N-monomethyl-L-arginine Research Articles

The relationship of asymmetric dimethylarginine levels with uncontrolled arterial hypertension and hypertension disease stage

Background: Patients with treatment-resistant hypertension have a worse cardiovascular prognosis compared to those who achieve their target levels of blood pressure (BP). One of biochemical and clinical markers of a decreased response to antihypertensive therapy can be asymmetric dimethylarginine (ADMA), a molecule that reduces formation of nitric oxide and promotes endothelial dysfunction and vasoconstriction.
 Aim: To assess the status of the nitrogen oxide synthesis system and clinical and laboratory profile in patients, depending on the hypertensive disease stage, who achieved or not achieved their target BP levels during hospitalization.
 Materials and methods: We performed аn observational retrospective uncontrolled study in a consecutive sample of 192 patients aged 45–65 years who were admitted to the City Clinical Hospital No. 25 (city of Novosibirsk) with a diagnosis of uncomplicated hypertensive crisis. On the day of discharge, the patients were retrospectively divided into 2 groups. Group 1 included patients who achieved their target BP during hospitalization (target BP group, n = 116). Group 2 included patients with uncontrolled hypertension, in whom the administration of three antihypertensive drugs including a diuretic in optimal or maximal tolerated doses did not result in the achievement of the target BP below 140 and/or 90 mm Hg during hospitalization (non-target BP group, n = 76).
 The following parameters were measured: ADMA, symmetrical dimethylarginine (SDMA), N-monomethyl-L-arginine (NMMA), and total nitric oxide.
 Results: Serum ADMA concentrations (Me [Q25%; Q75%] were increasing with the stage of hypertension: stage I, 0.75 µmol/L [0.66; 0.78], stage II, 1.14 µmol/L [0.87; 1.39], stage III, 1.38 µmol/L [1.22; 1.49] (p 0.0001, Kruskal-Wallis test). In the pairwise comparison, the difference between all these subgroups was significant at p 0.01. In the patients with uncontrolled arterial hypertension ADMA levels were increased compared to those in the target BP group: 1.2 µmol/L [0.99; 1.47] vs 1.07 [0.79; 1.34] µmol/L (p = 0.002). The proportion of patients with type 2 diabetes mellitus among those with uncontrolled arterial hypertension was 31.2% (24/76), while in the target BP group there were only 3.5% of such patients (4/116) (odds ratio 12.57, 95% confidence interval 4.15–38.05; p = 0.00001).
 Conclusion: ADMA measurement may help identify patients with a potential poor response to antihypertensive therapy. This should be taken into account when choosing a treatment regimen and BP monitoring. In addition, ADMA seems to be a promising target for the development of new drug classes.

Pyrrolidine dithiocarbamate in combination with L-N-monomethyl arginine alleviates Staphylococcus aureus infection via regulation of CXCL8/CXCR1 axis in peritoneal macrophages in vitro

The CXCL8/CXCR1 axis in conjoint with the free radicals and anti-oxidants dictates the severity of inflammation caused by the bacteria, Staphylococcus aureus. S.aureus mediated inflammatory processes is regulated by NF-κB and its product, iNOS. The objective of this study was to examine the effects of inhibition of NF-κB and iNOS on CXCL8/CXCR1, alteration in M1/M2 polarization of macrophages and associated inflammatory responses during S.aureus infection in vitro. For this, the murine peritoneal macrophages were pretreated with NF-κB inhibitor, Pyrrolidine dithiocarbamate (PDTC) and iNOS inhibitor, L-N-monomethyl arginine (LNMMA), either alone or in combination, followed by time-dependent S.aureus infection. The chemotactic migrations of macrophages were determined by the agarose spot assay. The iNOS, NF-κB and CXCR1 protein expressions were evaluated. The ROS level (superoxide, H2O2, NO) and antioxidant activities (SOD, CAT, GSH, arginase) were measured. The intra-macrophage phagoctyic activity had been analyzed by confocal microscopy. S.aureus activated macrophages showed increased iNOS expression that symbolizes M1 characterization of macrophages. The results suggest that the combination treatment of LNMMA + PDTC was effective in diminution of CXCL8 production and CXCR1 expression through downregulation of NF-κB and iNOS signaling pathway. Consequently, there was decrement in macrophage migration, reduced ROS generation, elevated antioxidant enzyme activity as well as bacterial phagocytosis at 90 min post bacterial infection. The increased arginase activity further proves the switch from pro-inflammatory M1 to anti-inflammatory M2 polarization of macrophages. Concludingly, the combination of PDTC + LNMMA could resolve S.aureus mediated inflammation through mitigation of CXCL8/CXCR1 pathway switching from M1 to M2 polarization.

Effects of tofacitinib therapy on arginine and methionine metabolites in association with vascular pathophysiology in rheumatoid arthritis: A metabolomic approach.

Rheumatoid arthritis (RA) has been associated with changes in lipid, arginine and NO metabolism with increased cardiovascular (CV) risk. The aim of this study is to evaluate the effect of tofacitinib, a Janus kinase (JAK) inhibitor, on arginine and methionine metabolism in correlation with inflammation, functional and pathological vascular changes during one-year treatment of patients with RA. Thirty RA patients with active disease were treated with either 5 mg bid or 10 mg bid tofacitinib for 12 months. We determined DAS28, CRP, IgM rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) levels. We assessed brachial artery flow-mediated vasodilation (FMD), carotid intima-media thickness (IMT) and pulse-wave velocity (PWV) by ultrasound at baseline and after 6 and 12 months. We also determined plasma L-arginine, L-citrulline, L-ornithine, inducible nitric oxide synthase (iNOS), asymmetric (ADMA) and symmetric dimethylarginine (SDMA), L-N-monomethyl-arginine (L-NMMA), cysteine, homocysteine, and methionine levels at these time points. Twenty-six patients (13 on each arm) completed the study. CRP, ESR and DAS28 decreased significantly during one-year treatment with tofacitinib. Arginine and ADMA showed a negative univariate correlation with CRP but not with FMD, PWV or IMT. Tofacitinib at 10 mg bid significantly increased L-arginine, L-ornithine, iNOS and methionine levels after 12 months. ADMA and SDMA levels did not change in our study. Methionine showed negative correlation with FMD at baseline and positive correlation with PWV after 12 months. No change was observed in FMD and PWV but a significant increase was measured in IMT at 6 and 12 months. Multivariate analysis indicated variable correlations of L-arginine, L-citrulline, ADMA, L-NMMA, homocysteine and methionine with DAS28, CRP, ESR and RF but not with anti-CCP after one-year treatment. With respect to vascular pathophysiology, only PWV and methionine correlated with each other. One-year tofacitinib treatment suppressed systemic inflammation and improved functional status in RA. FMD, PWV have not been affected by one-year tofacitinib treatment., while IMT increased further despite treatment. Increased arginine and methionine might contribute to the anti-inflammatory effects of tofacitinib. Increased arginine availability with no changing ADMA may protect FMD and PWV from deterioration. The increase of IMT in the anti-inflammatory environment cannot be explained by arginine or methionine metabolism in this study.

POS0383 EFFECTS OF TOFACITINIB THERAPY ON ARGININE AND METHIONINE METABOLITES IN ASSOCIATION WITH VASCULAR PATHOPHYSIOLOGY IN RHEUMATOID ARTHRITIS: A METABOLOMIC APPROACH

Background:Rheumatoid arthritis (RA) has been associated with increased cardiovascular (CV) risk and metabolic changes.Objectives:We wished to determine how the Janus kinase (JAK) inhibitor tofacitinib influences vascular pathophysiology and metabolites of the arginine and methionine-homocysteine pathways.Methods:Thirty RA patients with active disease were treated with either 5 mg bid or 10 mg bid tofacitinib and evaluated at baseline and after 6 and 12 months. We determined DAS28, CRP, IgM rheumatoid factor (RF) and anti-cyclic citrullinated peptide (aCCP) levels. We assessed brachial artery flow-mediated vasodilation (FMD), carotid intima-media thickness (IMT) and pulse-wave velocity (PWV) by ultrasound. We also determined plasma L-arginine, L-citrulline, L-ornithine, inducible nitric oxide synthase (iNOS), asymmetric (ADMA) and symmetric dimethylarginine (SDMA), L-N-monomethyl-arginine (L-NMMA), cysteine, homocysteine, and methionine levels.Results:Twenty-six patients completed the study. Tofacitinib treatment maintained FMD and PWV. Ten mg bid tofacitinib significantly increased L-arginine, L-ornithine, iNOS and methionine levels after 12 months. Tofacitinib transiently increased L-citrulline and L-NMMA and decreased homocysteine levels after 12 months. Based on L-citrulline, L-ornithine, ADMA and SDMA levels, L-arginine remained highly available for endothelial NO production. Multivariate analysis indicated variable correlations of L-arginine, L-citrulline, ADMA, L-NMMA, homocysteine and methionine with DAS28, CRP, ESR and RF but not with aCCP. Regarding vascular pathophysiology, only PWV and methionine correlated with each other after 12 months.Conclusion:Tofacitinib suppressed systemic inflammation in RA yielding stabilization of vascular function. It may exert CV protective effects in RA, at least in part, by shifting L-arginine metabolism to high arginine availability and decreasing homocysteine levels.Acknowledgements:This research was supported by the European Union and the State of Hungary and co-financed by the European Social Fund in the framework of TAMOP-4.2.4.A/2-11/1-2012-0001 ‘National Excellence Program ’(Z.S.); by the European Union grant GINOP-2.3.2-15-2016-00015 (Z.S.) and by the WI188341 investigator-initiated research (IIR) grant obtained from Pfizer US (Z.S.).Disclosure of Interests:Boglárka Soós: None declared, Attila Hamar: None declared, Anita Pusztai: None declared, Monika Czókolyová: None declared, Edit Végh: None declared, Szilvia Szamosi Speakers bureau: Roche, Zsófia Pethö: None declared, Katalin Gulyás: None declared, György Kerekes: None declared, Éva Szekanecz: None declared, Sándor Szántó Speakers bureau: Abbvie, MSD, Novartis, Consultant of: Abbvie, Novartis, Gabriella Szücs Speakers bureau: Actelion, Roche, Sager, Boehringer, Consultant of: Boehringer, Actelion, Sager, Uwe Christians: None declared, Jelena Klawitter: None declared, Tamas Seres: None declared, Zoltán Szekanecz Speakers bureau: Pfizer, Abbvie, Roche, Lilly, Novartis, Boehringer, Consultant of: Pfizer, Abbvie, Novartis, Grant/research support from: Pfizer

Serum ADMA levels were positively correlated with EDSS scores in patients with multiple sclerosis

Multiple sclerosis (MS) is an autoinflammatory, chronic central nervous system disease. In the pathogenesis of the disease increased nitric oxide (NO) levels play an important role. Nitric oxide (NO) has neuroprotective effects in physiological conditions, however, it is thought that excessive NO formation in MS may lead to demyelination and axonal damage. Derivatives of methylarginine including asymmetric dimethyl arginine (ADMA), L-N monomethyl arginine (L-NMMA), symmetric dimethyl arginine (SDMA) directly or indirectly reduce NO production. Our aim was to measure the levels of methylarginine derivatives and citrulline, ornithine, arginine, homoarginine levels, which are metabolites associated with NO metabolism, in MS subgroups.

Developing a robust, fast and reliable measurement method for the analysis of methylarginine derivatives and related metabolites

BackgroundNitric oxide (NO) plays an important role in endothelial homeostasis. Asymmetric dimethyl arginine (ADMA), L-N monomethyl arginine (L-NMMA) and symmetric dimethyl arginine (SDMA), which are derivatives of methylarginine, directly or indirectly reduce NO production. Therefore, these metabolites are an important risk factor for various diseases, including cardiovascular diseases. Numerous methods have been developed for the measurement of methylarginine derivatives, but various difficulties have been encountered. This study aimed to develop a reliable, fast and cost-effective method for the analysis and measurement of methylarginine derivatives (ADMA, SDMA, L-NMMA) and related metabolites (arginine, citrulline, homoarginine, ornithine), and to validate this method according to Clinical and Laboratory Standards Institute (CLSI) protocols. MethodsFor the analysis of ADMA, SDMA, L-NMMA, arginine, homoarginine, citrulline, ornithine, 200 µl of serum were precipitated with methanol, and subsequently derivatized with a butanol solution containing 5% acetyl chloride. Butyl derivatives were separated using a C18 reverse phase column with a 5 min run time. Detection of analytes was achieved by utilising the specific fragmentation patterns identified through tandem mass spectrometry. ResultsThe method was linear for ADMA, SDMA, L-NMMA, ornithine, arginine, homoarginine and citrulline in the ranges of 0.023–6.0, 0.021–5.5, 0.019–5.0, 0.015–250, 0.015–250, 0.019–5 and 0.015–250 µM, respectively. The inter-assay CV% values for all analytes was less than 9.8%. ConclusionsData obtained from method validation studies shows that the developed method is highly sensitive, precise and accurate. Short analysis time, cost-effectiveness, and multiplexed analysis of these metabolites, with the same pretreatment steps, are the main advantages of the method.

HPLC and LC-MS/MS measurement methods for the quantification of asymmetric dimethylarginine (ADMA) and related metabolites

Abstract Methyl arginine derivatives such as asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), L-N-monomethyl arginine (L-NMMA) are formed by proteolytic catalysis following methylation of arginine residues in proteins. These metabolites reduce NO production. Methylated arginines are an important biomarker for various diseases such as cardiovascular and renal diseases. Therefore, many methods have been developed to reliably and accurately measure the levels of these metabolites. This review, HPLC and LC-MS/MS methods developed for the measurement of methylarginine derivatives are discussed. In HPLC methods, solid phase extraction, derivatization and subsequent separation by reverse phase chromatography were performed. Since these metabolites are polar, they are difficult to retain in conventional reverse phase columns. In addition, as serum levels of these metabolites are low, sensitivity problems have been observed in HPLC methods. Derivatization has been applied to eliminate these problems. However, there have been problems with the stability of derivatives formed. Another important problem is that the separation of stereoisomer ADMA and SDMA can only be achieved chromatographically. Tandem mass spectrometric methods are accurate, selective, sensitive and rapid since analytes are separated depending on m/z ratios rather than chromatographic separation. Therefore, tandem mass spectrometry methods might be considered as the goal standard for these analytes.

Red raspberry (Rubus idaeus) consumption restores the impaired vasoconstriction and vasorelaxation response in the aorta of the obese Zucker rat, a model of the Metabolic Syndrome

Metabolic Syndrome (MetS) increases the risk of cardiovascular disease. Whole red raspberry (WRR) consumption on vascular function was investigated in the obese Zucker rat (OZR), model of MetS. Male OZR and their lean littermates (LZR) were placed on a control (C) or an 8% w/w WRR-enriched diet for 8 weeks. Phenylephrine (Phe)-induced vasoconstriction and acetylcholine (Ach)-induced vasorelaxation were measured in aortic rings in the presence or absence of L-N-monomethyl-arginine (L-NMMA) and mefenamic acid (MFA). Phe-induced vasoconstriction was lower in the OZR-C compared to LZR-C (p < 0.05). The WRR diet partially restored aortic response in the OZR-WRR aorta (p < 0.05) compared to OZR- C. The OZR-WRR group pre-treated with L-NMMA increased compared to OZR-C (p < 0.05). Pre-treatment with L-NMMA, maximal relaxation response was higher in the OZR compared to the LZR (p < 0.05). With L-NMMA, maximal relaxation response in OZR-WRR (p < 0.05) was lower compared to the OZR-C. Prostacyclin I2 concentration was higher in the OZR compared to the LZR (p < 0.05) and was attenuated in the OZR-WRR (p < 0.05). Aortic expression of eNOS and COX-2 were downregulated in the OZR-WRR (p < 0.05). In conclusion, WRR restores the impaired vascular tone of the OZR by enhancing Phe-induced vasoconstriction and attenuating Ach-induced vasorelaxation.

Assessment of potential cardiovascular risk in trichloroethylene exposure by serum methylated arginine levels

ABSTRACT Trichloroethylene (TCE) is a widely used solvent in industrial applications and has toxic effects on various systems. Methylated arginine amino acids (eg asymmetric dimethyl arginine (ADMA), symmetric dimethyl arginine (SDMA)) cause the development of cardiovascular disease by inhibiting NO synthesis, which is considered to be heart-protector. The aim of this study is to determine the risk of cardiovascular diseases in TCE exposure by methylated arginine biomarkers. About 98 controls and 100 TCE-exposed male subjects were included in the study. Trichloroacetic acid (urinary metabolite of TCE), arginine, homoarginine, citrulline ADMA, SDMA, and N-monomethyl L-arginine (L-NMMA) levels were found significantly higher than control group (p < 0.001). The strongest correlation was found between ADMA and Trichloroacetic acid (TCA) level (r = 0.453, p < 0.01). Long-term TCE exposure, may be an important risk factor for cardiovascular diseases by increasing methylated arginine levels.

Vascular endothelium dysfunction: a conservative target in metabolic disorders.

Vascular endothelium plays a role in capillary transport of nutrients and drugs and regulates angiogenesis, homeostasis, as well as vascular tone and permeability as a major regulator of local vascular homeostasis. The present study has been designed to investigate the role of endothelium in metabolic disorders. The endothelium maintains the balance between vasodilatation and vasoconstriction, procoagulant and anticoagulant, prothrombotic and antithrombotic mechanisms. Diabetes mellitus causes the activation of aldose reductase, polyol pathway and advanced glycation-end-product formation that collectively affect the phosphorylation status and expression of endothelial nitric oxide synthatase (eNOS) and causes vascular endothelium dysfunction. Elevated homocysteine levels have been associated with increase in LDL oxidation, generation of hydrogen peroxides, superoxide anions that increased oxidative degradation of nitric oxide. Hyperhomocysteinemia has been reported to increase the endogenous competitive inhibitors of eNOS viz L-N-monomethyl arginine (L-NMMA) and asymmetric dimethyl arginine (ADMA) that may contribute to vascular endothelial dysfunction. Hypercholesterolemia stimulates oxidation of LDL cholesterol, release of endothelins, and generation of ROS. The increased cholesterol and triglyceride level and decreased protective HDL level, decreases the activity and expression of eNOS and disrupts the integrity of vascular endothelium, due to oxidative stress. Hypertension also stimulates release of endothelins, vasoconstrictor prostanoids, angiotensin II, inflammatory cytokines, xanthine oxidase and, thereby, reduces bioavailability of nitric oxide. Thus, the cellular and molecular mechanisms underlying diabetes mellitus, hyperhomocysteinemia, hypercholesterolemia hypertension and hyperuricemia leads to an imbalance of phosphorylation and dephosphorylation status of lipid and protein kinase that cause modulation of vascular endothelial L-arginine/nitric oxide synthetase (eNOS), to produce vascular endothelium dysfunction.

ADMA and arginine derivatives in relation to non-invasive vascular function in the general population

ObjectiveNitric oxide produced from l-arginine is central to vascular homeostasis. Little is known about the relationship between arginine derivatives including asymmetric dimethylarginine (ADMA) and non-invasive vascular function measures in the general population. Approach and resultsIn 5000 individuals (median age 56; 25th/75th percentile: 46, 65; 49% women) taking part in the population-based Gutenberg Health Study (Mainz area, Germany), we measured the relationship between the arginine derivatives asymmetric dimethylarginine (ADMA), N-monomethyl l-arginine (NMMA), symmetric dimethylarginine (SDMA) and l-arginine with flow-mediated dilation (FMD) and peripheral arterial tonometry (PAT). Weak bivariate correlations were observed between all measured arginine derivatives and vascular function measures, except of l-arginine and FMD and SDMA and PAT ratio. In multivariate adjusted linear regression analyses we could show statistically significant relationships between arginine derivatives and vascular function measures, which were influenced by age, sex and body mass index (BMI). Thus, a negative relationship between ADMA and FMD in females who were normal (beta: −0.095, P < 0.001) to overweight (beta: −0.071, P < 0.001) and a negative association of SDMA and FMD for middle-aged females was seen. The relationship between ADMA and PAT was negative for males who were normal (beta: −0.089, P < 0.001) to overweight (beta: −0.051, P = 0.007) and positive for obese females (beta: 0.073, P = 0.021). ConclusionsWe showed small but significant correlations between ADMA and related arginine derivatives and non-invasive vascular function measures representative of different vascular regions. The associations were markedly influenced by age, sex and BMI. These findings support a complex interplay of arginine metabolism and vascular function.

Impaired Increase of Retinal Capillary Blood Flow to Flicker Light Exposure in Arterial Hypertension

We hypothesized that the increase of retinal capillary blood flow (RCF) to flicker light exposure is impaired in subjects with arterial hypertension. In 146 nondiabetic untreated male subjects with (n=50) or without (n=96) arterial hypertension, RCF was measured before and after flicker light exposure noninvasively and in vivo using scanning laser Doppler flowmetry. In addition, in a subgroup of 28 subjects, the change of RCF to flicker light exposure was again assessed during parallel infusion of nitric oxide synthase inhibitor N-monomethyl-L-arginine (L-NMMA). The increase of RCF to flicker light exposure was lower in patients with untreated hypertension compared with normotensive subjects when expressed in absolute terms (7.69±54 versus 27.2±44 AU; P adjusted=0.013) or percent changes (2.95±14 versus 8.33±12%; P adjusted=0.023). Systolic (β=-0.216; P=0.023) but not diastolic blood pressure (β=-0.117; P=0.243) or mean arterial pressure (β=-0.178; P=0.073) was negatively related to the percent change of RCF to flicker light exposure, independently of other cardiovascular risk factors. In the subgroup of 28 subjects, the increase of RCF to flicker light exposure was similar at baseline and during parallel infusion of L-NMMA when expressed in absolute terms (20.0±51 versus 22.6±56 AU; P=0.731) or percent changes (7.12±16 versus 8.29±18%; P=0.607). The increase of RCF to flicker light exposure is impaired in arterial hypertension. In the subgroup of the total study cohort, nitric oxide was not a major determinant of the increase of RCF to flicker light exposure.

The toxic effect of fluoride on MG-63 osteoblast cells is also dependent on the production of nitric oxide

Some soda-lime-phospho-silicate glasses, such as Hench's Bioglass ® 45S5, form bone-like apatite on their surface when bound to living bone. To improve their osteointegration for clinical purposes, the fluoride insertion in their structure has been proposed, but we recently showed that fluoride causes oxidative damage in human MG-63 osteoblasts, via inhibition of pentose phosphate oxidative pathway (PPP) and its key enzyme glucose 6-phosphate dehydrogenase (G6PD). In the same cells we have now investigated the role of nitric oxide (NO) in these effects. Fluoride-containing bioactive glasses and NaF caused, as expected, release of lactate dehydrogenase in the extracellular medium, accumulation of intracellular malonyldialdehyde, inhibition of PPP and G6PD: we have now observed that these effects were significantly reverted not only by superoxide dismutase (SOD) plus catalase (scavengers of reactive oxygen species), but also by N-monomethyl l-arginine ( l-NMMA, a NOS inhibitor) and 2-phenyl-4,4,5,5,-tetramethylimidazoline-1oxyl 3-oxide (PTIO, a NO scavenger). Moreover the two highest concentrations of both fluoride-containing bioglasses and NaF caused increase of nitrite (a stable derivative of NO) levels in the culture supernatant, which was inhibited by l-NMMA, erythrocytes, PTIO and SOD/catalase, and increase of intracellular NO synthase (NOS) activity. The incubation with bioglasses or NaF increased also the phosphorylation of Ser 1177 in the endothelial NOS isoform. Furthermore, the NO donor spermine NONOate was able to inhibit G6PD activity in vitro, and this effect was partly reverted by PTIO. Therefore our results suggest that most cytotoxic effects of fluoride are mediated by the production of NO: reactive oxygen species are important, causing NOS phosphorylation. We also observed, for the first time, that Tempol, but not SOD/catalase, besides inhibiting the oxidative stress induced by fluoride, also scavenges fluoride ions. For this reason it is not a selective inhibitor of the oxidative effects of fluoride.

Abstract: P1307 L-ARGININE DOES NOT IMPROVE ENDOTHELIAL FUNCTION IN PATIENTS WITH METABOLIC SYNDROME

Nitric oxide produced from l-arginine is central to vascular homeostasis. Little is known about the relationship between arginine derivatives including asymmetric dimethylarginine (ADMA) and non-invasive vascular function measures in the general population.In 5000 individuals (median age 56; 25th/75th percentile: 46, 65; 49% women) taking part in the population-based Gutenberg Health Study (Mainz area, Germany), we measured the relationship between the arginine derivatives asymmetric dimethylarginine (ADMA), N-monomethyl l-arginine (NMMA), symmetric dimethylarginine (SDMA) and l-arginine with flow-mediated dilation (FMD) and peripheral arterial tonometry (PAT). Weak bivariate correlations were observed between all measured arginine derivatives and vascular function measures, except of l-arginine and FMD and SDMA and PAT ratio. In multivariate adjusted linear regression analyses we could show statistically significant relationships between arginine derivatives and vascular function measures, which were influenced by age, sex and body mass index (BMI). Thus, a negative relationship between ADMA and FMD in females who were normal (beta: −0.095, P < 0.001) to overweight (beta: −0.071, P < 0.001) and a negative association of SDMA and FMD for middle-aged females was seen. The relationship between ADMA and PAT was negative for males who were normal (beta: −0.089, P < 0.001) to overweight (beta: −0.051, P = 0.007) and positive for obese females (beta: 0.073, P = 0.021).We showed small but significant correlations between ADMA and related arginine derivatives and non-invasive vascular function measures representative of different vascular regions. The associations were markedly influenced by age, sex and BMI. These findings support a complex interplay of arginine metabolism and vascular function.

Effect of caffeine on the secretion of peroxidase in rat submandibular gland: A study of its mechanism of action

Objetive In this work, we analysed the mechanism of action of caffeine on peroxidase secretion in the female rat submandibular gland. The signaling molecules cAMP and nitric oxide were monitored as potential mediators. Design The salivary gland peroxidase secretion of female albino Wistar rats was assessed by a spectroscopic method. Results Caffeine was found to exert an increase on peroxidase secretion in a concentration-response manner: the peroxidase secretion stimulation index (SI) (secreted peroxidase from treated/secreted peroxidase from basal) for caffeine 10 μg/ml: 2.2 ± 0.18 ( P < 0.05); caffeine 100 μg/ml alone: 3 ± 0.18 ( P < 0.01); +LNMMA (LN monomethyl arginine): 1 ± 0.1 ( P < 0.05); caffeine 1000 μg/ml alone: 5 ± 0.35 ( P < 0.01); +LNMMA: 2 ± 0.2 ( P < 0.05). These results were associated with an increase in cAMP and total nitrites production. Total nitrites, SI caffeine 100 μg/ml alone: 2.8 ± 0.2 ( P < 0.01); +LNMMA: 1 ± 0.08 ( P < 0.05); caffeine 1000 μg/ml alone: 4.8 ± 0.3 ( P < 0.01); +LNMMA: 2.3 ± 0.18 ( P < 0.05). Conclusion It could thus be concluded that cAMP and NO are involved in the mechanism of action displayed by caffeine. This is the first report on the mechanism of action of caffeine on peroxidase secretion.

Differential mechanisms of intracellular killing of Mycobacterium avium and Listeria monocytogenes by activated human and murine macrophages. The role of nitric oxide.

Murine peritoneal macrophages activated with interferon-gamma (IFN-gamma) produce large quantities of nitric oxide and are efficient in the killing of certain intracellular pathogens. To examine the role of this mechanism in the killing of Mycobacterium avium by murine and human macrophages, we infected mouse peritoneal macrophages and human monocyte-derived macrophages with M. avium and Listeria monocytogenes and stimulated the cells with recombinant tumour necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF) or IFN-gamma, in the presence or absence of N-monomethyl-L-arginine (NMA) or arginase. Neither competitive inhibition with NMA nor depletion of arginine by arginase had any effect on the inhibition of growth/intracellular killing of M. avium by activated human and murine macrophages. In contrast, activation of murine but not human macrophages infected with L. monocytogenes by IFN-gamma was significantly inhibited by the addition of NMA/arginase. Furthermore, murine macrophages produced large concentrations of nitric oxide following stimulation with recombinant cytokines, although no significant increase of nitric oxide production was observed with human monocyte-derived macrophages.

Basal nitric oxide synthase activity is a major determinant of glomerular haemodynamics in humans

Experimental data suggest that increased nitric oxide synthase (NOS) activity contributes to preglomerular vasodilation and subsequent glomerular hyperfiltration. Whether such a relationship also exists in the human renal vasculature has not yet been adequately determined. We assessed systemic, renal, and intragomerular haemodynamics in 310 subjects with normal renal function [glomerular filtration rate (GFR) > 60 ml/min per 1.73 m], before and after NOS inhibition with L-Nmonomethyl arginine (L-NMMA). Subjects were arbitrarily divided into tertiles according to their basal NOS activity, as assessed by the decrease in renal plasma flow in response to L-NMMA (high -23.4 +/- 8.1 versus medium -10.8 +/- 2.2 versus low -1.0 +/- 4.8%). Resting GFR differed significantly between tertiles: high 114 +/- 21 versus medium 109 +/- 19 versus low 104 +/- 21 ml/min per 1.73 m; analysis of variance P = 0.003. In a multiple stepwise regression analysis, basal NOS activity was the major factor to explain resting GFR (beta = -0.344; P < 0.001). Body weight (beta = -0.295; P < 0.001) and age (beta = -0.164; P < 0.001) emerged as additional factors, whereas body mass index and blood pressure did not enter the final model. The close relationship between resting GFR and basal NOS activity was also mirrored in the renal microcirculation, as demonstrated by an exaggerated effect of L-NMMA on the increase in arteriolar resistance, particularly that of the afferent (RA) arteriole, with higher basal nitric oxide (NO) activity (RA: high +1542 +/- 1065 versus medium +1024 +/- 718 versus low +675 +/- 861 dyne/s per cm; P < 0.001). Our data clearly support experimental evidence linking NOS activity with increased glomerular filtration, and suggest that basal NO activity is also a major determinant of glomerular haemodynamics in the human renal vasculature.

Nitric Oxide Synthase Inhibition Prevents Leptin Induced Gn-RH Release in Prepubertal and Peripubertal Female Rats

The aim of the present paper was to study the role of NO as a mediator of leptin action at the hypothalamic level during sexual maturation. First, we analyzed the effect of different leptin concentrations (10 (-13), 10 (-11) and 10 (-9) M) on Gn-RH release from anterior preoptic area and medio basal hypothalamus (APOA-MBH) of prepubertal (15 days old) and peripubertal (30 days old) female rats. Leptin 10 (-13) M was the most effective concentration in releasing Gn-RH in both groups of animals. Since glutamate (GLU) and GABA are involved in the hypothalamic control of Gn-RH neurons and also in the neuroendocrine mechanism of puberty, in a second serie of experiments, we evaluated the effect of a competitive inhibitor of nitric oxide synthase (NOS), N-monomethyl-L-arginine (NMMA) on Gn-RH, GLU and GABA release in response to leptin. Co incubation of APOA-MBH with NMMA 0.5 mM, completely blocked Gn-RH and GLU release induced by leptin 10 (-13) M in prepubertal and peripubertal rats. NMMA also blocked the stimulation of GABA release in prepubertal rats, as well as the inhibition of GABA release induced by leptin in peripubertal rats. It can be proposed that the different effect of NO on GABA release, could be related to ontogenic changes, e.g, maturation of receptors and/or interneuronal connections during sexual development. Present results provide evidence that leptin acts at the hypothalamic level to stimulate NO release, which in turn modifies the release of amino acid neurotransmitters involved in Gn-RH control.

Manganese stimulates luteinizing hormone releasing hormone secretion in prepubertal female rats: hypothalamic site and mechanism of action

We have shown recently that Mn2+ stimulates gonadotropin secretion via an action at the hypothalamic level, and a diet supplemented with a low dose of the element is capable of advancing the time of female puberty. In this study, we used an in vitro approach to investigate the mechanism by which Mn2+ induces luteinizing hormone-releasing hormone (LHRH) secretion from prepubertal female rats. The medial basal hypothalamus from 30-day-old rats was incubated in Locke solution for 30 min to assess basal LHRH secretion, then incubated with buffer alone or buffer plus either a nitric oxide synthase (NOS) inhibitor (N-monomethyl-L-arginine (NMMA); 300 or 500 microM) or a soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); 100 or 250 microM) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl2 (50 or 250 microM) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 microM MnCl2 stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300-500 microM NMMA to the medium did not block Mn2+-stimulated release of LHRH, even with the higher dose of MnCl2. Furthermore, while 50, 100 and 250 microM MnCl2 all significantly induced LHRH release, the two lowest doses did not stimulate total nitrite released from the same tissue, an effect only observed with the highest dose. Taken together, these data suggest that Mn2+ is not an effective stimulator of NO. Conversely, inhibiting sGC with ODQ blocked the Mn2+-stimulated secretion of LHRH in a dose-dependent manner, indicating that GC is the site of action of Mn2+. Additionally, we showed that Mn2+ stimulated cGMP and LHRH from the same tissues, and that downstream blocking of protein kinase G formation with KT5823 (10 microM) inhibited Mn2+-induced LHRH release. These data demonstrate that the principal action of Mn2+ within the hypothalamus is to activate sGC directly and/or as a cofactor with available NO, hence generating cGMP and resulting in prepubertal LHRH release.

Comparison of Plackett–Burman and supersaturated designs in robustness testing

An optimized FIA assay of l- N-monomethylarginine (LNMMA) was validated. The linearity, precision, accuracy and range of the analytical method were evaluated and robustness testing was performed. Several experimental designs for robustness testing containing different numbers of experiments ( N) were compared. Both Plackett–Burman ( N = 8 or 12) and supersaturated designs ( N = 6) were examined. The latter design results were analyzed with the Fixing Effects and Adding Rows (FEAR) method, based on the initial addition of zero effect rows to the model matrix, which then are iteratively replaced by fixed effects. It was evaluated whether by reducing the number of experiments from 12 to 8 or 6, similar effects are estimated and considered (non-)significant. The FIA method was found linear in a range of 70–130% of the LNMMA concentration in the samples, and precise and accurate in a range of 80–120%. The estimated factor effects and the critical effects were found comparable for all examined designs, though there also are some indications that some from the supersaturated designs tend to be overestimated. The method was considered robust, since no significant effects occurred for the response describing the quantitative aspect of the method. For other responses, such as peak height and residence time, significant effects occur. However, only the most important effects are found with all designs. The effects reported from a supersaturated design based on the FEAR method still can be subject of further research.