron/kg body weight) or normal saline as a control, and then given an intraperitoneal injection of LPS (5.0 mg/kg body weight). Rats, preloaded with iron, had evidence of both iron deposition and strong iNOS induction in liver Kupffer cells upon injection of LPS; phagocytic cells in the spleen and lung had similar findings. LPS-induced NO production in iron-preloaded rats was significantly higher than control rats as accessed by NO-hemoglobin levels measured by ESR (electron spin resonance) and NOx (nitrate plus nitrite) levels. Western blot analysis showed that iron preloading significantly enhanced LPS-induced iNOS induction in the liver, but not in the spleen or lung. LPS-induced plasma levels of IL-6, IL-1beta, and TNF-alpha were also significantly higher in iron-preloaded rats as shown by ELISA, but IFN-gamma levels were unchanged. We conclude that colloidal-iron phagocytosed by liver Kupffer cells enhanced LPS-induced NO production in vivo, iNOS induction in the liver, and release of IL-6, IL-1beta, and TNF-alpha.     

Preventive Effects of Tocotrienol on Stress-Induced Gastric Mucosal Lesions and Its Relation to Oxidative and Inflammatory Biomarkers

This study aimed to investigate the possible gastroprotective effect of tocotrienol against water-immersion restraint stress (WIRS) induced gastric ulcers in rats by measuring its effect on gastric mucosal nitric oxide (NO), oxidative stress, and inflammatory biomarkers. Twenty-eight male Wistar rats were randomly assigned to four groups of seven rats. The two control groups were administered vitamin-free palm oil (vehicle) and the two treatment groups were given omeprazole (20 mg/kg) or tocotrienol (60 mg/kg) orally. After 28 days, rats from one control group and both treated groups were subjected to WIRS for 3.5 hours once. Malondialdehyde (MDA), NO content, and superoxide dismutase (SOD) activity were assayed in gastric tissue homogenates. Gastric tissue SOD, iNOS, TNF-α and IL1-β expression were measured. WIRS increased the gastric MDA, NO, and pro-inflammatory cytokines levels significantly when compared to the non-stressed control group. Administration of tocotrienol and omeprazole displayed significant protection against gastric ulcers induced by exposure to WIRS by correction of both ulcer score and MDA content. Tissue content of TNF-α and SOD activity were markedly reduced by the treatment with tocotrienol but not omeprazole. Tocotrienol significantly corrected nitrite to near normal levels and attenuated iNOS gene expression, which was upregulated in this ulcer model. In conclusion, oral supplementation with tocotrienol provides a gastroprotective effect in WIRS-induced ulcers. Gastroprotection is mediated through 1) free radical scavenging activity, 2) the increase in gastric mucosal antioxidant enzyme activity, 3) normalisation of gastric mucosal NO through reduction of iNOS expression, and 4) attenuation of inflammatory cytokines. In comparison to omeprazole, it exerts similar effectiveness but has a more diverse mechanism of protection, particularly through its effect on NO, SOD activity, and TNF-α.     

Mechanistic studies on AMD6221: a ruthenium-based nitric oxide scavenger

Nitric oxide is a mediator of many disease states. Previous studies have demonstrated that ruthenium(III) polyaminocarboxylates can react with NO to form stable complexes reducing the levels of nitrite in the culture medium of stimulated RAW264 macrophages and reverse the NO-mediated hypotension in animal models of septic shock. It was necessary to confirm that these observations were due to NO scavenging and not inhibition of the NO metabolic pathway. Using RAW264 cells it was confirmed that [Ru(H(3)dtpa)(Cl)] (AMD6221) was neither acting at the level of iNOS induction, nor as an inhibitor of iNOS by measuring iNOS mRNA by RT-PCR and protein by Western blot and enzyme activity. Using HPLC, the nitrosyl adduct of reaction of AMD6221, [Ru(H(2)dtpa)NO], was identified in the medium of stimulated RAW264 cells co-incubated with AMD6221, concomitant with a stoichiometric reduction in nitrite/nitrate levels, thus confirming that the ruthenium(III) polyaminocarboxylates exert their pharmacological effect by scavenging NO.     

Role of inducible nitric oxide synthase in leukocyte extravasation in vivo

Several recent studies have suggested that nitric oxide (NO) derived from the inducible isoform of NO synthase (NOS) may act as an endogenous modulator of the inflammatory response by inhibiting adhesion of leukocytes to endothelial cells in vitro. Few studies have addressed specifically the role of iNOS in regulating leukocyte recruitment in vivo in a model of acute inflammation. Thus, the objective of this study was to assess the role of iNOS in modulating neutrophil (PMN) extravasation in an oyster glycogen-induced model of acute peritonitis in rats. Data obtained in the present study demonstrates that injection (IP) of oyster glycogen induces massive and selective PMN recruitment into the peritoneal cavity of rats at 6 hrs following OG administration. These extravasated cells were found to contain significant amounts of iNOS protein as assessed by Western blot analysis. Treatment of rats with the selective iNOS inhibitor L-iminoethyl-lysine (L-NIL) dramatically reduced NO levels in lavage fluid as measured by decreases in nitrate and nitrite concentrations without significantly affecting iNOS protein levels. Although L-NIL inhibited NO production by >70%, it did not alter oyster glycogen-induced PMN recruitment when compared to vehicle-treated rats. We conclude that PMN-associated, iNOS-derived NO does not play an important role in modulating extravasation of these leukocytes in this model of acute inflammation.     

Proinflammatory agents,IL-8 and IL-10, upregulate inducible nitric oxide synthase expression and nitric oxide production in avian osteoclast-like cells

Nitric oxide synthase (NOS) isoenzymes generate nitric oxide (NO), a sensitive multifunctional intercellular signal molecule. High NO levels are produced by an inducible NOS (iNOS) in activated macrophages in response to proinflammatory agents, many of which also regulate local bone metabolism. NO is a potent inhibitor of osteoclast bone resorption, whereas inhibitors of NOS promote bone resorption both in vitro and in vivo. The possibility that osteoclasts, like macrophages, express a regulated iNOS and produce NO as a potential autocrine signal following inflammatory stimulation was investigated in well-characterized avian marrow-derived osteoclast-like cells. NO production (reflected by medium nitrite levels) was markedly elevated in these cells by the proinflammatory agents lipopolysaccharide (LPS) and the synergistic action of IL-1α, TNFα, and IFNγ. Inhibitors of NOS activity (aminoguanidine, L-NAME) or iNOS induction (dexamethasone, TGFβ) reduced LPS-stimulated nitrite production. LPS also increased the NOS-associated diaphorase activity of these cells and their reactivity with anti-iNOS antibodies. RT-PCR cloning, using avian osteoclast-like cell RNA and human iNOS primers, yielded a novel 900 bp cDNA with high sequence homology (76%) to human, rat, and mouse iNOS genes. In probing osteoclast-like cell RNA with the PCR-derived iNOS cDNA, a 4.8 kb mRNA species was detected whose levels were greatly increased by LPS. Induction of iNOS mRNA by LPS, or by proinflammatory cytokines, occurred prior to the rise of medium nitrite in time course studies and was diminished by dexamethasone. Moreover, osteoclast-like cells demonstrated an upregulation of NO production and iNOS mRNA by IL-8 and IL-10, regulatory mechanism's not previously described. It is concluded that osteoclast-like cells express a novel iNOS that is upregulated by inflammatory mediators, leading to NO production. Therefore, NO may serve as both a paracrine and autocrine signal for modulating osteoclast bone resorption. © 1996 Wiley-Liss, Inc.