Small Amounts Of Nitric Oxide Research Articles

Mitochondrial NO and reactive nitrogen species production: Does mtNOS exist?

It is more than 10 years now that mitochondria are suspected to be sources of nitric oxide (NO). This hypothesis is intriguing since NO has multiple targets within the organelle and it is even suggested that mitochondria are the primary targets of NO in the cell. Most remarkably, nanomolar concentrations of NO can inhibit mitochondrial respiration, so even a small amount of NO in the mitochondrial matrix may regulate ATP synthesis. Therefore, the idea that mitochondria themselves are capable of NO production is an important concept in several physiological and pathological mechanisms. However, this field of research generates surprisingly few original papers and the published studies contain conflicting results. The reliability of the results is frequently questioned since they are seldom reproduced by independent investigators. Until 2003, all papers published in this field showed affirmative results but since then several studies directly challenged the existence of a mitochondrial nitric oxide synthase. The present review aims to summarize the most recent developments in mitochondrial NO production, highlights a few unsolved questions, and proposes new directions for future work in this research area.

Rapid and reactive nitric oxide production by astrocytes in mouse neocortical slices

Nitric oxide (NO), a cellular signaling molecule, is produced in the brain by both neurons and astrocytes. While neurons are capable of rapid release of small amounts of NO serving as neurotransmitter, astrocytic NO production has been demonstrated mainly as a slow reaction to various stress stimuli. Little is known about the role of astrocyte-produced NO. Using the NO indicator 4,5-diaminofluorescein-2 diacetate (DAF-2DA) and acute slices from mouse brain, we distinguished neurons from astrocytes based on their different fluorescence kinetics and pattern, cellular morphology, electrophysiology, and responses to selective nitric oxide synthase (NOS) inhibitors. Typically, astrocytic fluorescence followed neuronal fluorescence with a delay of 1-2 min and was dependent on the inducible NOS isoform (iNOS) activity. Western blot analysis established the presence of functional iNOS in the neocortex. An assay for cell death revealed that most DAF-2DA-positive neurons, but not astrocytes, were damaged. Whole cell recordings from astrocytes confirmed that these cells maintained their membrane potential and passive properties during illumination and afterward. Induction of excitotoxicity by brief application of glutamate triggered an immediate and intense astrocytic response, while high-frequency electrical stimulation failed to do so. The present study demonstrates, for the first time, rapid and massive iNOS-dependent NO production by astrocytes in situ, which appears to be triggered by acute neuronal death. These data may bear important implications for our theoretical understanding and practical management of acute brain insults.

Detection of endothelial nitric oxide release with the 2,3-diaminonapthalene assay.

The reliable measurement of nitric oxide (NO) production by endothelial cells in vitro has become an important tool for investigating mechanisms of endothelial dysfunction. This study evaluates measuring NO production by cultured porcine pulmonary artery endothelial cells (PAEC) using the assay based on the fluorometric detection of 1-(H)-naphthotriazole, the fluorescent product of the reaction between nitrite (NO2-) and 2,3-diaminonapthalene (DAN). To stimulate NO production, PAEC were treated for 60 min with agonists known to stimulate endothelial NO production. The DAN assay was unable to detect NO production from agonist-stimulated PAEC. In contrast, chemiluminescence analysis, which detects NO, NO2-, and nitrate (NO3-) (collectively referred to as NO(x)), detected significant increases in NO(x) from stimulated PAEC. Nitrate reductase-mediated reduction of NO3-to NO2- in media from stimulated PAEC enhanced the ability of the DAN assay to detect NO release from PAEC. These results provide the first direct comparison of the sensitivity of these two commonly employed assays. Our findings emphasize that NO3-reduction may be required to enable the DAN assay to detect small amounts of NO produced by cultured endothelial cells.

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H 2O 2, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- d-glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO 2 −, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.

Nitric oxide, oxidative stress, and dietary antioxidants

S ince nitric oxide (NO) was determined to be an important physiologic molecule in the late 1980s, its role in the pathophysiology of disease, therapeutic uses, and possible detrimental effects has occupied many researchers. In the process, the impact of NO on nutrition is being defined. The molecule and its metabolites affect gastrointestinal function, macronutrient metabolism and micronutrient status. Some of the actions of NO in gastrointestinal physiology and interactions with vitamins E and C in vascular tissue were recently covered in Nutrition.1 In the present article, the role of nutrients in the control of NO, in particular peroxynitrite defense, is discussed. The chemistry of NO is complex. The molecule reacts principally with guanylate cyclase and metaloproteins, such as those with iron or iron–sulfur centers.2 It is produced from arginine by nitric oxide synthase (NOS), which exists in three isoforms. Two of the isoforms are constitutive to endothelial tissue and neurons and produce small amounts of NO in a short time. Acting as a signal, this NO activates guanylate cyclase. In the case of endothelial NOS (eNOS), the end result is smooth muscle relaxation and increased blood flow. Much of the NO produced in this cycle is scavenged by hemoglobin and eventually reduced to nitrate.2 The third isoform of NOS (iNOS) is inducible by cytokines, notably tumor necrosis factor, interleukin-1, and lipolysaccharide. Actual NO production is evident a few hours later. In humans the enzyme is tightly regulated and at least two activators must be present.3 As part of the inflammatory response, iNOS produces large amounts of NO, which is lethal to pathogens and injurious to cells.4 NO AND OXIDATIVE STRESS

Nitric oxide and cardioprotection during ischemia-reperfusion.

Coronary artery reperfusion is widely used to restore blood flow in acute myocardial infarction and limit its progression. However, reperfusion of ischemic myocardium results in reperfusion injury and persistent ventricular dysfunction even when achieved after brief periods of ischemia. Normally, small amounts of nitric oxide (NO) generated by endothelial NO synthase (eNOS) regulates vascular tone. Ischemia-reperfusion triggers the release of oxygen free radicals (OFRs) and a cascade involving endothelial dysfunction, decreased eNOS and NO, neutrophil activation, increased cytokines and more OFRs, increased inducible NO synthase (iNOS) and marked increase in NO, excess peroxynitrite formation, and myocardial injury. Low doses of NO appear to be beneficial and high doses harmful in ischemia-reperfusion. eNOS knock-out mice confirm that eNOS-derived NO is cardioprotective in ischemia-reperfusion. iNOS overexpression increases peroxynitrite but did not cause severe dysfunction. Increased angiotensin II (AngII) after ischemia-reperfusion inactivates NO, forms peroxynitrite and produces cardiotoxic effects. Beneficial effects of angiotensin-converting-enzyme inhibition and AngII type 1 (AT(1)) receptor blockade after ischemia-reperfusion are partly mediated through AngII type 2 (AT(2)) receptor stimulation, increased bradykinin and NO. Interventions that enhance NO availability by increasing eNOS might be beneficial after ischemia-reperfusion.

Protection of Human Keratinocyte mtDNA by Low-Level Nitric Oxide

This study was designed to evaluate the DNA damaging effects of nitric oxide and to determine whether the endogenous generation of nitric oxide at low levels in the cell exerts a protective effect against this damage. Damage to mitochondrial and nuclear DNA in normal human epidermal keratinocytes (NHEK) was assessed after treatment of these cells with varying concentrations of S-nitroso-N-acetylpenicillamine, which decomposes to release nitric oxide. The results showed that mitochondrial DNA was more vulnerable to nitric oxide-induced damage than was a similarly sized fragment of the β-globin gene. To evaluate the effects on DNA damage by pretreatment of cells with low-levels of nitric oxide, NHEK cells were treated with the prodrug V-PYRRO/NO. This agent is metabolized inside these cells and releases small quantities of nitric oxide. The cells then were exposed to damaging amounts of nitric oxide produced by S-nitroso-N-acetylpenicillamine. The results of these studies showed that pretreatment of NHEK cells with V-PYRRO/NO attenuated the mtDNA damage and loss of cell viability produced by exposure to S-nitroso-N-acetylpenicillamine.

− OONO

Inhaled nitric oxide is an elegantly simple therapy for idiopathic pulmonary hypertension. Breathing small amounts of nitric oxide directly activates guanylyl cyclase in pulmonary resistance vessels to counteract hypertension. In patients who respond to inhaled nitric oxide, either the endogenous synthesis of nitric oxide is not sufficient to overcome the hypertensive stress or nitric oxide is being inactivated too rapidly to act on guanylate cyclase. Certainly, both processes could be operating simultaneously. Although nitric oxide has a reputation for being highly toxic, in reality the risk of toxicity with inhaled nitric oxide is minor because nitric oxide itself is unreactive with most biological molecules and the amounts administered are low enough to minimize the formation of nitrogen dioxide. Nitric oxide becomes toxic when converted to secondary reactive nitrogen species. Nitric oxide itself is swept away from the vasculature by rapid reactions with hemoglobin. In the high oxygen environment of the lung, nitric oxide will also not significantly inhibit mitochondrial respiration. By now, many patients have been breathing nitric oxide for weeks and even months without overt harm. However, sudden termination of inhaled nitric oxide occasionally causes a potentially life-threatening hypertensive rebound, even when treated for a few hours. Hypertensive rebound can occur in individuals who showed no initial vasodilation in response to nitric oxide. In this issue of Circulation Research , Wedgwood et al1 have shown that endothelin, the most …

Heat Shock Restores Insulin Secretion after Injury by Nitric Oxide by Maintaining Glucokinase Activity in Rat Islets

Heat shock protein (hsp), including hsp70, has been reported to restore the glucose-induced insulin release suppressed by nitric oxide (NO). However, the mechanism underlying this recovery remains unclear. In the present study, we examine the effects, in rat islets, of heat shock on insulin secretion inhibited by a small amount of NO and also on glucose metabolism, the crucial factor in insulin release. Exposure to a higher dose (15 U/ml) of interleukin-1β (IL-1β) abolished the insulin release by stimulation of glucose or KCl in both control and heat shocked islets. In rat islets exposed to a lower dose (1.5 U/ml) of IL-1β, insulin secretion in response to glucose, but not to glyceraldehydes (GA), ketoisocaproate (KIC), or KCl, was selectively impaired, concomitantly with lower ATP concentrations in the presence of 16.7 mM glucose, while such suppression of insulin secretion and ATP content was not observed in heat shock-treated islets. NO production in islets exposed to 1.5 U/ml IL-1β was significantly, but only partly, decreased by heat shock treatment. The glucose utilization rate measurement using [5-3H]-glucose and [2-3H]-glucose and the glucokinase activity in vitro were reduced in islets treated with 1.5 U/ml IL-1β. In heat shock-treated islets, glucose utilization and glucokinase activity were not affected by 1.5 U/ml IL-1β. These data suggest that heat shock restores glucose-induced insulin release inhibited by NO by maintaining glucokinase activity and the glucose utilization rate in islets in addition to reducing endogenous NO production.

Effects of cytokines on the production of nitric oxide in a chondrogenic cell line established from human osteogenic sarcoma.

The purpose of this study was to examine the effects of various cytokines and/or lipopolysaccharide (LPS) on nitric oxide (NO) production from USAC, a newly established clonal cell line derived from human osteogenic sarcoma that expressed chondrocytic phenotypes. No production was measured by Griess method. Inducible nitric oxide synthase (iNOS) mRNA was detected by PCR analysis. Western blotting analysis and immunocytochemistry was used to detect iNOS protein. Although USAC cells treated without any stimulants produced only small amounts of NO, exposure to cytokines and/or LPS induced iNOS in USAC cells and produced high levels of NO. The stimulatory effects of cytokines and/or LPS on NO production required TNF-alpha. TNF-alpha alone neither induced iNOS in USAC cells nor caused production of NO, but addition of TNF-alpha to USAC cells pretreated with LPS and IFN-gamma enhanced the expression of iNOS mRNA, induced iNOS protein and produced NO. Dexamethasone inhibited the stimulatory effect of TNF-alpha. The responsiveness of USAC cells to cytokines and/or LPS and steroid hormone on NO production was quite different from that reported for rabbit and human articular cartilaginous cells. The differences in responsiveness between articular cartilaginous chondrocytes and USAC cells might have been because USAC cells were established from a malignant tumor.

Expression of nitric oxide synthases in subcutaneous adipose tissue of nonobese and obese humans

Studies have shown evidence of production of nitric oxide (NO) in adipose tissue, as well as inhibition of lipolysis by NO. We have analyzed nitric oxide synthase (NOS) expression in subcutaneous adipose tissue from 13 nonobese and 18 obese male subjects. Using a competitive reverse transcription polymerase chain reaction method, endothelial (eNOS) and inducible (iNOS), but not neuronal (nNOS), nitric oxide synthase mRNA expression was detected in isolated fat cells and pieces of adipose tissue. Tissue mRNA levels for eNOS were 3,814 +/- 825 and 5,956 +/- 476 amol/mg RNA (P = 0.043), and for iNOS 306 +/- 38 and 332 +/- 48 amol/mg RNA, for nonobese and obese individuals, respectively. Western blotting revealed similar eNOS protein levels in isolated fat cells and adipose tissue pieces. Protein levels for eNOS in nonobese and obese individuals, respectively, were (in optical density [OD] units per mm(2) per 100 microgram of total protein) 0.11 +/- 0.08 and 2.80 +/- 1.30 (P = 0.043). iNOS protein was detectable, but not measurable, at low levels in a subset of obese patients (3 of 10). iNOS protein levels could not be detected in nonobese individuals. Hormone-sensitive lipase (HSL), the key regulating enzyme in lipolysis, is reduced in obesity. The expression of HSL protein in subcutaneous adipose tissue was studied in the same subset of patients; in agreement with previous results, HSL levels were reduced in obese subjects: 4.64 +/- 1.10 and 1.27 +/- 0.35 (P = 0.012) in nonobese and obese subjects, respectively. In conclusion, this study shows that eNOS and iNOS, but not nNOS, are present in human subcutaneous adipose tissue. Gene expression and protein levels of eNOS are increased, whereas HSL protein levels are decreased in obesity. It is speculated that increased NO production, preferably by eNOS, and decreased HSL levels may cause decreased subcutaneous adipose tissue lipolysis in obesity. synthases in subcutaneous adipose tissue of nonobese and obese humans.

Inhibitory effect of sodium salicylate on nitric oxide production from TM4 Sertoli cells

Nitric oxide (NO) has been proposed to play a role in a variety of inflammatory diseases. Sodium salicylate (NaSal) is the most commonly used anti-inflammatory agent. We investigated whether NaSal can diminish the production of NO in TM4 Sertoli cells. TM4 Sertoli cells produced a small amount of NO upon treatment with recombinant interferon-γ (rIFN-γ). The effect of rIFN-γ was enhanced markedly by the addition of recombinant TNF-α (rTNF-α) in a dose-dependent manner. NaSal (10 and 20 mM) significantly inhibited NO production from TM4 Sertoli cells induced by rIFN-γ plus rTNF-α. In addition, rIFN-γ in combination with rTNF-α showed a marked increase of the expression of inducible NO synthase (iNOS) protein. Western blot analysis revealed that NaSal (10 and 20 mM) blocked a step of iNOS protein synthesis. The rIFN-γ plus rTNF-α-induced nuclear factor-κB (NF-κB) activation was significantly blocked by NaSal (10 and 20 mM). On the other hand, neither staurosporine nor polymyxin B significantly inhibited NO production from TM4 Sertoli cells induced by rIFN-γ plus rTNF-α. The present results indicate that NaSal inhibits rIFN-γ plus rTNF-α-induced NO production in TM4 Sertoli cells via the signal transduction pathway of NF-κB activation.

The role of nitric oxide in arthritic joints: a therapeutic target?

Nitric oxide (NO) is produced by many cell types in the joint, and its expression is delicately regulated. Depending on its concentration and cellular origin, NO appears to have both pro- and anti-inflammatory potential in the joint. Constitutively expressed nitric oxide synthase (NOS) produces small amounts of NO, which is essential for normal physiological homeostasis. However, inflammatory stimuli such as endotoxins, cytokines, and growth factors promote inducible NOS (iNOS) expression, initially as an anti-inflammatory response, and catalyse a high output of NO. Excessive NO can amplify inflammatory pathways and contribute to the development and maintenance of arthritis. Consequently, proper regulation of NO synthesis can lead to a novel therapeutic approach for inflammatory joint diseases. Further careful study will be necessary to develop new drugs to regulate the NO pathway and to determine the dosage, timing of administration, and duration of treatment in order to avoid both undesirable immunostimulatory effects and immunosuppressive effects.

New NO donors with antithrombotic and vasodilating activities, Part 29. N-(1-cyanocyclohexyl)-C-phenylnitrones and glyoxaldinitrones.

Six N-(1-cyanocyclohexyl)-C-phenylnitrones 4a-f (4b-f for the first time) and 22 glyoxaldinitrones 7a-v were prepared and tested for antithrombotic (p.o. administration to rats, 60 mg/kg) effects. Both classes of compounds exhibit considerable antithrombotic activities. Maximum inhibition of thrombus formation in arterioles (21%) was observed in N,N'-bis-2-phenylethylglyoxaldinitrone (7o) and N,N'-bis-4-nitrobenzylglyoxaldinitrone (7u). The compounds form only small amounts of nitric oxide in vitro by the addition of a Fe(3+)-porphyrine complex and an oxygen donor.

Selective inhibition of inducible nitric oxide synthase: A promising strategy in the therapy of septic shock?

Nitric oxide (NO) is a short-lived effector molecule which is produced from L-arginine by several NO synthase (NOS) isoforms. Physiologically, small amounts of NO are produced by an endothelial constitutive NOS (ecNOS), which is involved in the regulation of vascular tone and blood flow distribution. On stimulation by bacterial products and various cytokines, an inducible NOS (iNOS) becomes diffusely expressed, producing large amounts of NO for extended periods of time, which are implicated in the pathogenesis of septic shock. The pharmacological inhibition of NO synthesis has been, therefore, proposed as a new therapy in this setting. Unfortunately, such inhibition has been frequently reported to be detrimental, and recent evidence reveals that this deleterious potential is a consequence of ecNOS blockade by nonselective NOS inhibitors. Thus, interest is now focusing in the identification of compounds able to selectively inhibit iNOS activity. Although the effects of such selective agents have been only poorly investigated so far, they appear extremely promising. Indeed, in various animal models of septic shock, selective iNOS inhibitors have produced a marked improvement in hemodynamics, tissue oxygenation, and organ function, leading to a reduced mortality. These favorable results, which markedly contrast with the deleterious influence of nonselective NOS inhibitors in similar conditions, suggest that selective iNOS inhibitors might become useful adjuncts to septic shock therapy in the future.

Limitations of exogenous L-arginine in exerting a cytoprotective effect on hepatic ischemia/reperfusion injury.

To test whether or not the L-arginine/nitric oxide (NO) pathway induces a protective effect, we investigated the effect of exogenous L-arginine on hepatic ischemia/reperfusion (I/R) injury, using ex vivo perfusion of the isolated rat liver. The rat liver was removed and preserved in cold saline for 60 min, followed by 120 min of reperfusion with oxygenated perfusate at 37 degrees C. Either 600 mg/kg of L-arginine (groups 1 and 4), D-arginine (group 2), N(G)-nitro-L-arginine methyl ester (L-NAME) (group 3), or saline (group 5) were administered through the portal vein starting from 5 min before reperfusion to 5 min after reperfusion. In group 4, 600 mg/kg of L-NAME was preadministered at 10 min prior to the administration of L-arginine. The intrahepatic nitric oxide (NO) levels showed only a temporal elevation (227% +/- 70% of the pre-reperfusion levels at 5 min) after reperfusion in group 1. Pretreatment with L-NAME suppressed the elevation of the NO levels immediately after reperfusion in group 4. The lactate dehydrogenase release to the effluent perfusate significantly decreased and the histological findings showed that the sinusoidal damage observed after reperfusion was mitigated in group 1 more than in the other groups. These results thus suggest that exogenous L-arginine produced a relatively small amount of NO and therefore resulted in a slight decrease of hepatic I/R injury.

Modification of the Cadmium Reduction Assay for Detection of Nitrite Production Using Fluorescence Indicator 2,3-Diaminonaphthalene

The measurement of nitric oxide (NO) is important for characterizing the regulatory roles of NO in various biological systems. In this communication we report that cadmium (Cd) reduction of nitrate (NO−3) to nitrite (NO−2) can be quantitated by using the fluorescence indicator, 2,3-diaminonaphthalene (DAN) to detect the sum of NO−3 and NO−2 (NO−x) from endothelial cells. This assay is at least 10-fold more sensitive than when Cd reduction is coupled with the spectrophotometric Greiss reaction and can be used to quantitate the small amounts of NO−x generated from the constitutive form of endothelial nitric oxide synthase (eNOS). In addition various P2 purinoceptor agonists and antagonists do not interfere the Cd reduction/DAN assay. Thus the Cd reduction/DAN assay can be used not only to characterize P2 purinoceptor release of NO−x from cultured endothelial cells but also to quantitate NO−x levels in serum.

Protein Kinase C Independent Activation of Inducible Nitric Oxide Synthase by Tumor Necrosis Factor-α in TM4 Sertoli Cells

To investigate the nitric oxide (NO) production and its signalling mechanism in TM4 Sertoli cells, the cells were treated with recombinant tumor necrosis factor-α (rTNF-α), recombinant interleukin-1 α (rIL-lα), or lipopolysaccharide (LPS), either alone or in combination with recombinant interferon-γ (rIFN-γ), and NO production was measured by using the Griess method. TM4 Sertoli cells produced a small amount of NO upon treatment with rIFN-γ. The effect of rIFN-γ was drastically increased by cotreatment with rTNF-α in a dose-dependent manner. However, combination of rIL-lα or LPS with rIFN-γ did not synergize to activate cells. RIFN-γ in combination with rTNF-α showed marked increase of the expression of iNOS protein. Protein kinase C inhibitors did not inhibit the production of NO induced by rIFN-α plus rTNF-α. These results suggest that the role of TNF-α is to provide TM4 Sertoli cells with the active cofactor for NO production and TNF-α-induced signaling for induction of NO synthesis is not dependent on protein kinase C activation.

Practical methods for detection of nitric oxide

Nitric oxide (NO), which is generated in vivo through conversion of L-arginine to L-citrulline by NO synthase (NOS), mediates many physiological and pathophysiological processes. At least two distinct isoforms of NOS have been identified, constitutive NOS (cNOS) and inducible NOS (iNOS). The cNOS, which is constitutively expressed in endothelial cells and central and peripheral neuronal cells, requires both calcium and calmodulin for its activation. Cells expressing cNOS generally produce small amounts of NO, because of their low levels of cNOS protein. On the other hand, iNOS, which is induced in cells stimulated with endotoxins, produced larger amounts of NO. Because of the short half-life, it is difficult to detect NO in situ directly, especially from cells expressing cNOS. In this review, we discuss practical methods for NO detection, which are useful for the detection of small amounts of NO from cNOS and for the bioimaging of living cells and cultured tissues.

Nitric oxide mediates the mitogenic effects of insulin and vascular endothelial growth factor but not of leptin in endothelial cells.

The regulation of vascular wall homeostasis by nitric oxide (NO) generated by endothelium is being intensively studied. In the present paper, the involvement of NO in the vascular endothelial growth factor (VEGF), insulin or leptin-stimulated proliferation of human endothelial cells (HUVEC) was measured by [3H]thymidine or bromodeoxyuridine incorporation. VEGF and insulin, but not leptin, increased NO generation in HUVEC, as detected with ISO-NO electrode. Proliferation of HUVEC induced by leptin was not changed or was higher in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) a nitric oxide synthase (NOS) inhibitor. In contrast, L-NAME blunted the proproliferative effect of VEGF and insulin. Furthermore, we demonstrated that, in human arterial smooth muscle cells (hASMC) transfected with endothelial NOS (eNOS) gene, the generation of biologically active VEGF protein was NO-dependent. Inhibition of NO generation by L-NAME decreased the synthesis of VEGF protein and attenuated HUVEC proliferation induced by conditioned media from transfected hASMC. Endothelium-derived NO seems to participate in VEGF and insulin, but not leptin, mitogenic activity. Additionally, the small amounts of NO released from endothelial cells, as mimicked by eNOS transfection into hASMC, may activate generation of VEGF in sub-endothelial smooth muscle cells, leading to increased synthesis of VEGF protein necessary for turnover and restitution of endothelial cells.