Stimulated murine macrophages as a bioassay for H. pylori-related inhibition of nitric oxide production

BACKGROUND: Interference with the L-arginine/nitric oxide pathway may be a virulence strategy for the gastric pathogen Helicobacter pylori. This study evaluates a bioassay for such inhibitory actions on nitric oxide synthase. METHODS: Cultured murine macrophages were stimulated by lipopolysaccharide and interferon-gamma. Nitric oxide synthesis and the expression of inducible nitric oxide synthase (iNOS) at increasing concentrations of L-arginine were analysed using chemiluminescence and Western blotting, respectively. RESULTS: The bioassay was evaluated against nitrite accumulation and two established NOS inhibitors. Bacterial extracts or whole cells of one H. pylori strain inhibited nitric oxide production at low L-arginine concentrations (2-20 microM). A higher concentration of L-arginine (200 microM) was not associated with such inhibition. The iNOS expression was not affected by any of the additives compared to stimulated controls. CONCLUSIONS: This bioassay is a reliable and simple method for analysing iNOS inhibition, resolving effects on enzyme activity or enzyme expression. H. pylori water extract and whole cells exert an L-arginine-dependent NOS inhibition, not influencing iNOS expression.     

L-Arginine is not the limiting factor for nitric oxide synthesis by human alveolar macrophages in vitro.

Unlike murine mononuclear phagocytes, human macrophages do not release high amounts of nitric oxide (NO) in vitro despite the presence of nitric oxide synthase (NOS). To determine whether this limited NO synthesis in vitro is due to limited availability of the NOS substrate L-arginine, and putative NOS inhibiting factors present in foetal serum preparations, both alveolar macrophages (AM) and monocyte derived macrophages (MDM) were incubated in various circumstances. Nitrite production measured using stimulated AM was typically <5 pmol x min(-1) x 10(-6) cells. A range of stimuli were tested, but without result. Furthermore, incubation of MDMs with normal human serum or purified bovine serum albumin instead of foetal calf serum failed to enhance NO production. Moreover, neither the use of arginase inhibitors nor the addition of surplus L-arginine resulted in an increased NO synthesis. Interestingly, addition of the NOS intermediate Nomega-hydroxy-L-arginine (100 microM) to AM led to nitrite release, which was unaffected by the NOS inhibitor amino guanidine showing that this effect is NOS independent. It is concluded that the limited nitric oxide production of human macrophages in vitro can neither be explained by limited availability of L-arginine, nor by nitric oxide synthase inhibiting substances in foetal serum. Furthermore, it is shown that nitrite release from Nomega-hydroxy-L-arginine by alveolar macrophages is nitric oxide synthase independent.     

Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures.

Exposure of primary cultures of neonatal rat cortical astrocytes to bacterial lipopolysaccharide (LPS) results in the appearance of nitric oxide synthase (NOS) activity. The induction of NOS, which is blocked by actinomycin D, is directly related to the duration of exposure and dose of LPS, and a 2-hr pulse can induce enzyme activity. Cytosol from LPS-treated astrocyte cultures, but not from control cultures, produces a Ca(2+)-independent conversion of L-arginine to L-citrulline that can be completely blocked by the specific NOS inhibitor NG-monomethyl-L-arginine. The induced NOS activity exhibits an apparent Km of 16.5 microM for L-arginine and is dependent on NADPH, FAD, and tetrahydrobiopterin. LPS also induces NOS in C6 glioma cells and microglial cultures but not in cultured cortical neurons. The expression of NOS in astrocytes and microglial cells has been confirmed by immunocytochemical staining using an antibody to the inducible NOS of mouse macrophages and by histochemical staining for NADPH diaphorase activity. We conclude that glial cells of the central nervous system can express an inducible form of NOS similar to the inducible NOS of macrophages. Inducible NOS in glia may, by generating nitric oxide, contribute to the neuronal damage associated with cerebral ischemia and/or demyelinating diseases.     

beta-Lapachone reduces endotoxin-induced macrophage activation and lung edema and mortality

beta-Lapachone, a 1,2-naphthoquinone, is a novel chemotherapeutic agent. It has been shown to be capable of suppressing inducible nitric oxide synthase expression and function in rat alveolar macrophages. The authors further performed experiments to examine the molecular mechanism of beta-lapachone on LPS-induced responses in rat alveolar macrophages and to evaluate its in vivo antiinflammatory effect. A significant increase in nitrite production and inducible nitric oxide synthase expression was elicited in macrophages treated with LPS that was inhibited by coincubation with beta-lapachone. beta-Lapachone could also inhibit the production of tumor necrosis factor-alpha induced by LPS. LPS induces protein tyrosine phosphorylation and nuclear factor-kappaB binding activity by gel mobility shift assay in macrophages. These events were significantly inhibited by beta-lapachone. Furthermore, beta-lapachone in vivo protected against the induction of lung edema, lung-inducible nitric oxide synthase protein expression and nuclear factor-kappaB activation, lethality, and increased plasma nitrite and serum tumor necrosis factor-alpha levels induced by LPS. These results indicate that beta-lapachone suppresses inducible nitric oxide synthase induction and tumor necrosis factor-alpha production mediated by the inhibition of protein tyrosine phosphorylation and nuclear factor-kappaB activation caused by LPS. This results in a beneficial effect in an animal model of sepsis.     

L-Arginine transport in disease

The importance of membrane transport in normal physiological cell function is unquestionable. However, to what extent alterations in the transport of amino acids are the cause and/or consequence of pathological changes observed in disease states is a question not yet completely clarified. Kinetic experiments with blood cells provide a simple and useful model for researching alterations in amino acid transport. The cationic amino acid L-arginine is the precursor of nitric oxide (NO), a key second messenger involved in functions such as endothelium-dependent vascular relaxation, immune defence and platelet activation. The transport of L-arginine, being rate-limiting for nitric oxide production, is extremely relevant to pathological conditions where NO synthesis and/or actions are affected. The current review provides an overview of L-arginine transport in disease, specifically in uraemia, heart failure, hypertension, diabetes mellitus, septic shock and sickle cell disease.     

Gastric nitric oxide synthase expression during endotoxemia: implications in mucosal defense in rats

BACKGROUND & AIMS: This study was performed to examine expression of gastric nitric oxide synthase (NOS) isoforms during endotoxemia in rats and to assess their role(s) in gastric injury from bile and ethanol. METHODS: Lipopolysaccharide (LPS) enhanced the expression and activity of inducible nitric oxide synthase in gastric mucosa in a dose- and time-dependent manner. RESULTS: Endothelial nitric oxide synthase and neural nitric oxide synthase expression did not significantly change, but constitutive nitric oxide synthase activity decreased over time. LPS alone caused injury to the gastric mucosa and disrupted F-actin filaments in the same cells with enhanced immunostaining for inducible nitric oxide synthase. LPS also exacerbated gastric injury from the mild irritants 5 mmol/L acidified taurocholate and 20% ethanol as did local intra-arterial infusion of the nitric oxide donor S-nitroso-N-acetyl-penicillamine. The selective inducible nitric oxide synthase inhibitor aminoguanidine negated LPS-induced exacerbation of gastric injury from these irritants. The nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester augmented the deleterious effects of LPS, an effect reversed by L-arginine but not D-arginine. Aminoguanidine, but not N(G)-nitro-L-arginine methyl ester, negated LPS-induced accumulation of gastric luminal nitrates. CONCLUSIONS: These data suggest that increased inducible NOS activity and decreased constitutive nitric oxide synthase activity are primarily responsible for exacerbating gastric injury from luminal irritants during endotoxemia. Moreover, septic patients may be more susceptible to gastric injury from bile during gastrointestinal ileus.     

Concomitant down-regulation of L-arginine transport and nitric oxide (NO) synthesis in rat alveolar macrophages by the polyamine spermine.

Polyamines can inhibit NO synthesis in activated macrophages (Mphi). Since NO synthesis in Mphi depends on cellular uptake of L-arginine, effects of polyamines on L-arginine uptake were studied. Rat alveolar Mphi (AMphi) were cultured in absence or presence of lipopolysaccharides (LPS) and/or different polyamines for up to 20 h. LPS increased nitrite accumulation about 10-fold and [(3)H]-L-arginine uptake about 2.5-fold, effects almost abolished by spermidine. Spermine had much weaker and putrescine no effects. The effects of spermine depended largely on the presence of serum in the culture medium, suggesting that spermine aldehyde might be involved. Spermine suppressed the mRNA for inducible nitric oxide synthase (iNOS) and that for a specific cationic amino acid transporter (CAT), CAT-2B. In conclusion, in Mphi spermine concomitantly down-regulates NO synthesis and cellular L-arginine uptake by suppressing the expression of iNOS and CAT-2B. By inhibiting specific functions of activated Mphi the polyamine oxidase-polyamine system may play a role as immuno-suppressive modulator.     

Nitric oxide: from endogenous vasodilator to biologic mediator

This paper review the actual knowledges about the physiological role of nitric oxide, sintetized from amino acid L-arginine. The nitric oxide sintetized in the vascular endothelium has a fundamental role in vascular tone, blood flow and arterial pressure control, acting stimulating guanylate cyclase on vascular smooth muscle. Nitric oxide could be considered the endogenous nitrovasodilator. Its action on the cardiovascular system are imitated by nitroglycerine, sodium nitroprusside and related compounds. Probably the disturbance in the synthesis or release of nitric oxide may be involved in the pathophysiology of hypertension, vasospasm and atherosclerosis. Recently has been shown that nitric oxide synthesis from L-arginine also occurs in other different cells like macrophages, central nervous system, liver, neutrophils, adrenal glands, playing different biological effects. Changes in nitric oxide synthesis or action in those systems, could be related to different pathological disorders as inflammation, atherosclerosis and cancer. The found of a substance as simple as nitric oxide, let suppose that we are in the presence of a biological mediator with a very early evolutionary origin, probably widespread in all the animal kingdom, and which represents the universal transduction system for activation of the soluble guanylate cyclase enzyme.     

Intrauterine growth retardation is associated with reduced activity and expression of the cationic amino acid transport systems y+/hCAT-1 and y+/hCAT-2B and lower activity of nitric oxide synthase in human umbilical vein endothelial cells

Intrauterine growth retardation (IUGR) is associated with vascular complications leading to hypoxia and abnormal fetal development. The effect of IUGR on L-arginine transport and nitric oxide (NO) synthesis was investigated in cultures of human umbilical vein endothelial cells (HUVECs). IUGR was associated with membrane depolarization and reduced L-arginine transport (V(max)= 5.8+/-0.2 versus 3.3+/-0.1 pmol/microg protein per minute), with no significant changes in transport affinity (K(m)=159+/-15 versus 137+/-14 micromol/L). L-Arginine transport was trans-stimulated (8- to 9-fold) in cells from normal and IUGR pregnancies. IUGR was associated with reduced production of L-[3H]citrulline from L-[3H] arginine, lower nitrite and intracellular L-arginine, L-citrulline, and cGMP. IUGR decreased hCAT-1 and hCAT-2B mRNA, and increased eNOS mRNA and protein levels. IUGR-associated inhibition of L-arginine transport and NO synthesis, and membrane depolarization were reversed by the NO donor S-nitroso-N-acetyl-L,D-penicillamine. In summary, endothelium from fetuses with IUGR exhibit altered L-arginine transport and NO synthesis (L-arginine/NO pathway), reduced expression and activity of hCAT-1 and hCAT-2B and reduced eNOS activity. Alterations in L-arginine/NO pathway could be critical for the physiological processes involved in the etiology of IUGR in human pregnancies.     

Vascular effects of dietary L-arginine supplementation

The vascular endothelium is acknowledged to play an important role in vascular physiology. Attention has focused on endothelial production of nitric oxide as a key element in many of the processes associated with the development of atherosclerosis. L-arginine is the substrate for the enzyme nitric oxide synthase (NOS), which is responsible for the endothelial production of nitric oxide. Therefore, many investigators have been interested in whether dietary L-arginine supplementation can augment nitric oxide production and thereby improve vascular health. The effects of oral L-arginine on vascular health and disease have been examined both in human beings and in various animal models. In this review, we summarize the results of studies of oral L-arginine supplementation on atherosclerotic lesion formation, as well as markers of endothelial function (e.g. macrophage function, platelet aggregation and adhesion, and in vitro vascular ring studies). Although results of oral L-arginine supplementation in hypercholesterolemic animals have generally shown beneficial effects, the data in humans are varied, possibly because of small sample sizes and brief periods of study. Long-term randomized clinical trials are needed to more definitively address whether oral L-arginine supplementation could be advantageous for vascular health.     

Mechanisms of disease: L-arginine in coronary atherosclerosis--a clinical perspective

L-arginine is the substrate of endothelial nitric oxide synthase and the main precursor of nitric oxide in the vascular endothelium, thus its effects are mediated largely by increases in nitric oxide production. L-arginine has antioxidant and antiapoptotic properties, increases smooth muscle relaxation, inhibits the expression of adhesion molecules and chemotactic peptides, decreases endothelin-1 expression, and inhibits platelet aggregation. This amino acid also improves endothelial function in patients with coronary artery disease and dilates human epicardial atheromatous coronary arteries. Despite the positive results from small case-control studies, it is still unclear whether chronic administration of L-arginine has any effect on clinical outcome in patients with coronary artery disease. In addition, other indirect strategies, such as the inhibition of arginase, could prove more effective at improving intracellular L-arginine bioavailability than exogenous L-arginine administration. The potential clinical usefulness of L-arginine, therefore, needs further evaluation in large, prospective clinical trials. Here, we present a critique of the existing literature about the role of L-arginine in the prevention of atherosclerosis.     

Role of nitric oxide signal transduction pathway in regulation of vascular tone.

Nitric oxide signal transduction pathway has been detected in a number of cell types including vascular endothelial cells, smooth muscle cells, and noncholinergic nonadrenergic nerve endings. Nitric oxide synthase is a key enzyme which produces nitric oxide from L-arginine. Three different types of this enzyme have been described: constitutive soluble, inducible soluble, and constitutive particulate. Nitric oxide is a lipophilic molecule that can rapidly diffuse through biological membranes. It provides communication between endothelial and smooth muscle cells as well as between nerve endings and smooth muscle cells. Decreased production of nitric oxide may lead to vasospasm, whereas its overproduction may cause pathological vasodilatation. Understanding of the role of nitric oxide signal transduction pathway in regulation of vascular tone will facilitate design of better strategies for the prevention and treatment of vascular diseases.     

NG-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implications for the involvement of nitric oxide.

Clinical assessment of the activity of tumor necrosis factor (TNF) against human cancer has been limited by a dose-dependent cardiovascular toxicity, most frequently hypotension. TNF is also thought to mediate the vascular collapse resulting from bacterial endotoxin. The present studies address the mechanism by which TNF causes hypotension and provide evidence for elevated production of nitric oxide, a potent vasodilator initially characterized as endothelium-derived relaxing factor. Nitric oxide is synthesized by several cell types, including endothelial cells and macrophages, from the guanidino nitrogen of L-arginine; the enzymatic pathway is competitively inhibited by NG-methyl-L-arginine. We found that hypotension induced in pentobarbital-anesthetized dogs by TNF (10 micrograms/kg, i.v., resulting in a fall in mean systemic arterial pressure from 124.7 +/- 7 to 62.0 +/- 22.9 mmHg; 1 mmHg = 133 Pa) was completely reversed within 2 min following administration of NG-methyl-L-arginine (4.4 mg/kg, i.v.). In contrast, NG-methyl-L-arginine failed to reverse the hypotensive response to an equivalent depressor dose of nitroglycerin, a compound that acts by forming nitric oxide by a nonenzymatic, arginine-independent mechanism. The effect of NG-methyl-L-arginine on TNF-induced hypotension was antagonized, and the hypotension restored, by administration of excess L-arginine (100 mg/kg, i.v.). Our findings suggest that excessive nitric oxide production mediates the hypotensive effect of TNF.     

The nitric oxide pathway is amplified in venular vs arteriolar cultured rat mesenteric endothelial cells.

To determine if there are differences in nitric oxide activity between pre- and postcapillary microvessels, we studied cultured rat mesenteric arteriolar and venular endothelial cells (RMAEC, RMVEC). We measured expression of endothelial nitric oxide synthase (eNOS), the activity of eNOS, and L-arginine transport in live RMAEC and RMVEC and the L-arginine content of RMAEC and RMVEC lysates. The abundance of eNOS was significantly greater in RMVEC vs RMAEC; this was also true for freshly harvested, pooled microvessels. Baseline NOS activity was higher in RMVEC than in RMAEC. NG-monomethyl-L-arginine (L-NMA; 5 mM) inhibited NOS activity by approximately 70-80% in both RMAEC and RMVEC, indicating that metabolism of l-arginine is largely via NOS. Intracellular L-arginine levels were higher in RMVEC vs RMAEC and well above the eNOS Km in both cell types. L-arginine levels increased with L-NMA in both RMAEC and RMVEC, presumably due to reduced substrate utilization. Since L-arginine transport was not higher in RMVEC vs RMAEC, this may reflect higher intracellular arginine synthesis. A higher intrinsic level of baseline NO production in the postcapillary microvascular endothelium may reflect both the contribution of venular derived NO to control of arteriolar tone and a key role of venular-derived NO in local thrombosis control.     

Bacterial DNA activates cell mediated immune response and nitric oxide overproduction in peritoneal macrophages from patients with cirrhosis and ascites

BACKGROUND AND AIMS: Translocation of intestinal bacteria to ascitic fluid is probably the first step in the development of episodes of spontaneous bacterial peritonitis in patients with cirrhosis. We have recently reported the detection of bacterial DNA in blood and ascitic fluid from patients with advanced cirrhosis, what we consider as molecular evidence of bacterial translocation. Several studies have shown the immunogenic role of bacterial DNA in vitro, and we hypothesised that the presence of bacterial DNA could activate the type I immune response in peritoneal macrophages from these patients, leading to greater cytokine synthesis (interleukin (IL)-2 and IL-12, tumour necrosis factor alpha, and interferon gamma) and effector molecules such as nitric oxide. METHODS: Peritoneal macrophages obtained from patients with cirrhosis and culture negative non-neutrocytic ascitic fluid were collected and characterised by flow cytometry. Inducible nitric oxide synthase, nitric oxide levels, and cytokine production were measured by immunoenzymometric assays in basal and harvested conditions according to the presence/absence of bacterial DNA. RESULTS: The ability of peritoneal macrophages to synthesise nitric oxide and levels of all cytokines were significantly increased in patients with bacterial DNA. There was a positive correlation between inducible nitric oxide synthase and nitric oxide levels. CONCLUSIONS: The presence of bacterial DNA in patients with decompensated cirrhosis is associated with marked activation of peritoneal macrophages, as evidenced by nitric oxide synthesising ability, together with enhanced cytokine production.