Role of glutamate NMDA receptors and nitric oxide located within the periaqueductal gray on defensive behaviors in mice confronted by predator

The midbrain periaqueductal gray (PAG) is part of the brain system involved in active defense reactions to threatening stimuli. Glutamate N-methyl-d-aspartate (NMDA) receptor activation within the dorsal column of the PAG (dPAG) leads to autonomic and behavioral responses characterized as the fear reaction. Nitric oxide (NO) has been proposed to be a mediator of the aversive action of glutamate, since the activation of NMDA receptors in the brain increases NO synthesis. We investigated the effects of intra-dPAG infusions of NMDA on defensive behaviors in mice pretreated with a neuronal nitric oxide synthase (nNOS) inhibitor [N omega-propyl-l-arginine (NPLA)], in the same midbrain site, during a confrontation with a predator in the rat exposure test (RET). Male Swiss mice received intra-dPAG injections of NPLA (0.1 or 0.4 nmol/0.1 mu l), and 10 min later, they were infused with NMDA (0.04 nmol/0.1 mu l) into the dPAG. After 10 min, each mouse was placed in the RET. NMDA treatment enhanced avoidance behavior from the predator and markedly increased freezing behavior. These proaversive effects of NMDA were prevented by prior injection of NPLA. Furthermore, defensive behaviors (e.g., avoidance, risk assessment, freezing) were consistently reduced by the highest dose of NPLA alone, suggesting an intrinsic effect of nitric oxide on defensive behavior in mice exposed to the RET. These findings suggest a potential role of glutamate NMDA receptors and NO in the dPAG in the regulation of defensive behaviors in mice during a confrontation with a predator in the RET.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Inhibition of in vivo leishmanicidal mechanisms by tempol: Nitric oxide down-regulation and oxidant scavenging

Tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) has long been known to protect experimental animals from the injury associated with oxidative and inflammatory conditions. In the latter case, a parallel decrease in tissue protein nitration levels has been observed. Protein nitration represents a shift in nitric oxide actions from physiological to pathophysiological and potentially damaging pathways involving its derived oxidants such as nitrogen dioxide and peroxynitrite. In infectious diseases, protein tyrosine nitration of tissues and cells has been taken as evidence for the involvement of nitric oxide-derived oxidants in microbicidal mechanisms. To examine whether tempol inhibits the microbicidal action of macrophages, we investigated its effects on Leishmania amazonensis infection in vitro (RAW 264.7 murine macrophages) and in vivo (C57B1/6 mice). Tempol was administered in the drinking water at 2 mM throughout the experiments and shown to reach infected footpads as the nitroxide plus the hydroxylamine derivative by EPR analysis. At the time of maximum infection (6 weeks), tempol increased footpad lesion size (120%) and parasite burden (150%). In lesion extracts, tempol decreased overall nitric oxide products and expression of inducible nitric oxide synthase to about 80% of the levels in control animals. Nitric oxide-derived products produced by radical mechanisms, such as 3-nitrotyrosine and nitrosothiol, decreased to about 40% of the levels in control mice. The results indicate that tempol worsened L. amazonensis infection by a dual mechanism involving down-regulation of iNOS expression and scavenging of nitric oxide-derived oxidants. Thus, the development of therapeutic strategies based on nitroxides should take into account the potential risk of altering host resistance to parasite infection. (c) 2008 Elsevier Inc. All rights reserved.

Stimulation of Acidic Reduction of Nitrite to Nitric Oxide by Soybean Phenolics: Possible Relevance to Gastrointestinal Host Defense

This study aimed to evaluate the potential of soybean-promoted acidic nitrite reduction and to correlate this activity with the content of phenolics and with the bactericidal activity against Escherichia coli O157:H7. Extracts of embrionary axes and cotyledons enriched in phenolics increased (center dot)NO formation at acidic pH at values that were 7.1 and 4.5 times higher, respectively, when compared to the reduction of the nonenriched extracts. Among the various phenolics accumulated in the soybean extracts, five stimulated nitrite reduction in the following decreasing order of potency: epicatechin gallate, chlorogenic acid, caffeic acid, galic acid and p-coumaric acid. Extracts of embrionary axes presented higher contents of epicatechin gallate and caffeic acid, compared to that of cotyledons, indicating a positive correlation between activity of the extracts and content of phenolics with regard to nitrite reducing activity. Soybean extracts enriched in phenolics interacted synergistically with acidified nitrite to prevent E. coli O157:H7 growth. The results suggest that soybean phenolics may interfere with the metabolism of (center dot)NO in an acidic environment by accelerating the reduction of nitrite, with a potential antimicrobial effect in the stomach.

Nitric oxide sensing by cytochrome c bonded to a conducting polymer modified glassy carbon electrode

A nitric oxide biosensor based on cytochrome c (an heme protein) covalently immobilized to poly(5-amino-1-naphthol) by using cyanuric chloride as a bridge was developed. The immobilization was studied by cyclic voltammetry and quartz crystal microbalance. The nitric oxide detection as a function of poly(5-amino-1-naphthol) amount was recorded, and the best result was obtained with the electrode prepared by 70 cycles. The sensitivity and detection limit were 0.015 mu A cm(-2)/mu mol L(-1) and 2.85 mu mol L(-1), respectively. (C) 2009 Elsevier B.V. All rights reserved.

Study of a gold electrode modified by trans-[Ru(NH(3))(4)(Ist)SO(4)](+) to produce an electrochemical sensor for nitric oxide

The modification of a gold electrode surface by electropolymerization of trans-[Ru(NH(3))(4)(Ist)SO(4)](+) to produce an electrochemical sensor for nitric oxide was investigated. The influence of dopamine, serotonin and nitrite as interferents for NO detection was also examined using square-wave voltammetry (SWV). The characterization of the modified electrode was carried out by cyclic voltammetry, electrochemical quartz crystal microbalance (EQCM) and SERS techniques. The gold electrode was successfully modified by the trans-[Ru(NH(3))(4)(Ist)SO(4)](+) complex ion using cyclic voltammetry. The experiments show that a monolayer of the film is achieved after ten voltammetric cycles, that NO in solution can coordinate to the metal present in the layer, that dopamine, serotonin and nitrite are interferents for the detection of NO, and that the response for the nitrite is much less significant than the responses for dopamine and serotonin. The proposed modified electrode has the potential to be applied as a sensor for NO. (C) 2011 Elsevier Ltd. All rights reserved.

Experimental Chemotherapy against Trypanosoma cruzi Infection Using Ruthenium Nitric Oxide Donors

The ruthenium NO donors of the group trans-[Ru(NO)(NH(3))(4)L](n+), where the ligand (L) is N-heterocyclic H(2)O, SO(3)(2 -), or triethyl phosphite, are able to lyse Trypanosoma cruzi in vitro and in vivo. Using half-maximal (50%) inhibitory concentrations against bloodstream trypomastigotes (IC(50)(try)) and cytotoxicity data on mammalian V-79 cells (IC(50)(V79)), the in vitro therapeutic indices (TIs) (IC(50)(V79)/IC(50)(try)) for these compounds were calculated. Compounds that exhibited an in vitro TI of >= 10 and trypanocidal activity against both epimastigotes and trypomastigotes with an IC(50)(try/epi) of <= 100 mu M were assayed in a mouse model for acute Chagas` disease, using two different routes (intraperitoneal and oral) for drug administration. A dose-effect relationship was observed, and from that, the ideal dose of 400 nmol/kg of body weight for both trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) (isn, isonicotinamide) and trans-[Ru(NO)(NH3) 4imN](BF4) 3 (imN, imidazole) and median (50%) effective doses (ED50) of 86 and 190 nmol/kg, respectively, were then calculated. Since the 50% lethal doses (LD(50)) for both compounds are higher than 125 mu mol/kg, the in vivo TIs (LD(50)/ED(50)) of the compounds are 1,453 for trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) and 658 for trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3). Although these compounds exhibit a marked trypanocidal activity and are able to react with cysteine, they exhibit very low activity in T. cruzi -glycosomal glyceraldehyde-3-phosphate dehydrogenase tests, suggesting that this enzyme is not their target. The trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) and trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) compounds are able to eliminate amastigote nests in myocardium tissue at 400-nmol/kg doses and ensure the survival of all infected mice, thus opening a novel set of therapies to try against trypanosomatids.

Role of brain nitric oxide in the thermoregulation of broiler chicks

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The role of nitric oxide in the regulation of the systemic and pulmonary vasculature of the rattlesnake, Crotalus durissus terrificus

The functional role of nitric oxide (NO) was investigated in the systemic and pulmonary circulations of the South American rattlesnake, Crotalus durissus terrificus. Bolus, intra-arterial injections of the NO donor, sodium nitroprusside (SNP) caused a significant systemic vasodilatation resulting in a reduction in systemic resistance (Rsys). This response was accompanied by a significant decrease in systemic pressure and a rise in systemic blood flow. Pulmonary resistance (Rpul) remained constant while pulmonary pressure (Ppul) and pulmonary blood flow (Qpul) decreased. Injection of L-Arginine (L-Arg) produced a similar response to SNP in the systemic circulation, inducing an immediate systemic vasodilatation, while Rpul was unaffected. Blockade of NO synthesis via the nitric oxide synthase inhibitor, L-NAME, did not affect haemodynamic variables in the systemic circulation, indicating a small contribution of NO to the basal regulation of systemic vascular resistance. Similarly, Rpul and Qpul remained unchanged, although there was a significant rise in Ppul. Via injection of SNP, this study clearly demonstrates that NO causes a systemic vasodilatation in the rattlesnake, indicating that NO may contribute in the regulation of systemic vascular resistance. In contrast, the pulmonary vasculature seems far less responsive to NO.

Involvement of nitric oxide in the gastroprotective effects of an aqueous extract of Pfaffia glomerata (Spreng) Pedersen, Amaranthaceae, in rats

The plants belonging to Pfaffia genus are used in folk medicine to treat gastric disturbances. This study examined the effects of an aqueous extract of Pfaffia glomerata (Spreng) Pedersen (AEP) on the gastrointestinal tract. Wistar rats were pretreated orally (p.o.) with the AEP (125, 250, 500 and 1000 mg.kg(-1)) before induction of ulcers by hypothermic restraint stress (HRS, 3 h restraint stress at 4 degreesC), ethanol (ET, 70%; 0.5 ml/animal; p.o.) or indomethacin (IND, 20 mg.kg(-1); s.c.). Control animals received water (C) or ranitidine (60 mg.kg(-1)) p.o. The AEP protected rats against HRS and ET-induced ulcers, but was not able to protect the gastric mucosa against IND-induced ulcers. When injected into the duodenal lumen, the AEP reduced total acidity and both basal and histamine-stimulated acid secretion in pylorus-ligated rats. In addition, gastric secretion from AEP-treated animals exhibited increased concentrations of nitrite and nitrate. Treatment of animals with L-NAME (120 mg.kg(-1), p.o.) prevented both the reduction of total acidity and the increase in NO, levels promoted by AEP treatment. In conclusion, AEP effectively protected the gastric mucosa and inhibited gastric acid secretion in rats, probably by involving the histaminergic pathway and an enhanced production of nitric oxide in the stomach. (C) 2003 Elsevier B.V. All rights reserved.

Nitric oxide release using natural rubber latex as matrix

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sporothrix schenckii lipid inhibits macrophage phagocytosis: Involvement of nitric oxide and tumour necrosis factor-alpha

The role of cell-wall compounds in the immune response to sporotrichosis is unknown. The effect of cell-wall compounds and exoantigen obtained from Sporothrix schenckii in macrophage/fungus interaction was analysed with respect to nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha). The lipid compound of the cell wall plays an important role in the pathogenesis of this mycosis and was found to inhibit the phagocytic process and to induce high liberation of NO and TNF-alpha in macrophage cultures in the present study. This is a very interesting result because it is the first report about one compound of the fungus S. schenckii that presents this activity.

Changes in sodium, potassium-ATPase induced by repeated fencamfamine: the roles of cyclic AMP-dependent protein kinase and the nitric oxide-cyclic GMP pathway

Fencamfamine (FCF) is an indirect dopamine agent with effects similar to amphetamine and cocaine. In the present study, we investigate changes in Na,K-ATPase, cyclic AMP-dependent protein kinase (PKA) and nitric oxide synthase (NOS) activity and cyclic GMP levels in the nucleus accumbens (NAc) and striatum (ST) of animals acutely or repeatedly treated with FCF (3.5 mg/kg). Na,K-ATPase had a similar activity in control and repeatedly treated animals, but was reduced in the NAc of the acute group. This enzyme was reduced in the ST in acute and repeatedly treated animals, compared to the control group. Expression of the alpha(1,2,3)-Na,K-ATPase isoforms in the NAc and the ST was not altered in all groups studied. Acute FCF induced a significant increase in PKA activity in both the ST and the NAc. Repeatedly treated animals showed a higher increase in PKA activity in the NAc, but not in the ST, when compared to the acute group. There was also an increase in both NOS activity and cyclic GMP levels only in the NAc of FCF repeatedly treated animals compared to the acute and control groups. We suggest that chronic FCF treatment is linked to a modification in Na,K-ATPase activity through the PKA and NO-cyclic GMP pathway. (C) 2003 Elsevier Ltd. All rights reserved.