Treatment with nitrite prevents reactive oxygen species generation in the corpora cavernosa and restores intracavernosal pressure in hypertensive rats.

Hypertension is a risk factor for erectile dysfunction (ED) and both conditions are associated with oxidative stress. Given that nitrite is described to display antioxidant effects, we hypothesized that treatment with nitrite would exert antioxidant effects attenuating both reactive oxygen species (ROS) generation in the corpora cavernosa (CC) and ED induced by hypertension. Two kidney, one clip (2K1C) hypertension was induced in male Wistar rats. Treatment with sodium nitrite (15 mg/kg/day, p.o., gavage) was initiated two weeks after surgery to induce hypertension and maintained for four weeks. Nitrite abrogated both the decrease in intracavernosal pressure and endothelial dysfunction of the CC induced by hypertension. Treatment with nitrite decreased hypertension-induced ROS generation in the CC assessed in situ using the fluorescent dye dihidroethidium (DHE) and with the lucigenin assay. Western immunoblotting analysis revealed that nitrite prevented the increase in Nox1 expression in the CC from 2K1C rats. Decreased concentrations of hydrogen peroxide (H2O2) were found in the CC from hypertensive rats and treatment with nitrite prevented this response. Treatment with nitrite increased the fluorescence of DAF-2DA in the CC from sham-operated rats and restored nitric oxide (NO) levels in the CC from 2K1C rats. In summary, we found novel evidence that nitrite reversed the decrease in intracavernosal pressure induced by 2K1C hypertension. This response was partially attributed to the antioxidant effect of nitrite that blunted ROS generation and endothelial dysfunction in the CC. In addition, nitrite-derived NO may have promoted direct protective actions against hypertension-induced CC dysfunction.

Dysregulated mitogen-activated protein kinase and matrix metalloproteinase in ethanol-induced cavernosal dysfunction.

We evaluated the effects of ethanol consumption on the mitogen-activated protein kinases (MAPK) and metalloproteinases (MMP) pathways in the rat cavernosal smooth muscle (CSM). Male Wistar rats were treated with ethanol (20% v/v) for 6 weeks. Quantitative real-time polymerase chain reaction experiments showed that ethanol consumption did not alter mRNA levels of p38MAPK, SAPK/JNK, ERK1/2, MMP-2, or MMP-9 in the rat CSM. Western immunoblotting experiments revealed decreased protein expression of p38MAPK and phosphorylation of SAPK/JNK in the CSM from ethanol-treated rats. Additionally, ethanol consumption decreased the expression of MMP-2. Functional assays showed that SP600125, an inhibitor of SAPK/JNK, prevented the increase in endothelin (ET)-1-induced contraction in the CSM from ethanol-treated rats. Treatment with ethanol decreased MMP-2 activity, but did not change net MMP activity in the rat CSM. Ethanol consumption increased the circulating levels of MMP-2, MMP-9, and TIMP-2 as well as the MMP-9/TIMP-1 ratio. The major finding of our study is that ethanol consumption down-regulates both MAPK and MMP pathways in the rat CSM, whereas it increases the circulating levels of MMP-9. Additionally, we found that SAPK/JNK plays a role in ethanol-induced increase on ET-1 contraction in the isolated rat CSM.

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.

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 (30mg/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-α 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.

Reactive oxygen species derived from NAD(P)H oxidase play a role on ethanol-induced hypertension and endothelial dysfunction in rat resistance arteries.

Chronic ethanol consumption is a risk factor for cardiovascular diseases. We studied whether NAD(P)H oxidase-derived reactive oxygen species (ROS) play a role in ethanol-induced hypertension, vascular dysfunction, and protein expression in resistance arteries. Male Wistar rats were treated with ethanol (20 % v/v) for 6 weeks. Ethanol treatment increased blood pressure and decreased acetylcholine-induced relaxation in the rat mesenteric arterial bed (MAB). These responses were attenuated by apocynin (30 mg/kg/day; p.o. gavage). Ethanol consumption increased superoxide anion (O2-) generation and decreased nitrate/nitrite (NO x ) concentration in the rat MAB and apocynin prevented these responses. Conversely, ethanol did not affect the concentration of hydrogen peroxide (H2O2) and reduced glutathione (GSH) or the activity of superoxide dismutase (SOD) and catalase (CAT) in the rat MAB. Ethanol increased interleukin (IL)-10 levels in the rat MAB but did not affect the levels of tumor necrosis factor (TNF)-α, IL-6, or IL-1ß. Ethanol increased the expression of Nox2 and the phosphorylation of SAPK/JNK, but reduced eNOS expression in the rat MAB. Apocynin prevented these responses. However, ethanol treatment did not affect the expression of Nox1, Nox4, p38MAPK, ERK1/2, or SAPK/JNK in the rat MAB. Ethanol increased plasma levels of TBARS, TNF-α, IL-6, IL-1ß, and IL-10, whereas it decreased NO x levels. The major finding of our study is that NAD(P)H oxidase-derived ROS play a role on ethanol-induced hypertension and endothelial dysfunction in resistance arteries. Moreover, ethanol consumption affects the expression and phosphorylation of proteins that regulate vascular function and NAD(P)H oxidase-derived ROS play a role in such responses.

Data on the mechanisms underlying succinate-induced aortic contraction.

We describe the mechanisms underlying the vascular contraction induced by succinate. The data presented here are related to the article entitled "Pharmacological characterization of the mechanisms underlying the vascular effects of succinate" (L.N. Leite, N.A. Gonzaga, J.A. Simplicio, G.T. Vale, J.M. Carballido, J.C. Alves-Filho, C.R. Tirapelli, 2016) [1]. Succinate acts as a signaling molecule by binding to a G-protein-coupled receptor termed GPR91, "Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors" (W. He, F.J. Miao, D.C. Lin, R.T. Schwandner, Z. Wang, J. Gao, J.L. Chen, H. Tian, L. Ling, 2004) [2]. Here we include data on the contractile effect of succinate in the aorta. Succinate contracted both endothelium-intact and endothelium-denuded aortic rings isolated from male Wistar rats or C57BL/6 mice. Succinate was less effective at inducing contraction in arteries isolated from GPR91-deficient mice, when compared to its vascular effect in aortas from wild type mice. SB203508 (p38MAK inhibitor), SP600125 (JNK inhibitor) and Y27632 (Rho-kinase inhibitor) reduced succinate-induced contraction in both endothelium-intact and endothelium-denuded rat aortic rings, while PD98059 (ERK1/2 inhibitor) did not affect succinate-induced contraction. The contractile response induced by succinate on endothelium-intact and endothelium-denuded rat aortic rings was reduced by indomethacin (non-selective cyclooxygenase inhibitor), H7 (protein kinase C inhibitor), verapamil (Ca(2+) channel blocker) and tiron (superoxide anion scavenger).

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.

Ethanol consumption increases endothelin-1 expression and reactivity in the rat cavernosal smooth muscle.

AIMS: We investigated the effects of chronic ethanol consumption on the cavernosal smooth muscle (CSM) reactivity to endothelin-1 (ET-1) and the expression of ET system components in this tissue. METHODS: Male Wistar rats were treated with heavy dose of ethanol (20% v/v) for 6 weeks. Reactivity experiments were performed in the isolated rat CSM. Plasma and CSM nitrate generation and also superoxide anion generation in rat CSM were measured by chemiluminescence. Protein and mRNA levels of pre-pro-ET-1, endothelin-converting enzyme-1 (ECE-1), ETA and ETB receptors, eNOS, nNOS and iNOS were assessed by western immunoblotting and quantitative real-time polymerase chain reaction, respectively. RESULTS: Chronic ethanol consumption increased plasma ET-1 levels and the contractile response induced by this peptide in the isolated CSM. The relaxation induced by acetylcholine, but not IRL1620, a selective ETB receptor agonist, was reduced in CSM from ethanol-treated rats. BQ123, a selective ETA receptor antagonist, produced a rightward displacement of the ET-1 concentration-response curves in CSM from control, but not ethanol-treated rats. Reduced levels of nitrate were found in the plasma and CSM from ethanol-treated rats. Ethanol consumption increased superoxide anion generation in the rat CSM. The mRNA levels of pre-pro-ET-1, ECE-1, ETA and ETB receptors, eNOS, nNOS and iNOS were not altered by ethanol consumption. Protein levels of ET-1, ETA receptor and iNOS were higher in the CSM from rats chronically treated with ethanol. CONCLUSION: The major findings of the present study are that heavy ethanol consumption increases plasma ET-1 levels and the contraction induced by the peptide in the CSM. Increased CSM reactivity to ET-1 and altered protein levels of ET-1 and ETA receptors could play a role in the pathogenesis of erectile dysfunction associated with chronic ethanol consumption.