Data of Nebivolol on oxidative stress parameters in hypertensive patients.

Oxidative stress is a key feature in hypertension, since reactive oxygen species are involved in all stages of cardiovascular diseases. Saliva is a body fluid that can be used to investigate alterations in the oxidative system with several specific advantages over blood. Nebivolol is a third-generation selective ß1-adrenergic receptor antagonist that promotes vasodilation and has been shown to reduce oxidative stress in pre-clinical and clinical studies. The use of Nebivolol in different periods of treatment demonstrated that it is an efficient anti-hypertensive drug. We evaluated the oxidative stress biomarkers and the enzymatic and non-enzymatic antioxidant systems in saliva of hypertensive patients before and after the use of anti-hypertensive therapeutic doses of Nebivolol, since saliva can be used as an auxiliary tool to analyze parameters of oxidative stress.

Comparative study between apocynin and protocatechuic acid regarding antioxidant capacity and vascular effects.

Reactive oxygen species (ROS) derived from NOX enzymes activity play an important role in the development of cardiovascular diseases. Compounds able to decrease oxidative stress damage are potential candidates as drugs and/or supplements for hypertension treatment. Here, we aimed to compare in vitro ROS scavenging potency, effective NOX inhibition and effects on vascular reactivity of apocynin to another phenolic compound, protocatechuic acid, in vascular cells from spontaneously hypertensive rat (SHR), where redox signaling is altered and contributes to the development and/or maintenance of hypertension. We evaluated the in vitro antioxidant capacity and free radical scavenging capacity of both phenolic compounds. Moreover, we investigated the effect of both compounds on lipid peroxidation, lucigenin chemiluminescence, nitric oxide (NO•) levels and ROS concentration in vascular cells of SHR or human umbilical vein endothelial cell (HUVEC). Apocynin and protocatechuic acid presented antioxidant capacity and ability as free radical scavengers, decreased thiobarbituric acid reactive substances (TBARS) in aortic cells from SHR, and increased NO• concentration in isolated HUVEC. Both compounds were able to reduce lucigenin chemiluminescence and increased the potency of acetylcholine in aorta of SHR. However, in SHR aortas, only apocynin diminished the contraction induced by phenylephrine. In conclusion, these results strongly reinforce the potential application of substances such as apocynin and protocatechuic acid that combine abilities as scavenging and/or prevention of ROS generation, establishment of NO bioactivity and modulation of vascular reactivity. Due to its phytochemical origin and low toxicity, its potential therapeutic use in vascular diseases should be considered.

Caveolin-1/Endothelial Nitric Oxide Synthase Interaction Is Reduced in Arteries From Pregnant Spontaneously Hypertensive Rats.

We have investigated the role caveolae/caveolin-1 (Cav-1) plays in endothelial nitric oxide synthase (eNOS) activation and how it impacts pregnancy-induced decreased vascular reactivity in normotensive (Wistar rats) and spontaneously hypertensive rats (SHR). Wistar rats and SHR were divided into non-pregnant (NP) and pregnant (P). Nitrite levels were assessed by the Griess method in the aorta and mesenteric vascular bed. In functional studies, arteries were incubated with methyl-ß-cyclodextrin (dextrin, 10mmol/L), which disrupts caveolae by depleting cholesterol, and concentration-response curves to phenylephrine (PE) and acetylcholine (ACh) were constructed. Electronic microscopy was used to determine endothelial caveolae density in the aorta and resistance mesenteric artery in the presence of vehicle or dextrin (10mmol/L). Western blot was performed to evaluate Cav-1, p-Cav-1, calmodulin (CaM), and heat shock protein 90 (Hsp90) expression. Cav-1/eNOS interaction in the aorta and mesenteric vascular bed was assessed by co-immunoprecipitation. Nitric oxide (NO) generation was greater in arteries from P groups compared to NP groups. Dextrin did not change vascular responses in the aorta from P groups or the number of caveolae in P groups compared to NP groups. Compared to NP Wistar rats, NP SHR showed smaller number of caveolae and reduced Cav-1 expression. Pregnancy did not alter Cav-1, CaM, or Hsp90 expression in the aorta or mesenteric vascular bed from Wistar rats or SHR. These results suggest that pregnancy does not alter expression of the main eNOS regulatory proteins, but it decreases Cav-1/eNOS interaction. Reduced Cav-1/eNOS interaction in the aorta and mesenteric vascular bed seems to be an important mechanism to increase eNOS activity and nitric oxide production in pregnant normotensive and hypertensive rats.

Pregnancy decreases O-GlcNAc-modified proteins in systemic arteries of normotensive and spontaneously hypertensive rats.

AIM: We determined the role played by O-linked N-acetylglucosamine (O-GlcNAc) of proteins in systemic arteries during late pregnancy in normotensive and hypertensive rats. MAIN METHODS: O-GlcNAc levels and O-GlcNAc modification of endothelial nitric oxide synthase (eNOS) were determined in aorta (conductance vessel) and mesenteric arteries (resistance vessels) of non-pregnant (NP) and pregnant (P) Wistar rats and spontaneously hypertensive rats (SHR). Vascular O-GlcNAc-modified proteins, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT) expression, and OGA activity were analyzed. Concentration-response to phenylephrine (PE) curves were constructed for arteries with and without endothelium. Arteries were treated with vehicle or PugNAc (OGA inhibitor, 100 µmol/L) in the presence of L-NAME (NOS inhibitor, 100 µmol/L). KEY FINDINGS: The content of vascular O-GlcNAc-modified proteins was lower, OGT and OGA expression did not change, and OGA activity was higher in arteries of P-Wistar rats and P-SHR compared to arteries of NP-groups. Reactivity to PE increased in arteries of P-Wistar rats treated with PugNAc compared to vehicle. O-GlcNAcylation of eNOS decreased in P-SHR compared to NP-SHR. PugNAc partially inhibited the effects of endothelium removal and L-NAME on reactivity to PE in arteries of P-Wistar rats. However, PugNAc did not alter reactivity to PE in arteries of P-SHR. Our data showed that pregnancy decreased the content of vascular O-GlcNAc-modified proteins. SIGNIFICANCE: Increased OGA activity and decreased O-GlcNAc modification of eNOS boosts eNOS activity in arteries of P-Wistar rats. In P-SHR, altered OGA activity may lower the content of O-GlcNAc-modified proteins, but decreased OGT activity seems a potential mechanism to reduce glycosylation.

Apocynin alters redox signaling in conductance and resistance vessels of spontaneously hypertensive rats.

Chronic treatment with apocynin reduces blood pressure and prevents endothelial dysfunction development in spontaneously hypertensive rats (SHR). Mechanisms underlying apocynin effects on SHR remain unclear. Compared to diapocynin and other drugs, apocynin is a weak antioxidant, which suggests that its effects on SHR are associated with other mechanisms besides its antioxidant capacity. Angiotensin (Ang) II regulates NOX, the major reactive oxygen species (ROS) source in the cardiovascular system. We hypothesized that, by inhibiting NOX, apocynin could alter Ang II pressor and vasoconstrictor effects on SHR. We analyzed how Ang II affects blood pressure and vascular reactivity in aorta and mesenteric resistance arteries and evaluated plasma antioxidant capacity, NOX isoforms and subunits, NOS isoforms, AT1 and AT2 receptors expression, ROS production, and NOS activity in apocynin-treated SHR blood vessels (30 mg/Kg/day, p.o.). In SHR, apocynin reduced Ang II pressor effects, increased plasmatic antioxidant capacity, and blunted aortic and mesenteric NOX-dependent oxidants production and NOX2 and p47phox overexpression, which demonstrated that apocynin inhibits NOX in SHR blood vessels. Moreover, apocynin raised plasmatic and aortic nitrate/nitrite levels, maintained NOS activity and eNOS, p-eNOS, nNOS, iNOS, sGC-α, and sGC-ß expression in mesenteric bed, diminished AT1 expression in aorta and mesenteric bed, and elevated AT2 expression in SHR aorta. Apocynin increased Ang II vasoconstriction endothelial modulation in SHR resistance arteries. All these results showed that in vivo treatment with apocynin alters several mechanisms that reduce Ang II pressor and vasoconstrictor effects on SHR. Such apocynin effects involve other mechanisms besides vascular ROS modulation, which improves NO availability in SHR vascular cells. These integrated data could help us to understand the promising apocynin activity as an antihypertensive drug that acts differently from the drugs that are currently being used in the clinical setting.

Endothelial modulation of a nitric oxide donor complex-induced relaxation in normotensive and spontaneously hypertensive rats.

We hypothesized that endothelium modulates relaxation induced by a nitric oxide (NO) donor ruthenium complex (TERPY, [Ru(terpy)(bdq)NO]3+) in mesenteric arteries of normotensive and spontaneously hypertensive (SHR) rats in different ways. We analyzed the mechanism involved in TERPY-induced relaxation in the second and third branches of mesenteric arteries and investigated how endothelium contributes to the TERPY vasodilator effect on SHR blood vessels. TERPY induced concentration-dependent relaxation in endothelium-denuded (E-) and endothelium-intact (E+) mesenteric arteries of normotensive rats and SHR. Pretreatment with ODQ (which inhibits soluble guanylyl cyclase) or TEA (tetraethylammonium, which blocks potassium channels) significantly reduced the TERPY vasodilator effect on E- mesenteric arteries of normotensive rats and SHR. The presence of endothelium shifted the concentration-effect curves for TERPY in E+ mesenteric arteries of normotensive rats to the right. Conversely, the presence of endothelium shifted the concentration-effect curves for TERPY in the case of SHR E+ mesenteric arteries to the left, which suggested increased potency. L-NNA, a more selective endothelial NO synthase (eNOS) inhibitor, reduced TERPY potency in SHR. The presence of endothelium and notably of NOS contributed to the TERPY vasodilator action in SHR: TERPY promoted eNOS Ser1177 phosphorylation with consequent NO production and increased soluble guanylyl cyclase activity, which may have directly activated potassium channels.

Hypotensive and vasorelaxant effect of Diapocynin in normotensive rats.

Nicotinamide adenine dinucleotide phosphate oxidase (NAD(P)H-oxidase) is a multicomponent enzyme system that generates superoxide anion by one-electron reduction of molecular oxygen and represents the major source of reactive oxygen species (ROS) in the vascular cells. Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in phagocytic cells and as an antioxidant in non-phagocytic cells. In phagocytes cells, due to the presence of myeloperoxidase, apocynin can be the converted to diapocynin, which is supposed to be the active form of this phytochemical. Moreover, apocynin was shown to induce hypotension and vasodilatation in many experimental animal models. However, there are no studies showing the effects of diapocynin on blood pressure or in vascular cells. In this present study, we used chemically synthesized diapocynin and analyzed its antioxidant capacity, effect on blood pressure and vascular reactivity. Moreover, it was evaluated the levels of nitric oxide (NO), ROS and calcium in aortic endothelial cells stimulated by diapocynin. All results were compared to apocynin. We found that diapocynin showed higher antioxidant capacity than apocynin. Apocynin and diapocynin, promoted hypotensive effects without changing the heart rate, however the effects of diapocynin were reversed faster than the effects of apocynin, which was long lasting. Diapocynin and apocynin induced endothelium dependent and independent vasodilatation, but diapocynin was less potent than apocynin regarding the capacity of induction of vasodilatation in mesenteric resistance arteries and aorta from Wistar rats. The relaxation induced by apocynin or diapocynin involves sGC and potassium channels in vascular smooth muscle cells and NOS participates of relaxation induced by apocynin or diapocynin in intact mesenteric rings. Apocynin and diapocynin increased NO and decreased ROS levels in endothelial cells, however diapocynin did not alter calcium levels in these cells. In conclusion, these results demonstrated that, similarly to apocynin, diapocynin also induces hypotensive and vasodilator effects in rats and vascular endothelium improves the diapocynin vasodilator effects by increases NO bioavailability.

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.

The PI3K-Akt-eNOS pathway is involved in aortic hyporeactivity to Phenylephrine associated with late pregnancy in spontaneously hypertensive rats.

AIM: This study aimed to evaluate the effects of Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), on aortic hyporeactivity to Phenylephrine (Phe) and nitric oxide bioavailability associated with pregnancy in hypertensive rats. MAIN METHODS: The intact aortic rings of pregnant and non-pregnant Wistar or spontaneously hypertensive rats (SHRs) were stimulated with Phe (1nmol/L to 10mmol/L) before and after incubation with Wortmannin (10nmol/L, 30min). Western blot experiments analyzed the expression of phosphorylated PI3K [p85-PI3K], Akt [p-Akt (Ser 473)] and eNOS [p-eNOS (Ser 1177)] in aorta homogenates of pregnant and non-pregnant Wistar rats or SHRs. The effect of Wortmannin (10nmol/L) on the cytosolic concentrations of nitric oxide (NO; measured using 4,5-diaminofluorescein diacetate [DAF-2DA], 10mmol/L), Ca(2+) (using Fluo 3-AM, 5µmol/L) and reactive oxygen species (ROS; using dihydroethidium [DHE], 2.5mmol/L) were measured fluorimetrically in freshly isolated endothelial cells. KEY FINDINGS: Wortmannin increases the reactivity of the aorta to Phe and decreases NO concentrations in the aortic endothelial cells of pregnant Wistar rats and SHR. SIGNIFICANCE: The PI3/AKT/endothelial nitric oxide synthase (eNOS) pathway contributes to aortic hyporeactivity to Phenylephrine associated with pregnancy in normo- and hypertensive rats.

The hypotensive effect of the ruthenium complex [Ru(terpy)(bdq)NO]³âº is higher in male than in female spontaneously hypertensive rats (SHR).

We have previously demonstrated that the hypotensive effect of the ruthenium complex [Ru(terpy)(bdq)NO](3+) (TERPY) is slow, long lasting, and does not lead to reflex tachycardia. TERPY's hypotensive effect is increased in hypertensive rats (SHR or 2 kidney-1clip) compared with normotensive rats. We hypothesized that sexual differences could interfere in the hypotensive effects of nitric oxide (NO) donors in SHR. Therefore, here we aimed to investigate the role of sexual differences and endogenous NO in the hypotension induced by TERPY. In conscious, unrestrained animals, we evaluated the hypotensive effect of TERPY before and after the administration of N-nitro-L-arginine methyl ester (L-NAME) (nonselective NO synthase inhibitor), APOCYNIN (NADPH/NOX inhibitor), and TEMPOL (superoxide dismutase mimetic). The hypotensive effect of TERPY was higher in male than in female SHR, but this difference was not observed in the normotensive Wistar group. The effect of TERPY increased after administration of L-NAME in Wistar rats; however, this effect was not altered by L-NAME in SHR. In SHR, sexual dimorphism in TERPY effect was still observed in animals treated with L-NAME. TEMPOL increases the effect of TERPY only in female SHR. After TEMPOL, the sexual dimorphism in TERPY effect was abolished in the SHR group. APOCYNIN increased the effect of TERPY in male and female Wistar and SHR, but maintained the previously observed difference between male and female SHR. Thus, this study shows that TERPY's hypotensive effect increased in male compared with female SHR and indicates that sexual dimorphism in TERPY effect is associated with oxidative stress.