Janaina A. Simplicio

Contribution of oxidative stress and prostanoids in endothelial dysfunction induced by chronic fluoxetine treatment.

OBJECTIVES The effects of chronic fluoxetine treatment were investigated on blood pressure and on vascular reactivity in the isolated rat aorta. METHODS AND RESULTS Male Wistar rats were treated with fluoxetine (10 mg/kg/day) for 21 days. Fluoxetine increased systolic blood pressure. Chronic, but not acute, fluoxetine treatment increased the contractile response induced by phenylephrine, serotonin (5-HT) and KCl in endothelium-intact rat aortas. L-NAME and ODQ did not alter the contraction induced by phenylephrine and 5-HT in aortic rings from fluoxetine-treated rats. Tiron, SC-560 and AH6809 reversed the increase in the contractile response to phenylephrine and 5-HT in aortas from fluoxetine-treated rats. Fluoxetine treatment increased superoxide anion generation (O2(-)) and the expression of cyclooxygenase (COX)-1 in the rat aorta. Reduced expression of nNOS, but not eNOS or iNOS was observed in animals treated with fluoxetine. Fluoxetine treatment increased prostaglandin (PG)F2α levels but did not affect thromboxane (TX)B2 levels in the rat aorta. Reduced hydrogen peroxide (H2O2) levels and increased catalase (CAT) activity were observed after treatment. CONCLUSIONS The major new finding of our study is that chronic fluoxetine treatment induces endothelial dysfunction, which alters vascular responsiveness by a mechanism that involves increased oxidative stress and the generation of a COX-derived vasoconstrictor prostanoid (PGF2α). Moreover, our results evidenced a relation between the period of treatment with fluoxetine and the magnitude in the increment of blood pressure. Finally, our findings raise the possibility that fluoxetine treatment increases the risk for vascular injury, a response that could predisposes to cardiovascular diseases.

Vitamin C prevents the endothelial dysfunction induced by acute ethanol intake

AIMS Investigate the effect of ascorbic acid (vitamin C) on the endothelial dysfunction induced by acute ethanol intake. MAIN METHODS Ethanol (1g/kg; p.o. gavage) effects were assessed within 30min in male Wistar rats. KEY FINDINGS Ethanol intake decreased the endothelium-dependent relaxation induced by acetylcholine in the rat aorta and treatment with vitamin C (250mg/kg; p.o. gavage, 5days) prevented this response. Ethanol increased superoxide anion (O2(-)) generation and decreased aortic nitrate/nitrite levels and these responses were not prevented by vitamin C. Superoxide dismutase (SOD) and catalase (CAT) activities as well as hydrogen peroxide (H2O2) and reduced glutathione (GSH) levels were not affected by ethanol. RhoA translocation as well as the phosphorylation levels of protein kinase B (Akt), eNOS (Ser(1177) or Thr(495) residues), p38MAPK, SAPK/JNK and ERK1/2 was not affected by ethanol intake. Vitamin C increased SOD activity and phosphorylation of Akt, eNOS (Ser(1177) residue) and p38MAPK in aortas from both control and ethanol-treated rats. Incubation of aortas with tempol prevented ethanol-induced decrease in the relaxation induced by acetylcholine. Ethanol (50mM/1min) increased O2(-) generation in cultured aortic vascular smooth muscle cells (VSMC) and vitamin C did not prevent this response. In endothelial cells, vitamin C prevented the increase on ROS generation and the decrease in the cytosolic NO content induced by ethanol. SIGNIFICANCE Our study provides novel evidence that vitamin C prevents the endothelial dysfunction induced by acute ethanol intake by a mechanism that involves reduced ROS generation and increased NO availability in endothelial cells.

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.

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.

Mechanisms underlying sodium nitroprusside-induced tolerance in the mouse aorta: Role of ROS and cyclooxygenase-derived prostanoids

Aims: To determine the role of reactive oxygen species (ROS) on sodium nitroprusside (SNP)‐induced tolerance. Additionally, we evaluated the role of ROS on NF‐&kgr;B activation and pro‐inflammatory cytokines production during SNP‐induced tolerance. Main methods: To induce in vitro tolerance, endothelium‐intact or ‐denuded aortic rings isolated from male Balb‐c mice were incubated for 15, 30, 45 or 60 min with SNP (10 nmol/L). Key findings: Tolerance to SNP was observed after incubation of endothelium‐denuded, but not endothelium‐intact aortas for 60 min with this inorganic nitrate. Pre‐incubation of denuded rings with tiron (superoxide anion (O2−) scavenger), and the NADPH oxidase inhibitors apocynin and atorvastatin reversed SNP‐induced tolerance. L‐NAME (non‐selective NOS inhibitor) and L‐arginine (NOS substrate) also prevented SNP‐induced tolerance. Similarly, ibuprofen (non‐selective cyclooxygenase (COX) inhibitor), nimesulide (selective COX‐2 inhibitor), AH6809 (prostaglandin PGF2&agr; receptor antagonist) or SQ29584 [PGH2/thromboxane TXA2 receptor antagonist] reversed SNP‐induced tolerance. Increased ROS generation was detected in tolerant arteries and both tiron and atorvastatin reversed this response. Tiron prevented tolerance‐induced increase on O2– and hydrogen peroxide (H2O2) levels. The increase onp65/NF‐&kgr;B expression and TNF‐&agr; production in tolerant arteries was prevented by tiron. The major new finding of our study is that SNP‐induced tolerance is mediated by NADPH‐oxidase derived ROS and vasoconstrictor prostanoids derived from COX‐2, which are capable of reducing the vasorelaxation induced by SNP. Additionally, we found that ROS mediate the activation of NF‐&kgr;B and the production of TNF‐&agr; in tolerant arteries. Significance: These findings identify putative molecular mechanisms whereby SNP induces tolerance in the vasculature.

The Labdane Ent-3-Acetoxy-Labda-8(17), 13-Dien-15-Oic Decreases Blood Pressure In Hypertensive Rats

Background Labdane-type diterpenes induce lower blood pressure via relaxation of vascular smooth muscle; however, there are no studies describing the effects of labdanes in hypertensive rats. Objective The present study was designed to investigate the cardiovascular actions of the labdane-type diterpene ent-3-acetoxy-labda-8(17), 13-dien-15-oic acid (labda-15-oic acid) in two-kidney 1 clip (2K-1C) renal hypertension. Methods Vascular reactivity experiments were performed in aortic rings isolated from 2K-1C and normotensive (2K) male Wistar rats. Nitrate/nitrite (NOx) measurement was performed in aortas by colorimetric assay. Blood pressure measurements were performed in conscious rats. Results Labda-15-oic acid (0.1-300 µmol/l) and forskolin (0.1 nmol/l - 1 µmol/l) relaxed endothelium-intact and endothelium-denuded aortas from both 2K-1C and 2K rats. Labda-15-oic acid was more effective at inducing relaxation in endothelium-intact aortas from 2K pre-contracted with phenylephrine when compared to the endothelium-denuded ones. Forskolin was more potent than labda-15-oic acid at inducing vascular relaxation in arteries from both 2K and 2K-1C rats. Labda-15-oic acid-induced increase in NOx levels was lower in arteries from 2K-1C rats when compared to 2K rats. Intravenous administration of labda-15-oic acid (0.3-3 mg/kg) or forskolin (0.1-1 mg/kg) induced hypotension in conscious 2K-1C and 2K rats. Conclusion The present findings show that labda-15-oic acid induces vascular relaxation and hypotension in hypertensive rats.

Chronic ethanol consumption increases vascular oxidative stress and the mortality induced by sub-lethal sepsis: Potential role of iNOS

Abstract We hypothesized that long‐term ethanol consumption would increase the mortality and aggravate the deleterious effects of sub‐lethal cecal ligation and puncture (SL‐CLP) in the vasculature by inducing the expression of inducible nitric oxide (NO) synthase (iNOS). Male C57BL/6J wild‐type (WT) or iNOS‐deficient mice (iNOS‐/‐) were treated with ethanol (20% v/v) for 12 weeks and then subjected to SL‐CLP. Mice were killed 24 h post‐operatively or followed six days for survival. Septic ethanol‐treated mice showed a higher mortality than septic WT mice. However, septic iNOS‐deficient mice treated with ethanol showed a decreased mortality rate when compared to ethanol‐treated WT mice. Ethanol and SL‐CLP augmented superoxide anion (O2‐) generation in the mesenteric arterial bed (MAB) of both WT and iNOS‐deficient mice. Treatment with ethanol and SL‐CLP enhanced lipoperoxidation in the MAB of WT, but not iNOS‐deficient mice. SL‐CLP enhanced nitrate/nitrite (NOx) concentrations in the MAB of WT, but not iNOS‐deficient mice. Both, ethanol and SL‐CLP increased TNF‐&agr; and IL‐6 levels in the MAB. Treatment with ethanol as well as SL‐CLP up‐regulated the expression of iNOS in the MAB of WT mice. The major finding of our study is that chronic ethanol consumption increases the mortality induced by SL‐CLP and that iNOS plays a role in such response. Although ethanol led to vascular alterations, it did not aggravate the vascular injury induced by SL‐CLP. Finally, iNOS mediated the increase in oxidative stress and pro‐inflammatory cytokines induced by SL‐CLP in the vasculature.