Asymmetric dimethylarginine does not inhibit arginase activity and is pro‐proliferative in pulmonary endothelial cells

Asymmetric dimethylarginine (ADMA) is an endogenously produced nitric oxide synthase (NOS) inhibitor. l ‐Arginine can be metabolised by NOS and arginase, and arginase is the first step in polyamine production necessary for cellular proliferation. We tested the hypothesis that ADMA would inhibit NOS but not arginase activity and that this pattern of inhibition would result in greater l ‐arginine bioavailability to arginase, thereby increasing viable cell number. Bovine arginase was used in in vitro activity assays with various concentrations of substrate (l ‐arginine, ADMA, N^G‐monomethyl‐l ‐arginine (L‐NMMA) and N^G‐nitro‐l ‐arginine methyl ester (l ‐NAME)). Only l ‐arginine resulted in measurable urea production (K_m = 6.9 ± 0.8 mmol/L; V_max = 6.6 ± 0.3 μmol/mg protein per min). We then incubated bovine arginase with increasing concentrations of ADMA, l ‐NMMA and l ‐NAME in the presence of 1 mmol/L l ‐arginine and found no effect of any of the tested compounds on arginase activity. Using bovine pulmonary arterial endothelial cells (bPAEC) we determined the effects of ADMA on nitric oxide (NO) and urea production and found significantly lower NO production and greater urea production (P < 0.003) with ADMA, without changes in arginase protein levels. In addition, ADMA treatment resulted in an approximately 30% greater number of viable cells after 48 h than in control bPAEC. These results demonstrate that ADMA is neither a substrate nor an inhibitor of arginase activity and that in bPAEC ADMA inhibits NO production and enhances urea production, leading to more viable cells. These results may have pathophysiological implications in disorders associated with higher ADMA levels, such as pulmonary hypertension.     

Antinociceptive and antidiarrheal effects of pioglitazone in a rat model of diarrhoea‐predominant irritable bowel syndrome: Role of nitric oxide

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l‐Cysteine enhances nutrient absorption via a cystathionine‐β‐synthase‐derived H2S pathway in rodent jejunum

Hydrogen sulphide (H₂S) is generated endogenously from l ‐cysteine (l ‐Cys) by the enzymes cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE). In addition, l ‐Cys is commonly used as a precursor in the food and pharmaceutical industries. The aim of the present study is to determine whether l ‐Cys regulates intestinal nutrient transport. To that end, the presence of CBS and CSE in the jejunum epithelium was assessed by immunohistochemistry, Western blotting and the methylene blue assay. In addition, in vivo l ‐Cys (100 mg/kg, administered immediately after the glucose load) significantly increased blood glucose levels 30 min after the oral administration of glucose to mice. This effect of l ‐Cys was completely blocked by amino‐oxyacetic acid (AOA; 50 mg/kg; administered at the same time as l ‐Cys) an inhibitor of CBS. Measurements of the short‐circuit current (I_sc) in the rat jejunum epithelium revealed that l ‐Cys (1 mmol/L; 6 min before the administration of l ‐alanine) enhances Na⁺‐coupled l ‐alanine or glucose transport, and that this effect is inhibited by AOA (1 mmol/L;10 min before the administration of l ‐Cys), but not d ,l ‐propargylglycine (PAG;1 mmol/L; 10 min before the administration of l ‐Cys), a CSE inhibitor. Notably, l ‐Cys‐evoked enhancement of nutrient transport was alleviated by glibenclamide (Gli;0.1 mmol/L; 10 min before the administration of l ‐Cys), a K⁺ channel blocker. Together, the data indicate that l ‐Cys enhances jejunal nutrient transport, suggesting a new approach to future treatment of nutrition‐related maladies, including a range of serious health consequences linked to undernutrition.     

Platelet hyperaggregability in obesity: is there a role for nitric oxide impairment and oxidative stress?

Epidemiological evidence has shown that platelet activation markers are consistently elevated in obesity, contributing to its prothrombotic state. In order to improve the understanding of the regulation of platelet function in obesity, the aim of this study was to investigate the l ‐arginine‐nitric oxide (NO) pathway in obese adults without other cardiovascular risk factor. Seventeen obese (body mass index [BMI] 35.9±1.0 kg/m²) and eighteen age‐matched normal weight subjects (BMI 22.0±0.6 kg/m²) were included in this study. l ‐arginine influx was measured with incubation of l ‐[³H]‐arginine. NO synthase (NOS) and arginase activities were determined by the citrulline assay and the conversion of l ‐[¹⁴C]‐arginine to [¹⁴C]‐urea, respectively. Cyclic guanosine monophosphate (cGMP) content was evaluated by enzyme‐linked immunosorbent assay. In addition, the study analyzed: platelet aggregation; intraplatelet antioxidant enzymes, via superoxide dismutase (SOD) and catalase activities; and systemic levels of l ‐arginine, fibrinogen, and C‐reactive protein (CRP). Obese patients presented a significant decrease of platelet l ‐arginine influx, NOS activity, and cGMP levels, along with platelet hyperaggregability. On the presence of NO donor, platelet aggregation was similar between the groups. The fibrinogen and CRP systemic levels were significantly higher and SOD activity was reduced in obesity. No significant differences were observed in plasma levels of l ‐arginine and intraplatelet arginase and catalase activities between groups. The diminished NO bioavailability associated with inflammatory status and impaired enzymatic antioxidant defence may contribute to future cardiovascular complications in obesity.     

Minocycline Attenuates Depressive‐Like Behaviour Induced by Rat Model of Testicular Torsion: Involvement of Nitric Oxide Pathway

Testicular torsion/detorsion (T/D) can induce depression in pre‐ and post‐pubertal patients. This study was conducted to investigate the psychological impact of testicular torsion and mechanism underlying its depressive‐like behaviour, as well as antidepressant‐like activity of minocycline and possible involvement of nitric oxide (NO)/cyclic GMP pathway in this paradigm in male rats undergoing testicular T/D. Unilateral T/D was performed in 36 male adult Wistar rats, and different doses of minocycline were injected alone or combined with N^ω‐nitro‐l ‐arginine methyl ester (l ‐NAME), non‐specific NO synthase (NOS) inhibitor; aminoguanidine (AG), specific inducible NOS inhibitor; l ‐arginine, an NO precursor; and selective PDE5I, sildenafil. After assessment of locomotor activity in open‐field test, immobility times were recorded in the forced swimming test (FST). Moreover, 30 days after testicular T/D, testicular venous testosterone and serum nitrite concentrations were measured. A correlation was observed between either a decrease in plasma testosterone or an increase in serum nitrite concentrations with prolongation in immobility time in the testicular T/D‐operated rats FST. Minocycline (160 mg/kg) exerted the highest significant antidepressant‐like effect in the operated rats in the FST (p < 0.001). Furthermore, combination of subeffective doses of minocycline (80 mg/kg) and either l ‐NAME (10 mg/kg) or AG (50 mg/kg) demonstrated a significant robust antidepressant‐like activity in T/D group (p < 0.01). Consequently, NO/cGMP pathway was involved in testicular T/D‐induced depressive‐like behaviour and antidepressant‐like activity of minocycline in the animal model. Moreover, a contribution was observed between either decreased testosterone or elevated serum nitrite levels and depressive‐like behaviour following testicular T/D.     

Morphine hyposensitivity in streptozotocin‐diabetic rats: Reversal by dietary l‐arginine treatment

Painful diabetic neuropathy (PDN) is a long‐term complication of diabetes. Defining symptoms include mechanical allodynia (pain due to light pressure or touch) and morphine hyposensitivity. In our previous work using the streptozotocin (STZ)‐diabetic rat model of PDN, morphine hyposensitivity developed in a temporal manner with efficacy abolished at 3 months post‐STZ and maintained for 6 months post‐STZ. As this time course mimicked that for the temporal development of hyposensitivity to the pain‐relieving effects of the furoxan nitric oxide (NO) donor, PRG150 (3‐methylfuroxan‐4‐carbaldehyde) in STZ‐diabetic rats, we hypothesized that progressive depletion of endogenous NO bioactivity may underpin the temporal loss of morphine sensitivity in STZ‐diabetic rats. Furthermore, we hypothesized that replenishment of NO bioactivity may restore morphine sensitivity in these animals. Diabetes was induced in male Dark Agouti rats by intravenous injection of STZ (85 mg/kg). Diabetes was confirmed on day 7 if blood glucose concentrations were ≥15 mmol/L. Mechanical allodynia was fully developed in the bilateral hindpaws by 3 weeks of STZ‐diabetes in rats and this was maintained for the study duration. Morphine hyposensitivity developed in a temporal manner with efficacy abolished by 3 months post‐STZ. Administration of dietary l ‐arginine (NO precursor) at 1 g/d to STZ‐diabetic rats according to a 15‐week prevention protocol initiated at 9 weeks post‐STZ prevented abolition of morphine efficacy. When given as an 8‐week intervention protocol in rats where morphine efficacy was abolished, dietary l ‐arginine at 1 g/d progressively rescued morphine efficacy and potency. Our findings implicate NO depletion in the development of morphine hyposensitivity in STZ‐diabetic rats.     

Nitric oxide signalling is involved in diarylheptanoid‐induced increases in femoral arterial blood flow in ovariectomized rats

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Endothelial and neuronal nitric oxide synthases variably modulate the oestrogen‐mediated control of blood pressure and cardiovascular autonomic control

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Changes in hepatic lipogenic and oxidative enzymes and glucose homeostasis induced by an acetyl‐l‐carnitine and nicotinamide treatment in dyslipidaemic insulin‐resistant rats

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l‐arginine and l‐NMMA for assessing cerebral endothelial dysfunction in ischaemic cerebrovascular disease: A systematic review

Endothelial dysfunction (ED), in particular cerebral ED, may be an essential biomarker for ischaemic cerebrovascular disease. However, there is no consensus on methods to best estimate cerebral ED. In this systematic review, we evaluate the use of l ‐arginine and N^G‐monomethyl‐l ‐arginine (l ‐NMMA) for assessment of cerebral ED. A systematic search of PubMed, EMBASE and the Cochrane Library was done. We included studies investigating cerebrovascular response to l ‐arginine or l ‐NMMA in human subjects with vascular risk factors or ischaemic cerebrovascular disease. Seven studies (315 subjects) were eligible according to inclusion and exclusion criteria. Studies investigated the effect of age (n=2), type 2 diabetes mellitus (DM) (n=1), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (n=1), leukoaraiosis (n=1), and prior ischaemic stroke or transient ischaemic attack (TIA) (n=2) on cerebral ED. Most studies applied transcranial Doppler to quantify cerebral ED. Endothelium‐dependent vasodilatation (EDV) induced by l ‐arginine was impaired in elderly and subjects with leukoaraiosis, but enhanced in CADASIL patients. Studies including subjects with prior ischaemic stroke or TIA reported both enhanced and impaired EDV to l ‐arginine. Responses to l ‐NMMA deviated between subjects with type 2 DM and the elderly. We found only few studies investigating cerebral endothelial responses to l ‐arginine and l ‐NMMA in subjects with vascular risk factors or ischaemic cerebrovascular disease. Inconsistencies in results were most likely due to variations in methods and included subject populations. In order to use cerebral ED as a prognostic marker, further studies are required to evaluate the association to cerebrovascular disease.     

Inhibition of nitrergic relaxations by the M3‐selective antagonist 4‐DAMP

The inhibitory effect of the selective M₃ musarinic acetylcholine receptor antagonist, 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP, 0.1–10 μM) on nicotine (100 μM)‐induced nitrergic relaxation was investigated in comparison to d‐tubocurarine (0.1–10 μM) and hexamethonium (0.1–10 μM) by using phenylephrine (1 μM)‐precontracted rat anococcygeus muscles in vitro. Nicotine produced a 60.1± 2.4% (n = 40) inhibition of phenylephrine precontractions. But this relaxant response was at a significantly lower magnitude of 20.2± 4.6% (n = 18, P < 0.01 vs. control) in the presence of the nitric oxide synthase (NOS) blocker N^G‐nitro‐L ‐arginine methyl ester (L ‐NAME, 30 μM), and it was 26.5± 5.5% (n = 8, P < 0.01 vs. control) in the presence of the soluble guanylate cyclase inhibitor methylene blue (30 μM). However, aminoguanidine (100 μM), a relatively selective blocker of the inducible nitric oxide synthase (iNOS), had no significant effect. Similarly, other iNOS inhibitors such as dexamethasone (5 mg/kg) or L ‐canavanine (100 mg/kg) did not modify contractile nor relaxant responses when they were given in vivo, concomitantly with Escherichia coli endotoxin (1 mg/kg, ip) 4 h before the isolation of the tissues. 4‐DAMP, hexamethonium, and d‐tubocurarine inhibited nicotine‐induced relaxation in a concentration‐dependent manner with the following order of potency: 4‐DAMP > hexamethonium > d‐tubocurarine with IC₅₀ values being 0.47± 0.04 μM, 0.75± 0.06 μM, and 1.02± 0.05 μM, respectively. Therefore, it was concluded that the selective M₃ muscarinic acetylcholine receptor antagonist 4‐DAMP also possesses potent antagonistic action on nicotinic receptors of peripheral nitrergic neurons that innervate the rat anococcygeus muscle. Drug Dev. Res. 46:148–154, 1999. © 1999 Wiley‐Liss, Inc.     

Pyridostigmine prevents peripheral vascular endothelial dysfunction in rats with myocardial infarction

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Erythrocyte nitric oxide synthase as a surrogate marker for mercury‐induced vascular damage: The modulatory effects of naringin

In this study, endothelial nitric oxide synthase activity and nitric oxide (NO) production by human erythrocytes in the presence and absence of mercuric chloride (HgCl₂), L ‐arginine (L ‐ARG), ‐ nitro‐L ‐arginine methyl ester (L ‐NAME), and naringin (NAR) were investigated. In addition, the levels of reduced glutathione (GSH) and related enzymes were estimated in erythrocytes hemolysate. The protein carbonyl content (PCC) and thiobarbituric acid‐reactive substances (TBARS) levels were also determined. The results of this study revealed that the treatment of erythrocytes with either HgCl₂ or L ‐NAME induced a significant decrease in NOS activity and nitrite levels compared with control cells. Furthermore, mercury exposure significantly increased the levels of PCC and TBARS but reduced the GSH level. The activities of glucose‐6‐phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, and glutathione‐S‐transferase (GST) were inhibited. The exposure of erythrocytes to HgCl₂ in combination with L ‐ARG, NAR, or both ameliorated the investigated parameters compared with erythrocytes incubated with HgCl₂ alone. These results indicate that mercury exposure decreased both erythrocyte NOS activity and nitrite production, and that these parameters might be indicative of mercury exposure. The data also suggest that concomitant treatment with NAR can restore NO bioavailability through either its metal‐chelating properties or its antioxidant activity. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1314–1322, 2014.     

Elevated vascular γ‐butyrobetaine levels attenuate the development of high glucose‐induced endothelial dysfunction

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Design,Synthesis, and Biological Evaluation of 1‐Benzyl‐1H‐pyrazole Derivatives as Receptor Interacting Protein 1 Kinase Inhibitors

Receptor interacting protein 1 (RIP1) kinase plays an important role in necroptosis, and inhibitors of the RIP1 kinase are thought to have a potential therapeutic value in the treatment of diseases related to necrosis. Herein, we report the structural optimization of a RIP1 kinase inhibitor, 1‐(2,4‐dichlorobenzyl)‐3‐nitro‐1H‐pyrazole ( 1a ). A number of 1‐benzyl‐1H‐pyrazole derivatives were synthesized and structure‐activity relationship (SAR) analysis led to the discovery of a potent compound, 4b , which showed a K_d value of 0.078 μm against the RIP1 kinase and an EC₅₀ value of 0.160 μm in a cell necroptosis inhibitory assay. Compound 4b also displayed considerable ability to protect the pancreas in an l ‐arginine‐induced pancreatitis mouse model.     

Increased brain l‐arginine availability facilitates cutaneous heat loss induced by running exercise

The effects of increased brain availability of l ‐arginine (l ‐arg), a precursor for nitric oxide synthesis, on core body temperature (T_core) and cutaneous heat loss were evaluated in running rats. One week prior to the experiments, adult male Wistar rats received the following implants: a chronic guide cannula in the lateral cerebral ventricle and a temperature sensor in the abdominal cavity. On the day of the experiments, the rats were assigned to receive a 2‐μL intracerebroventricular injection of either NaCl (0.15 mol/L) or l ‐arg solution (0.825, 1.65 or 3.30 mol/L); T_core and tail skin temperature were measured while the rats ran at a speed of 18 m/min until they were fatigued. l ‐arginine induced a dose‐dependent reduction in the threshold T_core required for cutaneous heat loss (38.09 ± 0.20°C for 3.30‐mol/L l ‐arg vs 38.61 ± 0.10°C for saline; P < 0.05), which attenuated the exercise‐induced hyperthermia. Although the rats treated with l ‐arg presented a lower T_core at the end of exercise (~0.7°C lower after treatment with the highest dose), no changes in the time to fatigue were observed relative to the control trial. These results suggest that brain l ‐arg controls heat loss during exercise, most likely by modulating the sympathetic vasoconstrictor tonus to skin vessels. Furthermore, despite facilitating cutaneous heat loss mechanisms, increased brain l ‐arg availability did not enhance physical performance.     

β‐asarone and levodopa co‐administration protects against 6‐hydroxydopamine‐induced damage in parkinsonian rat mesencephalon by regulating autophagy: down‐expression Beclin‐1 and light chain 3B and up‐expression P62

In this study, we investigated Beclin‐1, light chain (LC)3B, and p62 expression in 6‐hydroxydopamine (6‐OHDA)‐induced parkinsonian rats after β‐asarone and levodopa (l ‐dopa) co‐administration. Unilateral 6‐OHDA injection into the medial forebrain bundle was used to create the models, except in sham‐operated rats. Rats were divided into eight groups: sham‐operated group; 6‐OHDA model group; madopar group (75 mg/kg, per os (p.o.)); l ‐dopa group (60 mg/kg, p.o.); β‐asarone group (15 mg/kg, p.o.); β‐asarone + l ‐dopa co‐administered group (15 mg/kg + 60 mg/kg, p.o.); 3‐methyladenine group (500 nmol, intraperitoneal injection); and rapamycin group (1 mg/kg, intraperitoneal injection). Then, Beclin‐1, LC3B, and p62 expression in the mesencephalon were detected. The mesencephalon was also observed by transmission electron microscope. The results showed that Beclin‐1 and LC3B expression decreased and that p62 expression increased significantly in the madopar, l ‐dopa, β‐asarone, and co‐administered groups when compared with the 6‐OHDA model. Beclin‐1 and LC3B expression in the β‐asarone and co‐administered groups were less than in the madopar or l ‐dopa groups, whereas p62 expression in the β‐asarone and co‐administered groups was higher than in the madopar or l ‐dopa groups. In addition, a significant decrease in autophagosome was exhibited in the β‐asarone and co‐administered groups when compared with the 6‐OHDA group. Our findings indicate that Beclin‐1 and LC3B expression decreased, whereas p62 expression increased after co‐administration treatment. In sum, all data suggest that the co‐administration of β‐asarone and l ‐dopa may contribute to the treatment of 6‐OHDA‐induced damage in rats by inhibiting autophagy activity.     

Role of nitric oxide in the antifibrotic and anticholestatic actions of interferon‐α2b

Interferons possess important antifibrotic properties. In addition, there is evidence that they induce the production of nitric oxide (NO) and that it downregulates the synthesis of extracellular matrix by certain cells. The aim of the present work was to evaluate if L ‐arginine, the NO synthase substrate, is able to increase the antifibrotic properties of interferon‐α_2b and if L ‐NAME, an NO synthesis inhibitor, can prevent them. Fibrosis was induced by bile duct ligation (BDL) for 5 weeks in rats and interferon‐α_2b (IFN; 100,000 IU rat, s.c., daily) and/or L ‐arginine (500 mg/kg, p.o., twice daily) or L ‐NAME (100 mg/kg, p.o., twice daily) were administered. Collagen content was determined by measuring hydroxyproline in liver samples. Malondialdehyde (MDA) was used to estimate lipid peroxidation levels. Glycogen was measured colorimetrically. Serum enzyme activities and bilirubins were determined by standard procedures. Fibrosis was increased 6‐fold by BDL. L ‐arginine or IFN partially prevented the increment in collagen. Furthermore, administration of both drugs simultaneously showed an additive effect (P < 0.05), while L ‐NAME abolished the protective effect of IFN. The same effect was observed on the other markers of liver function or damage studied herein. The additive effects of L ‐arginine and IFN could be due to a synergism of both compounds by increasing NO concentration, which can act as an antifibrotic agent but also as a cytoprotective compound. These results also suggest that the protective effects of IFN are mediated by NO, since L ‐NAME prevented them. Drug Dev. Res. 48:45–52, 1999. © 1999 Wiley‐Liss, Inc.