Non-selective Nitric Oxide Synthase Inhibitor Research Articles

Involvement of the Nitric Oxide/Cyclic Guanylate Monophosphate Pathway in the Pilocarpine-Induced Seizure Model in Mice

The present study was designed to investigate the involvement of the nitric oxide (NO)/cyclic guanylate monophosphate pathway in pilocarpine-induced seizures in mice. Male Swiss mice (26–32 g) were used as the in vivo model. The following pharmacological tools were utilized: the non-selective NO synthase (NOS) inhibitor <smlcap>L</smlcap>-NAME (10 mg/kg, i.p.), a preferential inducible NOS (iNOS) inhibitor aminoguanidine (25 mg/kg, i.p.), a highly specific iNOS inhibitor 1400W (2.5 mg/kg, i.p.), the NO donor <smlcap>L-</smlcap>arginine (150 mg/kg, i.p.), and the soluble guanylyl cyclase inhibitor ODQ (10 mg/kg, i.p.). The animals were divided into groups (n = 8) and pretreated for 30 min before receiving pilocarpine (400 mg/kg, i.p.), while the control group received only pilocarpine. They were observed for 60 min to measure initial seizure latency, latency till death, and mortality. An administration of <smlcap>L</smlcap>-NAME or ODQ delayed the onset of initial seizure, increased latency till death, and produced a 25% survival rate. Aminoguanidine increased the initial seizure and latency until death, and administration of 1400W did not have an effect. Incremental increases of NO by <smlcap>L</smlcap>-arginine were capable of decreasing the seizure and death latency. These results support the idea that the constitutive NOS, probably neuronal NOS, followed by soluble guanylyl cyclase activation is involved in the convulsive responses caused by pilocarpine administration.

Effect of celecoxib and L-NAME on global ischemia-reperfusion injury in the rat hippocampus

Transient global ischemia continues to be an important clinical problem with limited treatment options. The present study aimed to investigate the possible protective effects of celecoxib [a selective cyclooxygenase (COX-2) inhibitor] and N-omega-nitro-L-arginine methyl ester (L-NAME) [a nonselective nitric oxide synthase (NOS) inhibitor] against global ischemia-reperfusion (IR) induced biochemical and histological alterations in the rat hippocampus. Global ischemia was induced by bilateral clamping of the common carotid arteries for 60 minutes. Hippocampal cysteinyl aspartate-specific protease-3 (caspase-3) activity, nitrite/nitrate contents (NOX), as well as COX-2 immunoreactivity in the hippocampal Cornu Ammonis 1 (CA1) subregion were dramatically increased 24 hours after global ischemia. After 72-hour of reperfusion, ischemia induced a selective, extensive neuronal loss in the hippocampus CA1 subregion. Celecoxib (3 and 5 mg/kg, intraperitoneally; i.p.), administered 30 minutes before ischemia and at 6, 12, and 22 hours of 24-hour reperfusion, caused significant reductions in hippocampal caspase-3 activity as well as the number of COX-2 immunoreactive (COX-2 ir) neurons in the CA1 subregion. Further, celecoxib (3 or 5 mg/kg, i.p.), administered 30 minutes before ischemia and at 6, 12, 22, and 48 hours of 72-hour reperfusion, provided a notable histological protection of hippocampal CA1 neurons. Meanwhile, L-NAME (3 mg/kg, i.p.), administered twice (immediately after ischemia and 45 minutes after starting the reperfusion period), effectively reduced the elevated NOX level, decreased hippocampal caspase-3 activity and COX-2 immumoreactivity, and ameliorated ischemia-induced damage in the hippocampal CA1 subregion. The present study indicates that celecoxib and L-NAME might be neuroprotective agents of potential benefit in the treatment of cerebral ischemia.

Endothelium-Derived Nitric Oxide Supports Renin Cell Recruitment Through the Nitric Oxide–Sensitive Guanylate Cyclase Pathway

Chronic challenge of renin-angiotensin causes recruitment of renin-producing cells in the kidney along the media layer of afferent arterioles and hypertrophy of cells in the juxtaglomerular apparatus. This study aimed to define the role of nitric oxide (NO) with regard to the recruitment pattern of renin-producing cells and to the possible pathways along which NO could act. We considered the hypothesis that endothelium-derived NO acts via NO-sensitive guanylate cyclase. Mice were treated with low-salt diet in combination with the angiotensin I-converting enzyme inhibitor enalapril for 3 weeks, which led to a 13-fold increase in renin expression associated with marked recruitment of renin cells in afferent arterioles and hypertrophy of the juxtaglomerular apparatus in wild-type mice. In wild-type mice additionally treated with the nonselective NO synthase inhibitor L-NAME, the recruitment of renin-expressing cells along the afferent arterioles was absent and juxtaglomerular hypertrophy was diminished. An almost identical attenuation of renin cell recruitment as with L-NAME treatment in wild-type mice was found in mice lacking the endothelial isoform of NO synthase. Treatment of mice lacking NO-sensitive guanylate cyclase in renin-expressing cells and preglomerular smooth muscle cells with low-salt diet in combination with the angiotensin I-converting enzyme inhibitor enalapril for 3 weeks produced juxtaglomerular hypertrophy like in wild-type mice, but no recruitment in afferent arterioles. These findings suggest that endothelium-derived NO and concomitant formation of cGMP in preglomerular renin cell precursors supports recruitment of renin-expressing cells along preglomerular vessels, but not in the juxtaglomerular apparatus.

Role of N-Nitro-L-Arginine-Methylester as anti-oxidant in transient cerebral ischemia and reperfusion in rats

BackgroundPrevious reports assessing the neuroprotective role of nonselective Nitric Oxide synthase (NOS) inhibitor N-nitro-L-arginine-methylester (L-NAME) following cerebral ischemia/reperfusion are contradictory. The aim of this work was to examine the potential benefits of L-NAME on rats subjected to transient focal cerebral ischemia/reperfusion.MethodsThe study involved 30 adult male Wistar rats divided into three groups 10 rats in each: First group was sham-operated and served as a control, a ischemia/reperfusion (I/R) group of rats infused with 0.9% normal saline intraperitoneally 15 minutes prior to 30 minutes of left common carotid artery (CCA) occlusion and a test group infused with L-NAME intraperitoneally 15 minutes prior to ischemia. Neurobehavioral assessments were evaluated and quantitative assessment of malondialdehyde (MDA), Nitric oxide (NO) metabolites and total antioxidant capacity (TAC) in both serum and the affected cerebral hemisphere were achieved.ResultsRats’ neurological deficit and TAC were significantly decreased while NO and MDA were significantly increased in the I/R compared with the control group (P < 0.001). Alternatively in the L-NAME group, neurological deficit and TAC were significantly improved while NO and MDA were significantly decreased compared to I/R group (P < 0.001).ConclusionsL-NAME pretreatment for rats undergoing cerebral ischemia/reperfusion significantly improves neurological deficit while reducing oxidative stress biomarkers in the affected cerebral hemisphere.

Differential effects of selective and non-selective nitric oxide synthase inhibitors on the blood perfusion of ischemia-reperfused myocardium in dogs

BackgroundNitric oxide (NO) is protective for the cardiovascular system, and excessive NO exerts negative effects on the circulatory system. This study aimed to compare the effects of selective or non-selective NO synthase (NOS) inhibitors on blood flow perfusion of ischemia-reperfused myocardium.Materials/MethodsMale mongrel dogs were randomly assigned to 4 groups: only ischemia-reperfusion (control), ischemia-reperfusion plus Nω-nitro-L-arginine methyl ester (NAME) treatment, ischemia-reperfusion plus aminoguanidine (AMD) treatment, and sham operation group. Myocardial contrast echocardiography (MCE) was performed. Blood samples were taken for measurement of NO. Background-subtracted peak videointensity (PVI) and PVI ratio in myocardium were measured.ResultsIn the NAME-treated group, the PVI at 5 min reperfusion did not significantly differ from pre-LAD-occlusion, but declined to and retained at a level obviously lower than the pre-LAD-occlusion. In the AMD-treated group, the PVI at 5 min reperfusion was significantly higher than at pre-LAD-occlusion, and then restored to and remained at the pre-LAD-occlusion level. The changes of PVI ratios in the 3 groups were similar to PVI values. In the AMD-treated group, the curve width increased in the early reperfusion, but returned to the pre-LAD-occlusion level at 90 min reperfusion. The plasma NO concentration in the NAME-treated group greatly decreased and remained low during the whole period of reperfusion. In the AMD-treated group, there were only slight increases in NO concentrations during reperfusion.ConclusionsNAME totally inhibited NO production and attenuated myocardial blood flow perfusion. Aminoguanidine significantly relieved the increase in NO production and alleviated the congestion of reperfused myocardium. Selective inhibitors of iNOS might be useful in the management of certain diseases associated with ischemia-reperfusion.

Risk of cardiovascular diseases in diabetes mellitus and serum concentration of asymmetrical dimethylarginine.

Introduction. Asymmetric dimethylarginine (ADMA) is a nonselective nitric oxide (NO) synthase inhibitor associated with cardiovascular and metabolic disorders. ADMA plays an important role in the regulation of vascular tone by acting as an endogenous inhibitor of NO synthesis. Objectives. This study aimed to investigate ADMA with respect to diabetes and its clinical relevance as an independent predictor of CAD (Coronary Artery Disease). Methodology. The present case control study includes two hundred and forty patients selected randomly. Serum ADMA was analyzed by using enzyme immunoassay for the quantitative determination of endogenous ADMA, and serum nitric oxide was estimated by the method of Cortes. Results. Elevated NO level levels was a strong predictor and significantly (t: 9.86, P < 0.001) associated with occurrence of CAD. Increased ADMA level was found to be another strong predictor and associated significantly (t: 8.02, P < 0.001) with CAD. On intra group analysis, the relationship between ADMA and NO in diseased group, is significant negative correlation (r = −0.743). P (0.001) was found between ADMA and NO. Conclusion. ADMA level was found to be one of the strong predictors for CAD. ADMA is an emerging independent risk marker for future CVD (cardiovascular disease) events.

Вплив каліксарену С-90 на скоротливу активність гладеньких м’язів міометрія щурів

It is known that calix[4]arene with cipher C-90 selectively and with high affinity inhibits Ca 2+ ,Mg 2+ -ATPase of smooth muscle cells plasma membrane preparations. The work wis devoted to investigation of the influence of calixarene C-90 (10 µM) on spontaneous and induced (high-potassium solution and oxytocin) contractions of rat uterus longitudinal smooth muscles. Contractile activity was studied tensometrically in the isometric mode, analysis of the kinetic properties of contractions was performed by the calculation of the normalized maximal velocity of contraction ( V n с ) and relaxation ( V nr ) phases. Calixarene C-90 changed the spontaneous contractile activity, causing a decrease in amplitude and has no significant effect on the frequency, while slowing down of the relaxation phase of individual contractions (decreasing parameter V nr ) occurred. In the presence of non-selective NO-synthase inhibitor L-NAME (100 µM), calixarene C-90 did not cause a reduction of the amplitude of spontaneous contractions and the speed of relaxation phase returned to the control level. Furthermore, calixarene C-90 was equally contributing factor to reduced force of both oxytocin-induced (0.1 IU) and K + -induced (80 mM) contractions without affecting the nature of the increase in contractile force responses (normalized maximal velocity of contraction phase stayed at control level). The relaxation velocity of caused contractions recieved opposite changes depending on the nature of the contractile stimulation: in case of oxytocin-evoked contractions – decreased, while for K + -induced contractions – increased. In the presence of L-NAME calixarene C-90 did not cause inhibition of the maximal force K + - and oxytocin-induced contractions, but evoked changes in the kinetical para­meters of contractile responses (decrease V nr ). Thus, blocking of NO synthesis resulted in the removal of inhibiting both spontaneous and evoked contractions of smooth muscle myometrium under the influence of calixarene C-90. These results suggest that inhibition force of uterus smooth muscle contractions under the influence of calixarene C-90 is by NO-dependent way, whereas slow relaxation (decrease in normalized maximal velocity V nr ) is caused by the inhibition of Ca 2+ -transport function of the plasma membrane calcium pump. Keywords : smooth muscle, uterus, contraction, kinetical parameters, calixarene C-90, plasma membrane calcium pump, nitric oxide.

Nerve growth factor-mediated regulation of low density lipoprotein receptor-related protein promoter activation.

The mechanisms by which nerve growth factor (NGF) increases the level of low density lipoprotein receptor-related protein (LRP1) are not known. Administration of nitric oxide synthase (NOS) inhibitors modulates several of the neurotrophic actions of NGF, including TrkA signalling pathway activation, increases in gene expression and neurite outgrowth. The present study investigated whether NGF regulates the transcription of LRP1 as well as the role of NO and the individual TrkA signalling pathways in this action of NGF. PC12 cells were transfected with luciferase reporter constructs containing various sized fragments of the LRP1 promoter and treated with NGF (50 ng/mL) to establish whether NGF altered LRP transcription. NGF significantly increased luciferase activity in all LRP1 promoter construct-transfected cells with the NGF-responsive region of the promoter identified to be present in the first 1000 bp. The non-selective NOS inhibitor N(ω)-nitro-L-arginine methylester (L-NAME; 20 mM) had no effect on the NGF-mediated increase in luciferase activity, while the inducible NOS selective inhibitor s-methylisothiourea (S-MIU; 2 mM) attenuated the NGF-induced activation of the LRP1 promoter. Pretreatment of PC12 cells with 10 μM bisindolylmaleimide 1 (BIS-1) prevented the NGF-mediated increase in LRP1 promoter activation while 50 μM U0126 partially inhibited this response. In combination with S-MIU, all of the TrkA signalling pathway inhibitors blocked the ability of NGF to increase LRP1 transcription. These data suggest the NGF-mediated increase in LRP1 levels occurs, at least in part, at the level of transcription and that NO and the TrkA signalling pathways cooperate in the modulation of LRP1 transcription.

Differential effects of LPS from Escherichia coli and Porphyromonas gingivalis on IL-6 production in human periodontal ligament cells

Objective. Periodontal ligament (PDL) cells produce IL-6 upon stimulation with inflammation promoters, but the signaling pathways involved have not been characterized. This study investigates underlying mechanisms behind regulation of PDL cell IL-6 production by E. coli and P. gingivalis LPS. Materials and methods: Human PDL cells, endothelial cells and monocytes were stimulated with E. coli or P. gingivalis LPS in the presence or absence of pharmacological agents in order to disclose pathways involved in LPS signaling. Gene expression and cellular protein levels were assessed by quantitative real-time PCR and ELISA, respectively. Results. Stimulation with LPS from E. coli (1 µg/ml) for 24 h enhanced PDL cell IL-6 expression several fold, demonstrated both on transcript and protein levels, but P. gingivalis LPS (1–5 µg/ml) had no effect. TLR2 mRNA was more highly expressed than TLR4 transcript in PDL cells. Treatment with the non-selective nitric oxide synthase inhibitor L-NAME (100 µM) reduced E. coli LPS-induced PDL cell IL-6 by 30%, while neither aminoguanidine (10 µM), an inhibitor of inducible nitric oxide synthase, nor estrogen (17β-estradiol, 100 nM) influenced IL-6. Treatment with the glucocorticoid dexamethasone (1 µM) totally prevented the E. coli LPS-induced PDL cell IL-6. In endothelial cells, neither E. coli LPS nor P. gingivalis LPS promoted IL-6 production. In monocytes, serving as positive control, both E. coli and P. gingivalis LPS stimulated IL-6. Conclusions. E. coli LPS but not P. gingivalis LPS stimulates PDL cell IL-6 production through a glucocorticoid-sensitive mechanism involving nitric oxide formation, probably via endothelial nitric oxide synthase.

Do cholinergic nerves innervating rat mesenteric arteries regulate vascular tone?

Vascular blood vessels have various types of cholinergic acetylcholine receptors (AChR), but the source of ACh has not been confirmed. Perivascular adrenergic nerves and nonadrenergic calcitonin gene-related peptide (CGRP)-containing (CGRPergic) nerves innervate rat mesenteric arteries and regulate vascular tone. However, function of cholinergic innervation remains unknown. The present study investigated cholinergic innervation by examining effects of cholinesterase inhibitor (neostigmine), a muscarinic AChR antagonist (atropine), and a nicotinic AChR antagonist (hexamethonium) on adrenergic nerve-mediated vasoconstriction and CGRPergic nerve-mediated vasodilation in rat mesenteric vascular beds without endothelium. In preparations treated with capsaicin (CGRP depletor) or in the presence of N(ω)-nitro-l-arginine methyl ester (nonselective nitric oxide synthase inhibitor), perivascular nerve stimulation (PNS; 2-12 Hz) evoked a frequency-dependent vasoconstriction. In the same preparations, exogenous norepinephrine induced a concentration-dependent vasoconstriction. Atropine, hexamethonium, and neostigmine had no effect on vasoconstrictor responses to PNS and norepinephrine injections. In denuded preparations, these cholinergic agents did not affect the PNS (12 Hz)-evoked release of norepinephrine in perfusate. In preconstricted preparations without endothelium in the presence of guanethidine (adrenergic neuron blocker), PNS (1-4 Hz) induced a frequency-dependent vasodilation, which was not affected by atropine, hexamethonium, and neostigmine. In denuded preparations treated with capsaicin and guanethidine, PNS did not induce vascular responses, and atropine, neostigmine, and physostigmine had no effect on PNS. Immunohistochemistry study showed choline acetyltransferase-immunopositive fibers, which were resistant to capsaicin and 6-hydroxydopamine (adrenergic toxin). These results suggest that rat mesenteric arteries have cholinergic innervation, which is different from adrenergic and capsaicin-sensitive nerves and not associated with vascular tone regulation.

Nitric oxide regulates cytokine induction in the diaphragm in response to inspiratory resistive breathing

Resistive breathing (encountered in chronic obstructive pulmonary disease and asthma) results in cytokine upregulation and decreased nitric oxide (NO) levels in the strenuously contracting diaphragm. NO can regulate gene expression. We hypothesized that endogenously produced NO downregulates cytokine production triggered by strenuous diaphragmatic contraction. Wistar rats treated with vehicle, the nonselective NO synthase inhibitor NG-nitro-l-arginine-methylester (l-NAME), or the NO donor diethylenetriamine-NONOate (DETA) were subjected to inspiratory resistive breathing (IRB; 50% of maximal inspiratory pressure) for 6 h or sham operation. Additional groups of rats were subjected to IRB for 6 h with concurrent administration of l-NAME and inhibitors of NF-κB (BAY-11-7082), ERK1/2 (PD98059), or P38 (SB203580). Inhibition of NO production (with l-NAME) resulted in upregulation of IRB-induced diaphragmatic IL-6, IL-10, IL-2, TNF-α, and IL-1β levels by 50%, 53%, 60%, 47%, and 45%, respectively. In contrast, the NO donor (DETA) attenuated the IRB-induced cytokine upregulation to levels characteristic of quietly breathing animals. l-NAME augmented IRB-induced activation of MAPKs (P38 and ERK1/2) and NF-κB, whereas DETA triggered the opposite effect. NF-κB and ERK1/2 inhibition in l-NAME-treated animals blunted the l-NAME-induced cytokine upregulation except IL-6, whereas P38 inhibition blunted all (including IL-6) cytokine upregulation. NO downregulates IRB-induced cytokine production in the strenuously contracting diaphragm through its action on MAPKs and NF-κB.

Long-lasting Hypotensive Effect in Renal Hypertensive Rats Induced by Nitric Oxide Released From a Ruthenium Complex

In this study, we investigated the effect of the ruthenium complex [Ru(terpy)(bdq)NO] (TERPY) on the arterial pressure from renal hypertensive 2 kidney-1 clip (2K-1C) rats, which was compared with sodium nitroprusside (SNP). The most interesting finding was that the intravenous bolus injection of TERPY (2.5, 5.0, 7 mg/kg) had a dose-dependent hypotensive effect only in 2K-1C rats. On the other hand, SNP (35 and 70 μg/kg) presented a similar hypotensive effect in both normotensive (2K) and 2K-1C although the effect of 70 μg/kg was >35 μg/kg. The injection of the nonselective NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) increased the arterial pressure in 2K and 2K-1C rats with a similar magnitude. After infusion of L-NAME, the hypotensive effect induced by TERPY and SNP was potentiated in both 2K and in 2K-1C rats. The administration of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl increased the hypotensive effect induced by TERPY or SNP in both 2K and 2K-1C rats. The hypotensive effect induced by TERPY was longer than that produced by SNP. Taken together, our results show that the TERPY has a long-lasting hypotensive effect, which has a dose dependence and higher magnitude in 2K-1C compared with in 2K rats. In comparison with SNP, TERPY is less potent in inducing arterial pressure fall, but it presents a much longer hypotensive effect.

Acute and Chronic Cardioprotection by the Enkephalin Analogue, Eribis Peptide 94, is Mediated via Activation of Nitric Oxide Synthase and Adenosine Triphosphate-Regulated Potassium Channels

Background/Aims: Eribis peptide 94 (EP 94) is a new enkephalin derivative which potently binds to the µ- and δ-opioid receptor. In this study, we determined the effects of EP 94 and potential mechanism(s) involved in cardioprotection of the rat heart. Methods and Results: An acute (5 and10 min into ischemia) and a chronic (24 h prior to ischemia) EP 94 administration produced a similar 30–40% reduction in infarct size/area at risk and the effects were blocked by the K_ATP channel antagonists, HMR 1098 and 5-HD. The cardioprotective effects were blocked by a nonselective nitric oxide synthase (NOS) inhibitor (L-NAME) following acute administration and by a selective iNOS inhibitor (1400W) following chronic administration. Conclusion: These results suggest that EP 94 may have potential for the treatment of ischemic heart disease via a nitric oxide (NO)-K_ATP-mediated mechanism.

The effect of the non-selective nitric oxide synthase inhibitor L-name on rotational behaviour and dopamine metabolism in 6-OHDA-lesioned rats treated chronically with L-DOPA

The strong evidence obtained from in vivo and ex-vivo studies suggests an interaction between the dopaminergic and nitrergic systems. The aim of this study was to assess whether the inhibition of nitric oxide synthesis in 6-OHDA-lesioned rats treated chronically with L-DOPA affects rotational behaviour and dopamine (DA) metabolism. Male Wistar rats were injected unilaterally with a single dose of 6-OHDA (8 μg/4 μl) into the left medial forebrain bundle. After two weeks, only rats exhibiting a substantial loss of nigrostriatal neurons were treated chronically with L-DOPA (25 mg/kg) once daily for 15 days. On day 14, the rats received single doses of L-NAME (50 or 100 mg/kg) ten minutes before L-DOPA injection, and rotational behaviour was recorded for more than 2 h. On the following day, 1 h after the last injections of L-NAME and L-DOPA, animals were killed. The levels of dopamine (DA) and its metabolites (DOPAC, HVA) were assayed in striatal and nigral homogenates using an HPLC method. A single dose of L-NAME (50 mg/kg), administered before the penultimate dose of L-DOPA, caused contralateral rotations whose intensity was lower than that produced by L-DOPA alone. Moreover, such a combined treatment decreased striatal and nigral DA, DOPAC and HVA levels on the lesioned side compared to administration of L-DOPA alone. On the contralateral side, decreases in the content of DA metabolites only were observed. The higher dose of L-NAME produced weaker effects. Our biochemical results suggest that L-NAME prevents DA formation from L-DOPA, which eventually leads to attenuation of the L-DOPA-induced rotations.

Induction of Selective Blood-Tumor Barrier Permeability and Macromolecular Transport by a Biostable Kinin B1 Receptor Agonist in a Glioma Rat Model

Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg9BK (LDBK) and SarLys[dPhe8]desArg9BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T 1-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites.

Enhanced vascular neuronal nitric-oxide synthase-derived nitric-oxide production underlies the pressor response caused by peripheral N-methyl-D-aspartate receptor activation in conscious rats.

Although the N-methyl-D-aspartate (NMDA) receptor (NMDAR) obligatory unit NMDAR1 is expressed in the vasculature and myocardium, the impact of peripheral NMDAR activation on blood pressure (BP) has received little attention. We demonstrate, for the first time, dose-related pressor responses elicited by systemic NMDA (125, 250, 500, and 1000 μg/kg) in conscious rats. The pressor response was peripheral NMDAR-mediated because: 1) it persisted after ganglion blockade (hexamethonium; 5 mg/kg i.v.); 2) it was attenuated by the selective NMDAR blocker DL-2-amino-5-phosphonopentanoic acid (5 mg/kg, i.v.) or the glycine/NMDAR antagonist R-(+)-3-amino-1-hydroxypyrrolid-2-one [R-(+)-HA-966; 10 mg/kg i.v.]; and 3) NMDA (1.25-10 mM) increased contractile force of rat aorta in vitro. It is noteworthy that ex vivo studies revealed enhanced nitric oxide (NO) and reactive oxygen species (ROS) generation in vascular tissues collected at the peak of the NMDAR-mediated pressor response. Pharmacological, ex vivo, and in vitro findings demonstrated attenuation of the NMDAR-mediated increases in BP and vascular NO and ROS by the nonselective NO synthase (NOS) inhibitor N(ω)-nitro-L-arginine methyl ester hydrochloride (10 mg/kg i.v.) or the neuronal NOS (nNOS) inhibitor N(ω)-propyl-L-arginine hydrochloride (150 μg/kg i.p.) but not by the endothelial NOS inhibitor N(5)-(1-iminoethyl)-L-ornithine (4 or 10 mg/kg i.v.). Furthermore, R-(+)-HA-966 attenuated NMDA-evoked generation of vascular NO and ROS. The findings suggest a pivotal role for enhanced vascular nNOS-derived NO in ROS generation and in the subsequent pressor response elicited by peripheral NMDAR in conscious rats.

D Endothelium specific insulin resistance leads to accelerated atherosclerosis: a role for reactive oxygen species

<h3>Background</h3> Global insulin resistance and endothelial dysfunction have been identified as predisposing factors for atherosclerosis. However, it is unclear whether selective insulin resistance in endothelial cells alone, is sufficient to promote atherosclerosis. We addressed this question by crossing Endothelial Specific Mutant Insulin Receptor Over-expressing (ESMIRO) mice with ApoE^−/− mice. ESMIRO mice over-express a human insulin receptor with an Ala-Thr1134 mutation in the tyrosine kinase domain (which disrupts insulin signalling) selectively in endothelial cells under the control of the tie-2 promoter/enhancer. <h3>Methods</h3> Male ApoE^−/−/ESMIRO mice were compared with sex-matched littermate ApoE^−/− mice (both on a C57Bl6 background) after feeding a Western-style diet for 12 weeks. <h3>Results</h3> ApoE^−/−/ESMIRO mice were morphologically indistinguishable from ApoE^−/− control littermates and showed normal development with no differences between groups in body mass. Heart rate, systolic blood pressure, glucose tolerance, insulin sensitivity and fasting glucose levels were similar in ApoE^−/−/ESMIRO and ApoE^−/− mice. ApoE^−/−/ESMIRO cultured endothelial cells demonstrated insulin resistance through significantly reduced insulin mediated eNOS activity (p=0.003). Aortic lipid deposition along the whole aorta, assessed by en-face oil red O staining, was similar in ApoE^−/−ESMIRO and ApoE^−/− mice (6.4%±0.5% vs 5.8%±0.5%; p=0.39). Analysis of lipid deposition along the lesser curvature of the aortic arch revealed a significant increase in ApoE^−/−ESMIRO when compared to controls (9.4±0.89 vs 12.43±1.19% p=0.035). Atherosclerotic lesion area in cross sections of aortic sinus was also significantly increased in ApoE^−/−/ESMIRO mice compared to ApoE^−/− controls (24.8%±2.4% vs 16.6%±2.4%; p=0.02). Vascular function assessed through relaxation responses of aortic rings in response to the endothelial specific vasodilator acetylcholine revealed that aortic rings from ApoE^−/−/ESMIRO mice had blunted relaxation responses to acetylcholine (Emax ApoE^−/− 102.88±6, Emax ApoE^−/−/ESMIRO 65±41%, p=0.02), which was restored by the superoxide dismutase mimetic and antioxidant MnTMPyP (Emax ApoE^−/−/ESMIRO without MnTMPyP 65±41%, with MnTMPyP 112±15% p=0.048). Endothelial cells from ApoE^−/−/ESMIRO mice had significantly increased basal generation of superoxide (1.87-fold increase compared to ApoE^−/− p&lt;0.05) which was blunted by the selective NADPH oxidase inhibitor gp91ds-tat (11% reduction ±0.02, p=0.03) and the non-selective NO synthase inhibitor L-NMMA (6% reduction ±0.01, p=0.03). <h3>Conclusions</h3> Endothelial specific insulin resistance is sufficient to promote atherosclerosis and increase lesion area in ApoE null mice potentially via the increased ROS displayed in this model. This suggests that enhancing endothelial insulin sensitivity may be an appropriate target to prevent atherosclerosis in insulin-resistant conditions.

Effects of Nerve Growth Factor and Nitric Oxide Synthase Inhibitors on Amyloid Precursor Protein mRNA Levels and Protein Stability

We determined previously that nitric oxide (NO) modulates the nerve growth factor (NGF)-mediated increases in amyloid precursor protein (APP) levels in PC12 cells. To elucidate potential mechanisms, the effects of NGF and NO synthase (NOS) inhibitors on APP mRNA levels and protein stability were evaluated. Surprisingly, treatment of PC12 cells with NGF resulted in decreased levels of APP695 and APP751/770 mRNA. Therefore, the effect of NGF on APP protein stability was examined using the translation inhibitor, cycloheximide. Under these conditions, NGF did not alter the rate of APP degradation, suggesting that NGF may be enhancing the translation rate of APP. Since NOS inhibitors attenuate the NGF-mediated increase in APP levels, their effect on APP mRNA levels and protein stability was also assessed. S-methylisothiourea (S-MIU), selective for inducible NOS, decreased both APP695 and APP751/770 mRNA levels while the non-selective NOS inhibitor, Nω-nitro-L-arginine methylester (L-NAME) had no effect. In both control and NGF-treated PC12 cells, S-MIU increased the half-life of APP, with the greatest effect observed with the APP695 isoform. Based on these data we propose that in PC12 cells, NGF increases APP levels through enhanced translation rate and that NO, which modulates the NGF-induced increase in APP protein, also regulates APP mRNA levels and could play a role in APP processing.

Endothelium-dependent and independent vasorelaxation induced by an n-butanolic fraction of bark of Scutia buxifolia Reiss (Rhamanaceae)

Ethnopharmacological relevanceScutia buxifolia has been widely used in Brazilian folk medicine as an anti-hypertensive agent.We evaluated the vascular effects and mechanism involved in the relaxation of aorta induced by an n-butanolic fraction (BuOH) from Scutia buxifolia. Materials and MethodsRat aortic rings precontracted by phenylephrine (1μM) were exposed to cumulative concentrations (3–3000μg/ml) of crude extracts or fractions obtained from bark or leaves of Scutia buxifolia. Classical receptor antagonists, channel and enzymatic inhibitors were used to check the mechanisms involved. ResultsThe crude extracts of both leaves and bark of Scutia buxifolia, as well as several fractions, were able to induce partial or total relaxation of rat aortic rings. The BuOH fraction of bark of Scutia buxifolia was the most potent in endothelium-intact (E+) preparations, and also induced a partial, but very significant relaxation in endothelium-denuded (E−) vessels. The non-selective nitric oxide synthase inhibitor L-NAME, as well as the soluble guanylate cyclase inhibitor ODQ, vanished the relaxation in E+. In E− preparations, K+ channel blockers, such as tetraethylammonium, glibenclamide, 4-aminopyridine, and the large-conductance calcium-activated K+ channel blocker iberiotoxin, were able to significantly reduce the maximum relaxation elicited by BuOH fraction. ConclusionOur results demonstrated that BuOH fraction obtained from barks of Scutia buxifolia induced both endothelium-dependent and -independent relaxation in rat aortic rings. The endothelium-dependent relaxation is fully dependent on NO/cGMP system, while direct activation of K+ channels may explain, at least in part, the endothelium-independent relaxation induced by BuOH fraction of Scutia buxifolia.

Cardiovascular effects of Angiotensin peptides are differentially mediated at the CVLM of hypertensive rats

In the present study we investigated the effect of the non‐selective nitric oxide (NO) synthase inhibitor, L‐NAME, or the antioxidant vitamin C (Vit C) on the cardiovascular effects triggered by angiotensin (Ang) II and Ang‐(1–7) at the caudal ventrolateral medulla (CVLM) of renovascular hypertensive rats (2K1C). CVLM microinjection of L‐Name induced similar fall in blood pressure in Sham or 2K1C, while Vit C produced a fall in BP (− 19±4 mmHg, n=5) only in 2K1C rats. L‐NAME did not alter the hypotensive effect induced by CVLM microinjection of Ang II in Sham or 2K1C. However, L‐NAME increased the hypotensive effect of Ang‐(1–7) in Sham and did not alter its effect in 2K1C. In contrast, Vit C abolished the hypotensive effect of Ang II in the CVLM of both groups and did not change the hypotensive effect of Ang‐(1–7) in the CVLM of 2K1C (−12±1 mmHg, n=6) or Sham (− 12±1 mmHg, n=5). These data suggest that different mechanisms are involved in the cardiovascular effects of Ang peptides at the CVLM. Further, our results suggest that part of these mechanisms may be impaired in 2K1C hypertension.