Gabriel T. do Vale

Bed nucleus of the stria terminalis NMDA receptors and nitric oxide modulate contextual fear conditioning in rats

ABSTRACT The bed nucleus of the stria terminalis (BNST) modulates anxiety‐like responses, including conditioned emotional responses. Evidence suggests that glutamatergic neurotransmission in the BNST plays a role in the modulation of defensive responses. However, little is known about the involvement of glutamate NMDA receptor activation within the BNST, and its resultant increase in nitric oxide (NO) levels, in the expression of contextual fear conditioning (CFC). We investigated whether the antagonism of NMDA receptors or the reduction of NO levels in the BNST would attenuate behavioral and autonomic responses (i.e. increase in arterial pressure and heart rate, and decrease in tail cutaneous temperature) of rats submitted to a CFC paradigm. Intra‐BNST infusion of AP7, an NMDA receptor antagonist, attenuated both behavioral and autonomic changes induced by CFC. Similar results were observed with NPLA and c‐PTIO, an nNOS inhibitor and an NO scavenger, respectively. A positive correlation between BNST NO levels and the time spent in freezing behavior was also observed for animals submitted to the CFC. These findings indicate that the expression of CFC involves a facilitation of BNST NMDA receptor‐NO signaling. This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’. HIGHLIGHTSBlockade of BNST NMDA receptors attenuated contextual fear conditioning (CFC).Re‐exposure to an aversive context increases NO levels in the BNST.Reduction of NO levels in the BNST attenuated CFC.NMDA receptor antagonist intra‐BNST reduced increased BNST NO levels induced by CFC.NMDA receptor‐NO signaling in the BNST modulates CFC.

Melatonin: antioxidant and modulatory properties in age‐related changes during Trypanosoma cruzi infection

The purpose of this study was to investigate the effects of melatonin on selected biomarkers of innate and humoral immune response as well as the antioxidant/oxidant status (superoxide dismutase—SOD and reduced glutathione levels (GSH) to understand whether age‐related changes would influence the development of acute Trypanosoma cruzi (T. cruzi) infection. Young‐ (5 weeks) and middle‐aged (18 months) Wistar rats were orally treated with melatonin (gavage) (05 mg/kg/day), 9 days after infection. A significant increase in both SOD activity and GSH levels was found in plasma from all middle‐aged melatonin‐treated animals. Melatonin triggered enhanced expression of major histocompatibility class II (MHC‐II) antigens on antigen‐presenting cell (APC) and peritoneal macrophages in all treated animals. High levels of CD4+CD28‐negative T cells (*P<.05) were detected in middle‐aged control animals. Melatonin induced a significant reduction (***P<.001) in CD28‐negative in CD4+ and CD8+ T cells in middle‐aged control animals. Contrarily, the same group displayed upregulated CD4+CD28+T and CD8+CD28+T cells. Melatonin also triggered an upregulation of CD80 and CD86 expression in all young‐treated groups. Significant percentages of B and spleen dendritic cells in middle‐aged infected and treated animals were observed. Our data reveal new features of melatonin action in inhibiting membrane lipid peroxidation, through the reduction in 8‐isoprostane, upregulating the antioxidant defenses and triggering an effective balance in the antioxidant/oxidant status during acute infection. The ability of melatonin to counteract the immune alterations induced by aging added further support to its use as a potential therapeutic target not only for T. cruzi infection but also for other immunocompromised states.

Apocynin reduces blood pressure and restores the proper function of vascular endothelium in SHR.

This study has evaluated how the vascular endothelium of hypertensive rats chronically treated with apocynin affects acetylcholine (ACh), sodium nitroprusside (SNP), and phenylephrine (PE) action on the nitric oxide (NO) signal transduction pathway in endothelial (EC) and vascular smooth muscle cells. Treatment with apocynin significantly reduced the mean arterial pressure in spontaneously hypertensive rats (SHR). In addition, apocynin improved the impaired ACh hypotensive effect on SHR. Although systemic oxidative stress was high in SHR, SHR treated with apocynin and normotensive rats presented similar systemic oxidative stress levels. Endothelium significantly blunted PE contractions in intact aortas of treated SHR. The ACh effect was impaired in resistance arteries and aortas of SHR, but this same effect was improved in treated SHR. The SNP potency was higher in intact resistance arteries of treated SHR than in intact resistance arteries of untreated SHR. NO and calcium concentrations increased, whereas reactive oxygen species levels decreased in EC of treated SHR. Aortas of untreated and treated SHR did not differ in terms of sGC alpha or beta units expression. Aorta of treated SHR expressed higher eNOS levels as compared to aorta of untreated SHR. The study groups did not differ with respect to NOX1, NOXO1, or NOX4 expression. However, treatment with apocynin normalized overexpression of NOX2 and its subunit p47phox in aortas of SHR. Based on all the results presented in this study, we suggest apocynin increases NO biovailability by different mechanisms, restoring the proper function of vascular endothelium in SHR.

Nebivolol prevents ethanol-induced reactive oxygen species generation and lipoperoxidation in the rat kidney by regulating NADPH oxidase activation and expression

Abstract We studied whether the &bgr;1‐adrenergic antagonist nebivolol would prevent ethanol‐induced reactive oxygen species generation and lipoperoxidation in the rat renal cortex. Male Wistar rats were treated with ethanol (20% v/v) for 2 weeks. Nebivolol (10 mg/kg/day; p.o. gavage) prevented both the increase in superoxide anion (O2‐) generation and thiobarbituric acid reactive substances (TBARS) concentration induced by ethanol in the renal cortex. Ethanol decreased nitrate/nitrite (NOx) concentration in the renal cortex, and nebivolol prevented this response. Nebivolol did not affect the reduction of hydrogen peroxide (H2O2) concentration induced by ethanol. Nebivolol prevented the ethanol‐induced increase of catalase (CAT) activity. Both SOD activity and the levels of reduced glutathione (GSH) were not affected by treatment with nebivolol or ethanol. Neither ethanol nor nebivolol affected the expression of Nox1, Nox4, eNOS, nNOS, CAT, Nox organizer 1 (Noxo1), c‐Src, p47phox or superoxide dismutase (SOD) isoforms in the renal cortex. On the other hand, treatment with ethanol increased Nox2 expression, and nebivolol prevented this response. Finally, nebivolol reduced the expression of protein kinase (PK) C&dgr; and Rac1. The major finding of our study is that nebivolol prevented ethanol‐induced reactive oxygen species generation and lipoperoxidation in the kidney by a mechanism that involves reduction on the expression of Nox2, a catalytic subunit of NADPH oxidase. Additionally, we demonstrated that nebivolol reduces NADPH oxidase‐derived reactive oxygen species by decreasing the expression of PKC&dgr; and Rac1, which are important activators of NADPH oxidase.

Ethanol-induced erectile dysfunction and increased expression of pro-inflammatory proteins in the rat cavernosal smooth muscle are mediated by NADPH oxidase-derived reactive oxygen species

Abstract Ethanol consumption is associated with an increased risk of erectile dysfunction (ED), but the molecular mechanisms through which ethanol causes ED remain elusive. Reactive oxygen species are described as mediators of ethanol‐induced cell toxicity/damage in distinctive tissues. The enzyme NADPH oxidase is the main source of reactive oxygen species in the endothelium and vascular smooth muscle cells and ethanol is described to increase NADPH oxidase activation and reactive oxygen species generation. This study evaluated the contribution of NADPH oxidase‐derived reactive oxygen species to ethanol‐induced ED, endothelial dysfunction and production of pro‐inflammatory and redox‐sensitive proteins in the rat cavernosal smooth muscle (CSM). Male Wistar rats were treated with ethanol (20% v/v) or ethanol plus apocynin (30 mg/kg/day; p.o. gavage) for six weeks. Apocynin prevented both the decreased in acetylcholine‐induced relaxation and intracavernosal pressure induced by ethanol. Ethanol increased superoxide anion (O2‐) generation and catalase activity in CSM, and treatment with apocynin prevented these responses. Similarly, apocynin prevented the ethanol‐induced decreased of nitrate/nitrite (NOx), hydrogen peroxide (H2O2) and SOD activity. Treatment with ethanol increased p47phox translocation to the membrane as well as the expression of Nox2, COX‐1, catalase, iNOS, ICAM‐1 and p65. Apocynin prevented the effects of ethanol on protein expression and p47phox translocation. Finally, treatment with ethanol increased both TNF‐&agr; production and neutrophil migration in CSM. The major new finding of this study is that NADPH oxidase‐derived reactive oxygen species play a role on chronic ethanol consumption‐induced ED and endothelial dysfunction in the rat CSM.

Data on the effects of losartan on protein expression, vascular reactivity and antioxidant capacity in the aorta of ethanol-treated rats

We describe the effects of losartan, a selective AT1 receptor antagonist on the alterations induced by treatment with ethanol in the rat aorta. The data shown here are related to the article entitled “Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress” (P. Passaglia, C.S. Ceron, A.S. Mecawi, J. Antunes-Rodrigues, E.B. Coelho, C.R. Tirapelli, 2015) [1]. Here we include new data on the protective effect of losartan against ethanol-induced oxidative stress. Male Wistar rats treated for 2 weeks with ethanol (20%, vol./vol.) exhibited increased aortic production of reactive oxygen species (ROS) and losartan (10 mg/kg/day; p.o. gavage) prevented this response. Ethanol did not alter the expression of eNOS in the rat aorta. Losartan prevented ethanol-induced increase in the aortic expression of nNOS. Neither ethanol nor losartan affected superoxide dismutase (SOD) or catalase (CAT) activities in the rat aorta. Treatment with ethanol increased the contraction induced by phenylephrine in both endothelium-intact and endothelium-denuded aortas and these responses were prevented by losartan. Conversely, neither ethanol nor losartan affected the endothelium-dependent relaxation induced by acetylcholine.

Decreased reactive oxygen species production and NOX1, NOX2, NOX4 expressions contribute to hyporeactivity to phenylephrine in aortas of pregnant SHR

AIMS We determined whether decreased reactive oxygen species (ROS) production in the aorta of pregnant spontaneously hypertensive rats (SHR) resulted in increased nitric oxide (NO) bioavailability and hyporeactivity to phenylephrine (PE). MAIN METHODS Systemic and aortic oxidative stress were measured in pregnant and non-pregnant Wistar rats and SHR. Furthermore, the hypotensive effects of apocynin (30 mg/kg) and Tempol (30 mg/kg) were analyzed. Intact aortic rings of pregnant and non-pregnant rats were stimulated with PE in the absence of or after incubation (30 min) with apocynin (100 μmol/L). The effect of apocynin on the concentrations of NO and ROS were measured in aortic endothelial cells (AEC) using DAF-2DA (10 mmol/L) and DHE (2.5 mmol/L), respectively. Western blotting was performed to analyze eNOS, NOX1, NOX2, NOX4 and SOD expression. ROS production was analyzed by the lucigenin chemiluminescence method. KEY FINDINGS Aortic oxidative stress and ROS concentration in AEC were reduced in pregnant Wistar rats and SHR, when compared to non-pregnant rats. ROS production and NOX1, NOX2 and NOX4 expression in the aortas were decreased in pregnant SHR, but not in pregnant Wistar rats. Increased eNOS expression in aortas and NO concentration in AEC were observed in pregnant Wistar rats and SHR. Apocynin reduced PE-induced vasoconstriction in the aortas of non-pregnant Wistar rats and SHR, and pregnant Wistar rats, but not in the aortas of pregnant SHR. SIGNIFICANCE Taken together, these results suggest that ROS production was decreased in the aortas of pregnant SHR and could contribute to higher NO bioavailability and hyporeactivity to PE in the aortas of pregnant SHR.

Pharmacological characterization of the mechanisms underlying the vascular effects of succinate.

We investigated the mechanisms underlying the vascular effects of succinate. Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats and C57BL/6 wild type (WT) or GPR91(-/-) mice. Nitrate/nitrite (NOx) was measured colorimetrically whereas 6-keto-prostaglandin F1α (stable product of prostacyclin) was measured by enzyme immunoassay (EIA). Phosphorylation of endothelial nitric oxide synthase (eNOS) was assessed by western immunoblotting. Functional assays revealed that the direct effect of succinate in the vasculature is biphasic. At lower concentrations succinate induced relaxation while at higher concentrations succinate induced vascular contraction. Succinate concentration dependently relaxed rat aortic rings with intact endothelium. Endothelial removal reduced, but not abolished succinate-induced relaxation. Similarly, succinate relaxed endothelium-intact and endothelium-denuded aortas isolated from both C57BL/6 and GPR91(-/-) mice. Pre-incubation of endothelium-intact, but not endothelium-denuded rat aortic rings with l-NAME, indomethacin and tetraethylammonium (TEA) reduced succinate-induced relaxation. In endothelium-intact rings, succinate-induced relaxation was attenuated by ODQ, haemoglobin, Rp-8-Br-Pet-cGMPS, thapsigargin, wortmannin and SC-560. Blockade of K(+) channels with 4-aminopyridine, apamin and charybdotoxin reduced succinate-induced relaxation. Succinate increased the concentration of NOx and 6-keto-prostaglandin F1α as well as eNOS phosphorylation at ser(1177) residue. CaCl2-induced contraction of endothelium-intact or endothelium-denuded aortas was not affected by succinate. The major finding of our study is that it first demonstrates a direct effect of succinate in the vasculature. Succinate displays a biphasic and concentration-dependent effect. The vascular relaxation induced by succinate is partially mediated by endothelial GPR91 receptors via the NO-cGMP pathway, a vasodilator cyclooxygenase (COX) product(s) and the opening of K(+) channels.

Acute Ethanol Intake Induces NAD(P)H Oxidase Activation and Rhoa Translocation in Resistance Arteries.

Background The mechanism underlying the vascular dysfunction induced by ethanol is not totally understood. Identification of biochemical/molecular mechanisms that could explain such effects is warranted. Objective To investigate whether acute ethanol intake activates the vascular RhoA/Rho kinase pathway in resistance arteries and the role of NAD(P)H oxidase-derived reactive oxygen species (ROS) on such response. We also evaluated the requirement of p47phox translocation for ethanol-induced NAD(P)H oxidase activation. Methods Male Wistar rats were orally treated with ethanol (1g/kg, p.o. gavage) or water (control). Some rats were treated with vitamin C (250 mg/kg, p.o. gavage, 5 days) before administration of water or ethanol. The mesenteric arterial bed (MAB) was collected 30 min after ethanol administration. Results Vitamin C prevented ethanol-induced increase in superoxide anion (O2-) generation and lipoperoxidation in the MAB. Catalase and superoxide dismutase activities and the reduced glutathione, nitrate and hydrogen peroxide (H2O2) levels were not affected by ethanol. Vitamin C and 4-methylpyrazole prevented the increase on O2- generation induced by ethanol in cultured MAB vascular smooth muscle cells. Ethanol had no effect on phosphorylation levels of protein kinase B (Akt) and eNOS (Ser1177 or Thr495 residues) or MAB vascular reactivity. Vitamin C prevented ethanol-induced increase in the membrane: cytosol fraction ratio of p47phox and RhoA expression in the rat MAB. Conclusion Acute ethanol intake induces activation of the RhoA/Rho kinase pathway by a mechanism that involves ROS generation. In resistance arteries, ethanol activates NAD(P)H oxidase by inducing p47phox translocation by a redox-sensitive mechanism.

Ageing is not associated with an altered immune response during Trypanosoma cruzi infection: Ageing and Trypanosoma cruzi infection

Abstract The aims of this work were to evaluate the influence of ageing on the magnitude of the immune response in male Wistar rats infected with the Y strain of Trypanosoma cruzi (T. cruzi). Infected young animals displayed enhanced CD4+ T cells as compared to uninfected counterparts. Ageing also triggered a significant reduction in CD8+ T cells compared to young and uninfected groups. The percentage of spleen NKT cells was reduced for all groups, regardless of the infection status. Significant decreased B‐cells was noted in aged controls and infected animals as compared to young counterparts. A significant decrease in MHC class II (RT1B) expression in all aged animals was observed, whether infected or not. The highest and significant levels of Thiobarbituric Acid Reactive Substances (TBARS) were noted in the aged and infected animals as compared to young‐infected ones (16 day). Consequently superoxide dismutase (SOD) activity was reduced for both control and infected aged animals. Significant elevation of 8‐isoprostane levels was found in aged control and infected animals. Plasma glutathione (GSH) concentration was reduced in aged control animals, as well as, in the young infected animals. NO production was increased in both infected and uninfected aged animals compared to young infected and uninfected animals. Corticosterone levels were elevated in aged animals, whether infected or not. Thus, our results are inedited since the immune response is not worsened by the simple fact of animals being older. Ageing by itself triggered a damaged immune response as well as enhanced reactive oxygen species, when compared to young counterparts, but it did not contribute to impair the immune response of T. cruzi infected and aged rats. HighlightsWe have investigated the influence of ageing on the magnitude of the immune responseThe highest and significant levels of TBARS (Thiobarbituric Acid Reactive Substances) and 8‐Isoprostane were observed in the aged and infected animals as compared to young counterpartsSuperoxide dismutase (SOD) activity and Glutathione (GSH) concentration was reduced in control aged animals as compared to young onesCorticosterone and NO levels were significantly higher in aged and infected animals than in young ratsOur present results demonstrated that senescence by itself triggered a damaged immune response as well as enhanced reactive oxygen species, but it did not contribute to impair the immune response of T. cruzi infected and aged rats.