Impact of endothelial nitric oxide synthase activation on accelerated liver regeneration in a rat ALPPS model.

BACKGROUND: Although the associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) induces more rapid liver regeneration than portal vein embolization, the mechanism remains unclear. AIM: To assess the influence of inflammatory cytokines and endothelial nitric oxide synthase (eNOS) activation on liver regeneration in ALPPS. METHODS: The future liver remnant/body weight (FLR/BW) ratio, hepatocyte proliferation, inflammatory cytokine expression, and activation of the Akt-eNOS pathway were evaluated in rat ALPPS and portal vein ligation (PVL) models. Hepatocyte proliferation was assessed based on Ki-67 expression, which was confirmed using immunohistochemistry. The serum concentrations of inflammatory cytokines were measured using enzyme linked immune-solvent assays. The Akt-eNOS pathway was assessed using western blotting. To explore the role of inflammatory cytokines and NO, Kupffer cell inhibitor gadolinium chloride (GdCl3), NOS inhibitor N-nitro-arginine methyl ester (L-NAME), and NO enhancer molsidomine were administered intraperitoneally. RESULTS: The ALPPS group showed significant FLR regeneration (FLR/BW: 1.60% ± 0.08%, P < 0.05) compared with that observed in the PVL group (1.33% ± 0.11%) 48 h after surgery. In the ALPPS group, serum interleukin-6 expression was suppressed using GdCl3 to the same extent as that in the PVL group. However, the FLR/BW ratio and Ki-67 labeling index were significantly higher in the ALPPS group administered GdCl3 (1.72% ± 0.19%, P < 0.05; 22.25% ± 1.30%, P < 0.05) than in the PVL group (1.33% ± 0.11% and 12.78% ± 1.55%, respectively). Phospho-Akt Ser473 and phospho-eNOS Ser1177 levels were enhanced in the ALPPS group compared with those in the PVL group. There was no difference between the ALPPS group treated with L-NAME and the PVL group in the FLR/BW ratio and Ki-67 labeling index. In the PVL group treated with molsidomine, the FLR/BW ratio and Ki-67 labeling index increased to the same level as in the ALPPS group. CONCLUSION: Early induction of inflammatory cytokines may not be pivotal for accelerated FLR regeneration after ALPPS, whereas Akt-eNOS pathway activation may contribute to accelerated regeneration of the FLR.

Perinatal N(G)-Nitro-L-arginine methyl ester administration decreases anxiety- and depression-like behaviors in adult mice

ABSTRACT Objective: We hypothesized that perinatal manipulations of the nitrergic system would affect adult animal behaviors. Methods: We tested this hypothesis by perinatally administering N(G)-Nitro-L-arginine methyl ester (L-NAME), a non-specific antagonist of nitric oxide synthase for 15 days and assessed anxiety- and depression-like behaviors in adult mice. At 70 days of age, the mice were subjected to a battery of tests consisting of the open-field, light/dark box, forced swim, and tail-flick tests. The tests were performed at two-day intervals, and the order of the tests within the battery was determined according to the progressive invasiveness degree. Results: L-NAME-treated animals exhibited decreased anxiety-like behavior in the light/dark box and open field tests, with no change in locomotor activity. Additionally, they demonstrated decreased depression-like behavior in the forced swim test and no change in pain perception in the tail-flick test. Conclusion: The nitrergic system is possibly involved in neural circuitry development that regulates behaviors since blocking perinatal nitric oxide production decreases anxiety- and depression-like behaviors in adult mice.

Protective effects of anandamide against cisplatin-induced peripheral neuropathy in rats

Background/aim: Cisplatin (CIS) is an effective antineoplastic agent used in the treatment of several cancer types. Peripheral neuropathy is a major dose-limiting side-effect in CIS therapy. Cannabinoids may alleviate this painful side effect. This study investigated the analgesic effects of anandamide (AN) on CIS-induced peripheral neuropathy, in vitro effects of AN in CIS neurotoxicity, and the contribution of nitric oxide (NO) in this effect. Materials and methods: This is an experimental animal study. Primary dorsal root ganglion (DRG) cultures were prepared from one-day-old rats for in vitro investigations. DRG cells were incubated with CIS (100­300 M), and AN (10, 50, 100, and 500 µM) was administered with the submaximal concentration of CIS. Female Sprague Dawley rats were divided into control, CIS, CIS+AN, CIS+AN+L-NG-nitro arginine methyl ester (LNAME). CIS was administered 3 mg/kg i.p once weekly for 5 weeks. AN (1 mg/kg i.p) or in combination with 10 mg/kg i.p LNAME was administrated 30 min before CIS injection. Mechanical allodynia, thermal hyperalgesia, and tail clip tests were performed. After intracardiac perfusion, sciatic nerves (SN), and DRGs were isolated and semi-thin sections were stained with toluidine blue and investigated histologically. SPSS v. 21.0 and Sigma STAT 3.5 were used for statistical analysis. One/two way ANOVA, Kruskal­Wallis, and Wilcoxon signed ranks tests were used. A p-value of 0.05 was accepted as significant. Results: CIS caused significant mechanical allodynia. AN and AN+LNAME significantly increased hind paw withdrawal latency in mechanical allodynia test. The degenerated axons significantly increased in CIS group, while decreased in AN group. The frequency of larger neurons seemed to be higher in CIS+AN group. Conclusion: AN may be a therapeutic alternative for the treatment of CIS-induced peripheral neuropathy. However, its central adverse effects must be considered.

Effects of oxidative stress on liver, brain and spinal cord of rats using L-NAME and treated with hydroxyurea. A model of sickle cell complication

Abstract Purpose: To analyze the serum levels of nitric oxide and correlate them with the levels of thiobarbituric acid reactive substances (TBARS) in liver, brain and spinal cord of animals using L-NAME and treated with hydroxyurea. Methods: Eighteen male albino Wistar rats were divided into three groups. NG-nitro-L-arginine methyl ester (L-NAME) was intraperitoneally administered to induce oxidative stress. TBARS and plasma nitric oxide levels were analyzed in all groups. Histopathology of the liver and vascular tissue was performed. Results: Statistically significant differences were seen in liver, brain and spinal cord TBARS levels. Conclusions: Following the use of L-NAME, hepatic tissue increased the number of Kupffer cells as oxidative stress and inflammatory response increased. The use of L-NAME caused an increase in lipid peroxidation products and, consequently, in oxidative stress in animals. Hydroxyurea doses of 35 mg / kg / day reduced TBARS values in liver, brain and spinal cord.

Cardiovascular risk and the effect of nitric oxide synthase inhibition in female rats: The role of estrogen.

It is known that autonomic modulation is responsive to ovarian hormone levels and that estrogen increases nitric oxide (NO) bioavailability. However, little is known about the interaction of nitric oxide synthase (NOS) isoforms with autonomic modulation, oxidative stress and cardiovascular risk in females. This study aimed to investigate cardiovascular, autonomic and oxidative parameters after selective NOS inhibition. A spectral analysis of systolic arterial pressure (SAP) and heart rate variability (HRV) was performed. NO levels, total antioxidant capacity (TRAP), lipid hydroperoxides (LOOH) and paraoxonase 1 (PON1) activity were measured in the plasma of rats treated with L-NG-nitroarginine methyl ester (L-NAME), S-methylisothiourea (SMT) or saline. Wistar rats, ovariectomized (OVX) with or without estradiol treatment (1mg/kg/day) or with a false ovariectomy (SHAM), were submitted to artery and vein catheterization. Cardiovascular parameters were evaluated before and after the administration of saline or NOS inhibitors. After 2h, plasma samples were collected for biochemical measurement. At baseline, cardiovascular and autonomic parameters were not different among the groups. L-NAME, the constitutive NOS isoform (cNOS) inhibitor, promoted an increase in mean arterial pressure (MAP) and a reduction in the low frequency band (LF) of SAP of SHAM rats, but this increase was smaller in OVX animals, which also showed a reduction in PON1 activity. The decreased activity of PON1 caused by L-NAME was prevented in the OVX+E group. SMT, an inducible NOS isoform (iNOS) inhibitor, promoted an increase in MAP and in the LF of SAP, in interbeat interval (IBI) parameters at LFnu and in LF/HF ratio of HRV in all groups, but the OVX+E had lower levels of NO when compared with the OVX group. Our data suggest that while cNOS contributes to maintaining the activity of PON1 in OVX rats, iNOS activity maintains the levels of NO in OVX+E rats.

Hyperbaric oxygen therapy may overcome nitric oxide blockage during cyanide intoxication.

PURPOSE: To determine the effects of a blockade of nitric oxide (NO) synthesis on hyperbaric oxygen (HBO2) therapy during cyanide (CN) intoxication. METHODS: 39 anesthetized female Sprague-Dawley rats were exposed to CN intoxication (5.4 mg/kg intra-arterially) with or without previous nitric oxide synthase (NOS) inhibition by L-NG-nitroarginine methyl ester (L-NAME) injection (40 mg/kg intraperitoneally). Subsequently, either HBO2 therapy (284 kPa/90 minutes), normobaric oxygen therapy (100% oxygen/90 minutes) or nothing was administered. Intracerebral microdialysis was used to measure the interstitial brain concentration of lactate, glucose, glycerol and lactate/pyruvate ratios. RESULTS: L-NAME potentiated CN intoxication by higher maximum and prolonged lactate (in mM: 0. 5 ± 0.3 vs. 0.7 ± 0.4, P ⟨ 0.005) concentrations compared with solely CN-intoxicated rats. The same trend was found for mean glucose, glycerol and lactate/pyruvate ratio levels. During HBO2 treatment a sustained reduction occurred in mean lactate levels (in mM: 0.5 ± 0.5 vs. 0.7 ± 0.4, P ⟨ 0.01) regardless of NOS blockade by L-NAME. The same trend was found for mean glucose and glycerol levels. CONCLUSION: The results suggest that blocking NOS using L-NAME can worsen acute CN intoxication. HBO2 treatment can partially overcome this block and continue to ameliorate CN intoxication.

Effects of nitric oxide inhibitors in mice with bladder outlet obstruction

ABSTRACT Purpose To investigate the lower urinary tract changes in mice treated with L-NAME, a non-selective competitive inhibitor of nitric oxide synthase (NOS), or aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), after 5 weeks of partial bladder outlet obstruction (BOO), in order to evaluate the role of constitutive and non-constitutive NOS in the pathogenesis of this experimental condition. Materials and Methods C57BL6 male mice were partially obstructed and randomly allocated into 6 groups: Sham, Sham + L-NAME, Sham + aminoguanidine, BOO, BOO + L-NAME and BOO + aminoguanidine. After 5 weeks, bladder weight was obtained and cystometry and tissue bath contractile studies were performed. Results BOO animals showed increase of non-voiding contractions (NVC) and bladder capacity, and also less contractile response to Carbachol and Electric Field Stimulation. Inhibition of NOS isoforms improved bladder capacity and compliance in BOO animals. L-NAME caused more NVC, prevented bladder weight gain and leaded to augmented contractile responses at muscarinic and electric stimulation. Aminoguanidine diminished NVC, but did not avoid bladder weight gain in BOO animals and did not improve contractile responses. Conclusion It can be hypothesized that chronic inhibition of three NOS isoforms in BOO animals leaded to worsening of bladder function, while selective inhibition of iNOS did not improve responses, what suggests that, in BOO animals, alterations are related to constitutive NOS.

Effects of nitric oxide inhibitors in mice with bladder outlet obstruction.

PURPOSE: To investigate the lower urinary tract changes in mice treated with L-NAME, a non-selective competitive inhibitor of nitric oxide synthase (NOS), or aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), after 5 weeks of partial bladder outlet obstruction (BOO), in order to evaluate the role of constitutive and non-constitutive NOS in the pathogenesis of this experimental condition. MATERIALS AND METHODS: C57BL6 male mice were partially obstructed and randomly allocated into 6 groups: Sham, Sham + L-NAME, Sham + aminoguanidine, BOO, BOO + L-NAME and BOO + aminoguanidine. After 5 weeks, bladder weight was obtained and cystometry and tissue bath contractile studies were performed. RESULTS: BOO animals showed increase of non-voiding contractions (NVC) and bladder capacity, and also less contractile response to Carbachol and Electric Field Stimulation. Inhibition of NOS isoforms improved bladder capacity and compliance in BOO animals. L-NAME caused more NVC, prevented bladder weight gain and leaded to augmented contractile responses at muscarinic and electric stimulation. Aminoguanidine diminished NVC, but did not avoid bladder weight gain in BOO animals and did not improve contractile responses. CONCLUSION: It can be hypothesized that chronic inhibition of three NOS isoforms in BOO animals leaded to worsening of bladder function, while selective inhibition of iNOS did not improve responses, what suggests that, in BOO animals, alterations are related to constitutive NOS.

Short-Term Administration of Serelaxin Produces Predominantly Vascular Benefits in the Angiotensin II/L-NAME Chronic Heart Failure Model.

In patients hospitalized with acute heart failure, temporary serelaxin infusion reduced 6-month mortality through unknown mechanisms. This study therefore explored the cardiovascular effects of temporary serelaxin administration in mice subjected to the angiotensin II (AngII)/L-NG-nitroarginine methyl ester (L-NAME) heart failure model, both during serelaxin infusion and 19 days post-serelaxin infusion. Serelaxin administration did not alter AngII/L-NAME-induced cardiac hypertrophy, geometry, or dysfunction. However, serelaxin-treated mice had reduced perivascular left ventricular fibrosis and preserved left ventricular capillary density at both time points. Furthermore, resistance vessels from serelaxin-treated mice displayed decreased potassium chloride-induced constriction and reduced aortic fibrosis. These findings suggest that serelaxin improves outcomes in patients through vascular-protective effects.

ÓXIDO NÍTRICO E DINÂMICA DE CA2+ EM CARDIOMIÓCITOS: INFLUÊNCIA DA CAPACIDADE DE EXERCÍCIO / NITRIC OXIDE AND CA2+ DYNAMICS IN CARDIOMYOCYTES: INFLUENCE OF EXERCISE CAPACITY / ÓXIDO NÍTRICO Y DINAMICA DE CA2+ EN CARDIOMIOCITOS: INFLUENCIA DE LA CAPACIDAD DE EJERCÍCIO

RESUMO Introdução: A capacidade intrínseca para o exercício aeróbico está relacionada com o inotropismo cardíaco. Por outro lado, a participação do óxido nítrico (NO) como mensageiro intracelular sobre a dinâmica do Ca2+ ainda permanece desconhecida em ratos com diferentes capacidades intrínsecas para o exercício. Objetivo: Avaliar se o NO modula diferentemente o transiente intracelular de Ca2+ e liberações espontâneas de Ca2+(sparks) em cardiomiócitos de ratos com diferentes capacidades intrínsecas para o exercício. Métodos: Ratos machos Wistar foram selecionados como desempenho padrão (DP) e alto desempenho (AD), de acordo com a capacidade de exercício até a fadiga, mensurada através de teste de esforço progressivo em esteira. Os cardiomiócitos dos ratos foram utilizados para determinar o transiente intracelular de Ca2+ e Ca2+sparks em microscópio confocal. Para estimar a contribuição do NO foi utilizado o inibidor das sínteses do NO (L-NAME, 100 µM). Os dados foram analisados através de ANOVA two-way seguido do pós-teste de Tukey e apresentados como médias ± EPM. Resultados: Os cardiomiócitos de ratos AD exibiram aumentos na amplitude do transiente de Ca2+ em comparação aos DP. Entretanto, o L-NAME aumentou a amplitude do transiente de Ca2+ somente em ratos DP. Não foram encontradas diferenças na constante de tempo de decaimento do transiente de Ca2+ (t) em cardiomiócitos de ratos com DP e AP, contudo, a administração do L-NAME diminuiu o t em cardiomiócitos em ambos os grupos. cardiomiócitos de ratos AD apresentaram menor amplitude e frequência de Ca2+sparks em comparação ao grupo DP. A administração de L-NAME aumentou a amplitude de Ca2+sparks em cardiomiócitos do grupo AD. Conclusão: O NO modula o transiente de Ca2+ e as sparks de Ca2+ em cardiomiócitos de ratos com diferentes capacidades intrínsecas para o exercício.

ABSTRACT Introduction: The intrinsic capacity to aerobic exercise is associated with cardiac inotropism. On the other hand, the contribution of nitric oxide (NO) as an intracellular messenger on Ca2+ dynamics remains unknown in rats with different intrinsic capacities to exercise. Objective: To evaluate whether NO modulates differently Ca2+ intracellular transient and spontaneous Ca2+ releases (sparks) in cardiomyocytes of rats with different intrinsic capacities to exercise. Methods: Male Wistar rats were selected as standard-performance (SP) and high-performance (HP), according to the exercise capacity until fatigue, assessed through a treadmill progressive stress test. Cardiomyocytes of rats were used to determine Ca2+ intracellular transient and Ca2+ sparks evaluated using confocal microscope. To estimate NO contribution, a NO synthase inhibitor (L-NAME, 100 µM) was used. Data were analyzed through two-way ANOVA followed by Tukey's post hoc test and expressed as means ± SEM. Results: Cardiomyocytes of HP rats exhibited higher Ca2+ transient amplitude compared to SP. However, L-NAME increased Ca2+ transient amplitude only in SP rats. No differences were found in Ca2+ transient decay time constant ( t) in cardiomyocytes of SP and HP rats. However, administration of L-NAME caused reduction of tin cardiomyocytes of both groups. Lower amplitude and frequency of Ca2+ sparks were found in cardiomyocytes of HS rats compared to SP group. Administration of L-NAME increased the amplitude of Ca2+ sparks in cardiomyocytes of the HP group. Conclusion: NO modulates Ca2+ transient and Ca2+ sparks in cardiomyocytes of rats with different intrinsic exercise capacities.

RESUMEN Introducción: La capacidad intrínseca para el ejercicio aeróbico está relacionada con el inotropismo cardiaco. Por otro lado, todavía se desconoce la contribución del óxido nítrico (ON) como mensajero intracelular sobre la dinámica del Ca2+ en ratones con diferentes capacidades intrínsecas para el ejercicio. Objetivo: Evaluar si el ON modula diferencialmente la variación transitoria intracelular de Ca2+ y las liberaciones espontaneas de Ca2+ (sparks) en cardiomiocitos de ratones con diferentes capacidades intrínsecas para el ejercicio. Métodos: Ratones machos Wistar fueron seleccionados como desempeño estándar (DE) y alto desempeño (AD), de acuerdo con la capacidad de ejercicio hasta la fatiga, medida a través del test de fuerza progresiva en la caminadora o cinta eléctrica. Los cardiomiocitos de los ratones fueron utilizados para determinar el tránsito intracelular y sparks de Ca2+ evaluados en microscopio confocal. Para estimar la contribución del ON fue utilizado un inhibidor de síntesis del ON (L-NAME, 100 µM). Los datos fueron analizados a través de un ANOVA two-way seguido de un post-test Tukey y presentados como promedios ± EPM. Resultados: Los cardiomiocitos de ratones AD mostraron aumento en la amplitud de la variación transitoria de Ca2+ en comparación con los DE. Así mismo, el L-NAME incremento la amplitud transitoria de Ca2+ solamente en ratones DE. No se encontraron diferencias en la constante del tiempo de decaimiento de la variación transitoria ( t ) de Ca2+ en cardiomiocitos de ratones DE e AD. Todavía, la administración de L-NAME mostro una reducción en el t en cardiomiocitos de ambos los grupos. Cardiomiocitos de ratones AD presentaron menor amplitud y frecuencia de sparks de Ca2+ en comparación al grupo DE. La administración de L-NAME incrementó la amplitud de sparks de Ca2+ en cardiomiocitos del grupo AD. Conclusión: El ON modula la variación de Ca2+ y sparks de Ca2+ en cardiomiocitos de ratones con diferentes capacidades intrínsecas para el ejercicio.

Effect of N-arachidonoyl-l-serine on human cerebromicrovascular endothelium.

N-arachidonoyl-l-serine (ARA-S) is an endogenous lipid, chemically related to the endocannabinoid, N-arachidonoyl ethanolamine (i.e., anandamide) and with similar physiologic and pathophysiologic functions. Reports indicate that ARA-S possesses vasoactive and neuroprotective properties resembling those of cannabinoids. However, in contrast to cannabinoids, ARA-S binds weakly to its known classical receptors, CB1 and CB2, and is therefore considered to be a 'cannabinoid-like' substance. The originally described ARA-S induced-endothelial-dependent vasorelaxation was not abrogated by CB1, CB2 receptor antagonists or TRPV1 competitive inhibitor. The present report demonstrates that ARA-S enhances the fluorescence staining of both cannabinoid receptors (CB1 and CB2) in human brain endothelial cells (HBEC). This reaction is specific since it was reduced by respective selective receptor antagonist (SR141716A and SR141728A). ARA-S alone or in the presence of ET-1 was shown to alter the cytoskeleton (actin). Both ARA-S stimulated phosphorylation of various kinases (MAPK, Akt, JNK and c-JUN) and alteration of cytoskeleton are mediated via CB1, CB2 and TRPV1 receptors. The findings also showed the involvement of Rho/Rock and PI3/Akt/NO pathways in the ARA-S-induced phosphorylation of kinases and actin reorganization in HBEC. All of the above mentioned ARA-S-induced effects were reduced by the treatment with LY294002 (inhibitor of PI3/Akt kinase), except MAPK kinase. In addition, MAPK, JNK, c-JUN phosphorylation were inhibited by H1152 (inhibitor of Rho/ROCK kinase), except Akt kinase. Furthermore, PI3/Akt pathway was inhibited by pretreatment with l-NAME (inhibitor of NOS). The findings suggest that ARA-S is a modulator of Rho kinase and may play a critical role in the regulation of its activity and subsequent effects on the cytoskeleton and its role in supporting essential cell functions like vasodilation, proliferation and movement.

Effects of L-arginine and L-NAME on ischemia-reperfusion in rat liver

PURPOSE: To evaluated the effects of L-arginine (a NO donor) and L-NAME (Nw-nitro-L-arginine methyl ester - a NOS inhibitor) on ischemia-reperfusion in rat livers. METHODS: One hundred fifty two male Wistar rats were divided into four groups: control (simulated surgery); hepatic IR; pretreatment with L-arginine plus hepatic IR; and L-NAME plus hepatic IR. The hepatocellular damage was evaluated at the first, third and seventh days after the procedures through the alanine-aminotransferase (ALT) and aspartate-aminotransaminase (AST) levels, as well as histopathological features: vascular congestion (VC); steatosis (STE); necrosis (NEC); and inflammatory infiltration (INF). The mortality rate was also evaluated. RESULTS: The pretreatment with L-NAME significantly worsened the AST levels after hepatic IR (p<0.05) at first day and L-arginine demonstrated an attenuating effect on ALT levels at seventh day (p<0.05). Furthermore, the administration of L-arginine was able to reduce the VC and STE in the seventh day after hepatic IR (p<0.05). The analysis of the mortality rates did not demonstrate any difference between the groups. Nevertheless, there was not effect of L-arginine and L-NAME on the mortality of the animals. CONCLUSION: L-arginine/NO pathway has a role in the hepatic IR because the pretreatment with L-arginine partially had attenuated the hepatocellular damage induced by hepatic IR in rats. .

Nitric oxide ineerferes with hypoxia signaling during colonic inflamaation / A interferência do óxido nítrico na sinalização da hipóxia durante a inflamação colônica

Context Intestinal inflammation can induce a local reduction in oxygen levels that triggers an adaptive response centered on the expression of hypoxia-inducible factors (HIFs). Nitric oxide, a well-described inflammatory mediator, may interfere with hypoxia signaling. Objectives We aimed to evaluate the role of nitric oxide in hypoxia signaling during colonic inflammation. Methods Colitis was induced by single (acute) or repeated (reactivated colitis) trinitrobenzenosulfonic acid administration in rats. In addition, one group of rats with reactivated colitis was also treated with Nw-Nitro-L-arginine methyl ester hydrochloride to block nitric oxide synthase. Colitis was assessed by macroscopic score and myeloperoxidase activity in the colon samples. Hypoxia was determined using the oxygen-dependent probe, pimonidazole. The expression of HIF-1α and HIF-induced factors (vascular endothelial growth factor - VEGF and apelin) was assessed using Western blotting. Results The single or repeated administration of trinitrobenzenosulfonic acid to rats induced colitis which was characterized by a high macroscopic score and myeloperoxidase activity. Hypoxia was observed with both protocols. During acute colitis, HIF-1α expression was not increased, but VEGF and apelin were increased. HIF-1α expression was inhibited during reactivated colitis, and VEGF and apelin were not increased. Nw-Nitro-L-arginine methyl ester hydrochloride blockade during reactivated colitis restored HIF-1α, VEGF and apelin expression. Conclusions Nitric oxide could interfere with hypoxia signaling during reactivated colitis inflammation modifying the expression of proteins regulated by HIF-1α. .

Contexto A inflamação intestinal pode induzir uma redução local nos níveis de oxigênio e ativar uma resposta adaptativa relacionada à expressão de fatores induzíveis por hipóxia (HIFs). O óxido nítrico, um mediador inflamatório bem descrito, pode interferir com a sinalização de hipóxia. Objetivos O objetivo foi avaliar o papel do óxido nítrico na sinalização de hipóxia durante a inflamação colônica. Métodos A colite foi induzida em ratos pela administração única (aguda) ou repetida (com reativações) de ácido trinitrobenzenosulfônico. Adicionalmente, um grupo de ratos de colite com reativações foi também tratado com Nw-Nitro-L-arginina metil éster para inibir a óxido nítrico sintase. A colite foi avaliada através do escore macroscópico e da atividade de mieloperoxidase em amostras de cólon. A hipóxia foi determinada usando uma sonda dependente de oxigênio, o pimonidazol. A expressão de HIF-1α e de fatores induzidos pelo HIF (factor de crescimento endotelial vascular - VEGF e apelina) foi avaliada pela técnica de Western blotting. Resultados A administração única ou repetida de ácido trinitrobenzenosulfônico a ratos induziu colite que foi caracterizada por um alto escore macroscópico e alta atividade de mieloperoxidase. Hipóxia foi observada em ambos os protocolos. Durante a colite aguda, a expressão de HIF-1α não aumentou, enquanto a de VEGF e apelina aumentou. A expressão de HIF-1α esteve inibida durante a colite com reativações e, a expressão de VEGF e apelina não se modificou. O bloqueio com Nw-Nitro-L-arginina metil éster durante a colite com reativações restabeleceu a expressão de HIF-1α, VEGF e ...

Berberine protects endothelial progenitor cell from damage of TNF-α via the PI3K/AKT/eNOS signaling pathway.

Endothelial progenitor cells (EPCs) dysfunction is closely correlated with the coronary artery injury induced by Kawasaki disease (KD). The level of tumor necrosis factor-α (TNF-α) elevated significantly in acute phase of KD which can damage the functions of EPCs. The aim of this study was to investigate whether berberine (BBR) can protect EPCs from the inhibition caused by TNF-α via the PI3K (Phosphatidyl Inositol 3-kinase) /AKT (Serine/threonine protein kinase B) /eNOS (endothelial Nitric Oxide synthase) signaling pathway. The cell proliferative ability of EPCs was determined by MTT (methyl thiazolyl tetrazolium) assays. Nitric oxide (NO) level was determined in supernatants. The mRNA level of eNOS, PI3K and AKT were measured by Real Time-Polymerase Chain Reaction (RT-PCR), and the protein levels of eNOS, phospho-eNOS (p-eNOS), Akt, phospho-Akt (p-Akt) and PI3K were analyzed using Western-blot. The results demonstrated that TNF-α inhibits the proliferative ability of EPCs. However, BBR improves the proliferative activity of EPCs inhibited by TNF-α. Blockade of PI3K by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) and blockade of eNOS by l-NAME (NG-Nitroarginine Methyl Ester) attenuates the effect of BBR. BBR can increase the level of PI3K/Akt/eNOS mRNA and the protein level of PI3K, p-Akt, eNOS and p-eNOS, which can be blocked by PI3K inhibitor (LY294002) and eNOS inhibitor (l-NAME). Therefore, we concluded that impaired EPCs proliferation could be reversed by BBR via the PI3K/AKT/eNOS signaling pathway.

AMPA receptor upregulation in the nucleus accumbens shell of cocaine-sensitized rats depends upon S-nitrosylation of stargazin.

Behavioral sensitization to cocaine is associated with increased AMPA receptor (AMPAR) surface expression in the nucleus accumbens (NAc). This upregulation is withdrawal-dependent, as it is not detected on withdrawal day (WD) 1, but is observed on WD7-21. Its underlying mechanisms have not been clearly established. Nitric oxide (NO) regulates AMPAR trafficking in the brain by S-nitrosylation of the AMPAR auxiliary subunit, stargazin, leading to increased AMPAR surface expression. Our goal was to determine if stargazin S-nitrosylation contributes to AMPAR upregulation during sensitization. First, we measured stargazin S-nitrosylation in NAc core and shell subregions on WD14 after 8 daily injections of saline or 15 mg/kg cocaine. Stargazin S-nitrosylation was markedly increased in NAc shell but not core. To determine if this is associated with AMPAR upregulation, rats received 8 cocaine or saline injections followed by twice-daily treatments with vehicle or the nitric oxide synthase inhibitor l-NAME (50 mg/kg) on WD1-6, the time when AMPAR upregulation is developing in cocaine-exposed rats. Cocaine/vehicle rats showed elevated stargazin and GluA1 surface expression on WD7 compared to saline/vehicle rats; the GluA1 increase was more robust in core, while stargazin increased more robustly in shell. These effects of cocaine were attenuated in shell but not core when cocaine injections were followed by l-NAME treatment on WD1-6. Together, these results indicate that elevated S-nitrosylation of stargazin contributes to AMPAR upregulation during sensitization selectively in the NAc shell. It is possible that AMPAR upregulation in core involves a different TARP, γ4, which also upregulates in the NAc of sensitized rats.

Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts; the roles of reactive oxygen and nitrogen species.

Adaptive mechanisms involving upregulation of cytoprotective genes under the control of transcription factors such as Nrf2 exist to protect cells from permanent damage and dysfunction under stress conditions. Here we explore of the hypothesis that Nrf2 activation by reactive oxygen and nitrogen species modulates cytotoxicity during hypoxia (H) with and without reoxygenation (H/R) in H9C2 cardiomyoblasts. Using MnTBap as a cell permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger and L-NAME as an inhibitor of nitric oxide synthase (NOS), we have shown that MnTBap inhibited the cytotoxic effects of hypoxic stress with and without reoxygenation. However, L-NAME only afforded protection during H. Under reoxygenation, conditions, cytotoxicity was increased by the presence of L-NAME. Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R. The increased cytotoxicity and inhibition of Nrf2 activation by the presence of L-NAME during reoxygenation suggests that NOS activity plays an important role in cell survival at least in part via Nrf2-independent pathways. In contrast, O2 (-•) scavenging by MnTBap prevented both toxicity and Nrf2 activation during H and H/R implying that toxicity is largely dependent on O2 (-•).To confirm the importance of Nrf2 for myoblast metabolism, Nrf2 knockdown with siRNA reduced cell survival by 50% during 4 h hypoxia with and without 2 h of reoxygenation and although cellular glutathione (GSH) was depleted during H and H/R, GSH loss was not exacerbated by Nrf2 knockdown. These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage.

The effect of L-arginine and L-NAME on myocardial capillary density in normal rats.

BACKGROUND: This study evaluated the effect of L-arginine (Nitric Oxide (NO) precursor) and L-NG-Nitroarginine Methyl Ester (L-NAME) (NO synthase inhibitor) on myocardial capillary density in normal rats. MATERIALS AND METHODS: EIGHTEEN MALE RATS WERE DIVIDED INTO THREE GROUPS: Group 1: Received L-NAME (10 mg/kg/day; ip), Group 2: Received L-arginine (50 mg/kg/day; ip), and Group 3 (control) received normal saline. After 3 weeks, blood samples were taken and myocardial capillary density was evaluated using immunohistochemistry method. RESULTS: Serum NO concentration in control group was 6.45 ± 0.44 µmol/lit. Treatment of animals with L-arginine increased serum NO concentration (7.90 ± 0.75 vs. 6.45 ± 0.44 µmol/lit, respectively) and L-NAME decreased (4.86 ± 0.40 vs. 6.45 ± 0.44 µmol/lit, respectively) compare to control group. L-arginine significantly increased serum vascular endothelial growth factor (VEGF) concentration (353.01 ± 7.03 vs. 100.5 ± 6.61 pg/ml; P < 0.05), however, did not change myocardial capillary density. CONCLUSION: Although L-arginine alters some serum angiogenic factors, either L-arginine or L-NAME could not improve myocardial capillary density in normal rats.

Agmatine induced NO dependent rat mesenteric artery relaxation and its impairment in salt-sensitive hypertension.

l-Arginine and its decarboxylated product, agmatine are important mediators of NO production and vascular relaxation. However, the underlying mechanisms of their action are not understood. We have investigated the role of arginine and agmatine in resistance vessel relaxation of Sprague-Dawley (SD) and Dahl salt-sensitive hypertensive rats. Second or 3rd-order mesenteric arterioles were cannulated in an organ chamber, pressurized and equilibrated before perfusing intraluminally with agonists. The vessel diameters were measured after mounting on the stage of a microscope fitted with a video camera. The gene expression in Dahl rat vessel homogenates was ascertained by real-time PCR. l-Arginine initiated relaxations (EC50, 5.8±0.7mM; n=9) were inhibited by arginine decarboxylase (ADC) inhibitor, difluoromethylarginine (DFMA) (EC50, 18.3±1.3mM; n=5) suggesting that arginine-induced vessel relaxation was mediated by agmatine formation. Agmatine relaxed the SD rat vessels at significantly lower concentrations (EC50, 138.7±12.1µM; n=22), which was compromised by l-NAME (l-N(G)-nitroarginine methyl ester, an eNOS inhibitor), RX821002 (α-2 AR antagonist) and pertussis toxin (G-protein inhibitor). The agmatine-mediated vessel relaxation from high salt Dahl rats was abolished as compared to that from normal salt rats (EC50, 143.9±23.4µM; n=5). The α-2A AR, α-2B AR and eNOS mRNA expression was downregulated in mesenteric arterioles of high-salt treated Dahl hypertensive rats. These findings demonstrate that agmatine facilitated the relaxation via activation of α-2 adrenergic G-protein coupled receptor and NO synthesis, and this pathway is compromised in salt-sensitive hypertension.

Leptin modulates enteric neurotransmission in the rat proximal colon: an in vitro study.

Leptin has been shown to modulate gastrointestinal functions including nutrient absorption, growth, and inflammation and to display complex effects on gut motility. Leptin receptors have also been identified within the enteric nervous system (ENS), which plays a crucial role in digestive functions. Although leptin has recently been shown to activate neurons in the ENS, the precise mechanisms involved are so far unknown. Therefore, the aim of the present study was to determine the effects of leptin on rat proximal colon smooth muscle and enteric neuron activities. The effects of exogenous leptin on tone and on responses to transmural nerve stimulation (TNS) of isolated circular smooth muscle of proximal colon in rats were investigated using an organ bath technique. The effects of a physiological concentration (0.1 µM) of leptin were also studied on tone and TNS-induced relaxation in the presence of atropine, hexamethonium, L-N(G)-nitroarginine methyl ester (L-NAME) and capsazepine. Leptin caused a slight but significant decrease in tone, TNS-induced relaxation and contraction in a concentration-dependent manner in colonic preparations. Cholinergic antagonists abolished the effects of 0.1 µM leptin on TNS-induced relaxation. This concentration of leptin had no further effect on relaxation in the presence of L-NAME. In the presence of capsazepine, leptin had no further effect either on tone or relaxation compared to the drug alone. In conclusion, leptin modulates the activity of enteric inhibitory and excitatory neurons in proximal colon. These effects may be mediated through nitrergic neurons. Intrinsic primary afferent neurons may be involved.

Effect of L-Arginine and L-NAME treatments on polymorphonuclear leukocytes and mononuclear cells influx during tumor growth / Efeito dos tratamentos com L-Arginina e L-NAME sobre o influxo de leucócitos polimorfonucleares e células mononucleares durante o desenvolvimento tumoral

PURPOSE: Evaluate polymorphonuclear leukocytes (PMN's) and mononuclear cells (MN's) involvement in the Ehrlich´s solid tumor (ET) growth. METHODS: 90 Swiss mice were inoculated with 10(7) tumor cells (sc), distributed in three groups and treated once a day, via intraperitoneal (ip), with 0.1ml of diluent, L-Arginine (20mg/Kg) or L-NAME (20mg/Kg). After 7, 15 and 30 days of treatment, ten animals of each group were euthanized, the tumor mass was removed, processed and fixed for HE. Later, a morphometric analysis of the total area, parenchyma, necrosis, tumor stroma and PMN's leukocytes and MN's cells influx was performed. RESULTS: The L-Arginine treatment increased PMN's influx in the initial stage, whereas L-NAME reduced it. Our data suggests that NO effect on PMN's migration is dose-dependent. On the other hand, the MN´s cells influx was reduced by L-NAME treatment at all evaluated periods and at the same periods an increase in tumor growth was observed. CONCLUSION: At initial stages of tumor implantation, both PMN's leukocytes and MN's cells act together to control ET development.

OBJETIVO: Avaliar o envolvimento de leucócitos polimorfonucleares (PMN's) e células mononucleares (MN's) no crescimento do Tumor Sólido de Ehrlich (TE). MÉTODOS: 90 camundongos Suíços foram inoculados com 10(7) células tumorais (sc), distribuídos em três grupos e tratados uma vez ao dia, via intraperitoneal (ip), com 0.1ml de diluente, L-Arginina (20mg/Kg) ou L-NAME (20mg/Kg). Após 7, 15 e 30 dias, dez animais de cada grupo foram eutanasiados, a massa tumoral foi removida, processada e corada pela HE. Posteriormente, foi realizada análise morfométrica das áreas total, parênquima, necrose, estroma e influxo de leucócitos PMN's e células MN's. RESULTADOS: O tratamento com L-Arginina favoreceu o influxo de PMN's em períodos iniciais, enquanto o tratamento com L-NAME o reduziu. Nosso estudo sugere que o efeito do ON sobre a migração de PMN's é dose-dependente. Por outro lado, o influxo de células MN´s foi contido pelo tratamento com L-NAME em todos os períodos avaliados, mesmos períodos em que se observou um aumento no crescimento tumoral. CONCLUSÃO: Em fases iniciais do implante tumoral, ambos, leucócitos PMN's e células MN's, atuam juntos no controle do desenvolvimento do TE.