Carolina Baraldi Araujo Restini

Acidosis induces relaxation mediated by nitric oxide and potassium channels in rat thoracic aorta

We investigated the mechanism by which extracellular acidification promotes relaxation in rat thoracic aorta. The relaxation response to HCl-induced extracellular acidification (7.4 to 6.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M) or KCl (45mM). The vascular reactivity experiments were performed in endothelium-intact and denuded rings, in the presence or absence of indomethacin (10(-5) M), L-NAME (10(-4) M), apamin (10(-6) M), and glibenclamide (10(-5) M). The effect of extracellular acidosis (pH 7.0 and 6.5) on nitric oxide (NO) production was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5μM). The extracellular acidosis failed to induce any changes in the vascular tone of aortic rings pre-contracted with KCl, however, it caused endothelium-dependent and independent relaxation in rings pre-contracted with Phe. This acidosis induced-relaxation was inhibited by L-NAME, apamin, and glibenclamide, but not by indomethacin. The acidosis (pH 7.0 and 6.5) also promoted a time-dependent increase in the NO production by the isolated endothelial cells. These results suggest that extracellular acidosis promotes vasodilation mediated by NO, K(ATP) and SK(Ca), and maybe other K(+) channels in isolated rat thoracic aorta.

The role of reactive oxygen species in the modulation of the contraction induced by angiotensin II in carotid artery from diabetic rat

The modulation played by reactive oxygen species on the angiotensin II-induced contraction in type I-diabetic rat carotid was investigated. Concentration-response curves for angiotensin II were obtained in endothelium-intact or endothelium-denuded carotid from control or streptozotocin-induced diabetic rats, pre-treated with tiron (superoxide scavenger), PEG-catalase (hydrogen peroxide scavenger), dimethylthiourea (hydroxyl scavenger), apocynin [NAD(P)H oxidase inhibitor], SC560 (cyclooxygenase-1 inhibitor), SC236 (cyclooxygenase-2 inhibitor) or Y-27632 (Rho-kinase inhibitor). Reactive oxygen species were measured by flow cytometry in dihydroethidium (DHE)-loaded endothelial cells. Cyclooxygenase and AT(1)-receptor expression was assessed by immunohistochemistry. Diabetes increased the angiotensin II-induced contraction but reduced the agonist potency in rat carotid. Endothelium removal, tiron or apocynin restored the angiotensin II-induced contraction in diabetic rat carotid to control levels. PEG-catalase, DMTU or SC560 reduced the angiotensin II-induced contraction in diabetic rat carotid at the same extent. SC236 restored the angiotensin II potency in diabetic rat carotid. Y-27632 reduced the angiotensin II-induced contraction in endothelium-intact or -denuded diabetic rat carotid. Diabetes increased the DHE-fluorescence of carotid endothelial cells. Apocynin reduced the DHE-fluorescence of endothelial cells from diabetic rat carotid to control levels. Diabetes increased the muscular cyclooxygenase-2 expression but reduced the muscular AT(1)-receptor expression in rat carotid. In summary, hydroxyl radical, hydrogen peroxide and superoxide anion-derived from endothelial NAD(P)H oxidase mediate the hyperreactivity to angiotensin II in type I-diabetic rat carotid, involving the participation of cyclooxygenase-1 and Rho-kinase. Moreover, increased muscular cyclooxygenase-2 expression in type I-diabetic rat carotid seems to be related to the local reduced AT(1)-receptor expression and the reduced angiotensin II potency.

The Effect of Extracellular pH Changes on Intracellular pH and Nitric Oxide Concentration in Endothelial and Smooth Muscle Cells from Rat Aorta

Aims It has been known for more than a century that pH changes can alter vascular tone. However, there is no consensus about the effects of pH changes on vascular response. In this study, we investigated the effects of extracellular pH (pHo) changes on intracellular pH (pHi) and intracellular nitric oxide concentration ([NO]i) in freshly isolated endothelial cells and cross sections from rat aorta. Main Methods The HCl was used to reduce the pHo from 7.4 to 7.0 and from 7.4 to 6.5; the NaOH was used to increase the pHo from 7.4 to 8.0 and from 7.4 to 8.5. The fluorescent dyes 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (SNARF-1) and diaminofluorescein-FM diacetate (DAF-FM DA) were employed to measure the pHi and [NO]i, respectively. The fluorescence intensity was measured in freshly isolated endothelial cells by flow cytometry and in freshly obtained aorta cross sections by confocal microscopy. Key Findings The endothelial and vascular smooth muscle pHi was increased at pHo 8.5. The extracellular acidification did not change the endothelial pHi, but the smooth muscle pHi was reduced at pHo 7.0. At pHo 8.5 and pHo 6.5, the endothelial [NO]i was increased. Both extracellular alkalinization and acidification increased the vascular smooth muscle [NO]i. Significance Not all changes in pHo did result in pHi changes, but disruption of acid-base balance in both directions induced NO synthesis in the endothelium and/or vascular smooth muscle.

Counter-regulatory effects played by the ACE - Ang II-AT1 and ACE2-Ang-(1-7) - Mas axes on the reactive oxygen species-mediated control of vascular function:perspectives to pharmacological approaches in controlling vascular complications

The Renin-Angiotensin system plays an important role in the regulation of systemic blood pressure as well as in fluid and electrolyte balance. It is divided into two described axes, the ACE - Ang II - AT1 receptor, with Ang II as the main mediator, and the ACE2 - Ang-(1-7) - Mas receptor, with Ang-(1-7) responsible for the main effects. The main vascular effect induced by Ang II is contraction, while Ang-(1-7) includes relaxation in several vascular beds. Ang II also activates several cytokines that are important in the genesis of vascular inflammation and hypertrophy. In this context, Ang-(1-7) seems to have a protective role. Both AT1 and Mas receptors modulate, in different ways, the generation of, which are involved in the control of vascular tone and the genesis of vascular dysfunction triggered by several diseases, including diabetes mellitus, arterial hypertension and atherosclerosis. Thereby, this review presents an overview of the modulation played by the whole Renin-Angiotensin system on the reactive oxygen species-mediated control of vascular tone and the oxidative stress-elicited vascular dysfunction.

Cross‐talk with β2‐adrenoceptors enhances ligand affinity properties from endothelial alpha1D‐adrenoceptors that mediates carotid relaxation

Our main objectives were to investigate the affinity properties of endothelial and muscular α1D‐adrenoceptors and to characterize the cross‐talk between endothelial α1D‐adrenoceptors and β2‐adrenoceptors in rat carotid.

Development of Anatomophysiologic Knowledge Regarding the Cardiovascular System: From Egyptians to Harvey.

Our knowledge regarding the anatomophysiology of the cardiovascular system (CVS) has progressed since the fourth millennium BC. In Egypt (3500 BC), it was believed that a set of channels are interconnected to the heart, transporting air, urine, air, blood, and the soul. One thousand years later, the heart was established as the center of the CVS by the Hippocratic Corpus in the medical school of Kos, and some of the CVS anatomical characteristics were defined. The CVS was known to transport blood via the right ventricle through veins and the pneuma via the left ventricle through arteries. Two hundred years later, in Alexandria, following the development of human anatomical dissection, Herophilus discovered that arteries were 6 times thicker than veins, and Erasistratus described the semilunar valves, emphasizing that arteries were filled with blood when ventricles were empty. Further, 200 years later, Galen demonstrated that arteries contained blood and not air. With the decline of the Roman Empire, Greco-Roman medical knowledge about the CVS was preserved in Persia, and later in Islam where, Ibn Nafis inaccurately described pulmonary circulation. The resurgence of dissection of the human body in Europe in the 14th century was associated with the revival of the knowledge pertaining to the CVS. The main findings were the description of pulmonary circulation by Servetus, the anatomical discoveries of Vesalius, the demonstration of pulmonary circulation by Colombo, and the discovery of valves in veins by Fabricius. Following these developments, Harvey described blood circulation.

Mas-Mediated Antioxidant Effects Restore the Functionality of Angiotensin Converting Enzyme 2-Angiotensin-(1–7)-Mas Axis in Diabetic Rat Carotid

We hypothesized that endothelial AT1-activated NAD(P)H oxidase-driven generation of reactive oxygen species during type I-diabetes impairs carotid ACE2-angiotensin-(1–7)-Mas axis functionality, which accounts for the impaired carotid flow in diabetic rats. We also hypothesized that angiotensin-(1–7) chronic treatment of diabetic rats restores carotid ACE2-angiotensin-(1–7)-Mas axis functionality and carotid flow. Relaxant curves for angiotensin II or angiotensin-(1–7) were obtained in carotid from streptozotocin-induced diabetic rats. Superoxide or hydrogen peroxide levels were measured by flow cytometry in carotid endothelial cells. Carotid flow was also determined. We found that endothelial AT1-activated NAD(P)H oxidase-driven generation of superoxide and hydrogen peroxide in diabetic rat carotid impairs ACE2-angiotensin-(1–7)-Mas axis functionality, which reduces carotid flow. In this mechanism, hydrogen peroxide derived from superoxide dismutation inhibits ACE2 activity in generating angiotensin-(1–7) seemingly by activating I Cl,SWELL, while superoxide inhibits the nitrergic Mas-mediated vasorelaxation evoked by angiotensin-(1–7). Angiotensin-(1–7) treatment of diabetic rats restored carotid ACE2-angiotensin-(1–7)-Mas axis functionality by triggering a positive feedback played by endothelial Mas receptors, that blunts endothelial AT1-activated NAD(P)H oxidase-driven generation of reactive oxygen species. Mas-mediated antioxidant effects also restored diabetic rat carotid flow, pointing to the contribution of ACE2-angiotensin-(1–7)-Mas axis in maintaining carotid flow.

Resveratrol improves vasoprotective effects of captopril on aortic remodeling and fibrosis triggered by renovascular hypertension

BACKGROUND Renin-angiotensin system triggers vascular remodeling and fibrosis during the renovascular hypertensive two-kidney, one-clip (2K1C) model by oxidative-stress-mediated mechanisms. Thus, we hypothesized that the chronic treatment with the polyphenolic antioxidant resveratrol would improve the vasoprotective effects promoted by the chronic treatment with the angiotensin-converting enzyme inhibitor (ACEi) captopril in 2K1C hypertensive rats. Our main objective was to evaluate the effects of the combined treatment with resveratrol and captopril on vascular remodeling and fibrosis in 2K1C rats. METHODS Male Wistar rats underwent to unilateral renal stenosis by 2K1C Goldblatt model. Six weeks after surgery, rat systolic blood pressure (SBP) was measured by indirect tail-cuff plethysmography. 2K1C rats were considered hypertensive when presenting SBP higher than 160 mmHg and underwent resveratrol (20 mg/kg), captopril (6 or 12 mg/kg), or resveratrol (20 mg/kg) combined with captopril (6 or 12 mg/kg) treatment for 3 weeks. Nine weeks after surgery, rat SBP was measured, and rat thoracic aorta was isolated for histological assays with hematoxylin/eosin or Picrosirius Red to evaluate aortic remodeling and fibrosis, respectively. RESULTS Oral treatment of 2K1C hypertensive rats with resveratrol (20 mg/kg) combined with the dose-dependent ACEi captopril (6 and 12 mg/kg) resulted in lesser aortic thickening and reduced aortic fibrosis. Resveratrol (20 mg/kg) promoted a more expressive hypotensive effect with captopril (12 mg/kg) in 2K1C rats than the treatment with isolated captopril (12 mg/kg). CONCLUSION Resveratrol improves the vasoprotective effects promoted by captopril on aortic remodeling and fibrosis during renovascular hypertension probably by synergic mechanisms involving antioxidant actions and nitric oxide generation.

Decreased number of caveolae in endothelial cells impairs the relaxation induced by acetylcholine in hypertensive rat aortas

The present study was designed to investigate the contribution of endothelial cell caveolae to vascular relaxation in aortas from a normotensive (2K) and renal hypertensive (2K-1C) rat. For that purpose, concentration-effect curves to acetylcholine were constructed in 2K and 2K-1C intact endothelium aortic rings, in the absence or in the presence of the caveolae disassembler methyl-beta-ciclodextrin. The potency (pD(2)) and the maximum relaxant effect to acetylcholine were greater in 2K than in 2K-1C aortas. Methyl-beta-ciclodextrin reduced the pD(2) in 2K and the maximum relaxant effect in both 2K and 2K-1C. The quantification of the caveolae number by electronic microscopy has shown a larger number of caveolae in 2K than in 2K-1C endothelial cells, which was reduced by methyl-beta-ciclodextrin in both 2K and 2K-1C. The production of NO stimulated with acetylcholine was greater in 2K than in 2K-1C endothelial cells, and this effect was impaired by methyl-beta-ciclodextrin in both 2K and 2K-1C. The cytosolic Ca(2+) concentration ([Ca(2+)]c) was simultaneously measured in endothelial and smooth muscle cells stimulated with acetylcholine by confocal image of aortic slices. Acetylcholine produced a greater [Ca(2+)]c increase in 2K than in 2K-1C endothelial cells, which response was inhibited by methyl-beta-ciclodextrin only in 2K cells. In smooth muscle cells the reduction of [Ca(2+)]c was higher in 2K than in 2K-1C. This effect was inhibited by methyl-beta-ciclodextrin only in 2K cells. Taken together, our results suggest that the decreased number of caveolae in the endothelial cells from 2K-1C rat aortas is involved in the impaired effect of acetylcholine on [Ca(2+)]c and NO.

Vascular Relaxation Induced by C-Type Natriuretic Peptide Involves the Ca2+/NO-Synthase/NO Pathway

Aims C-type natriuretic peptide (CNP) and nitric oxide (NO) are endothelium-derived factors that play important roles in the regulation of vascular tone and arterial blood pressure. We hypothesized that NO produced by the endothelial NO-synthase (NOS-3) contributes to the relaxation induced by CNP in isolated rat aorta via activation of endothelial NPR-C receptor. Therefore, the aim of this study was to investigate the putative contribution of NO through NPR-C activation in the CNP induced relaxation in isolated conductance artery. Main Methods Concentration-effect curves for CNP were constructed in aortic rings isolated from rats. Confocal microscopy was used to analyze the cytosolic calcium mobilization induced by CNP. The phosphorylation of the residue Ser1177 of NOS was analyzed by Western blot and the expression and localization of NPR-C receptors was analyzed by immunohistochemistry. Key Findings CNP was less potent in inducing relaxation in denuded endothelium aortic rings than in intact ones. L-NAME attenuated the potency of CNP and similar results were obtained in the presence of hydroxocobalamin, an intracellular NO0 scavenger. CNP did not change the phosphorylation of Ser1177, the activation site of NOS-3, when compared with control. The addition of CNP produced an increase in [Ca2+]c in endothelial cells and a decrease in [Ca2+]c in vascular smooth muscle cells. The NPR-C-receptors are expressed in endothelial and adventitial rat aortas. Significance These results suggest that CNP-induced relaxation in intact aorta isolated from rats involves NO production due to [Ca2+]c increase in endothelial cells possibly through NPR-C activation expressed in these cells. The present study provides a breakthrough in the understanding of the close relationship between the vascular actions of nitric oxide and CNP.