Pharmacological characterization of the calcium influx pathways involved in nitric oxide production by endothelial cells / Caracterização farmacológica das vias de influxo de cálcio envolvidas na produção de óxido nítrico por células endoteliais

ABSTRACT Objective: To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors. Methods: We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings. Results: Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3μM). Pyr3 (0.3 to 3μM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s−1) and thapsigargin (3.9±0.25mg.s−1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3μM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3μM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3μM). Conclusion: These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx.

RESUMO Objetivo: Caracterizar as vias do influxo de cálcio envolvidas no aumento sustentado da concentração intracelular de cálcio na célula endotelial, essencial para a síntese e a liberação de fatores relaxantes. Métodos: Analisamos o efeito de derivados pirazólicos sintetizados recentemente, descritos como inibidores seletivos para canais ORAI (BTP2/Pyr2 e Pyr6) e TRPC3 (Pyr3 e Pyr10), nos relaxamentos dependentes de endotélio e cálcio extracelular, produzidos por acetilcolina e tapsigargina, em anéis pré-contraídos da aorta torácica de rato. Resultados: As respostas de acetilcolina e tapsigargina foram completamente revertidas por Pyr2 e Pyr6 (1 a 3μM). Pyr3 (0,3 a 3μM) produziu reversão rápida dos relaxamentos de acetilcolina (6,2±0,08mg.s−1) e tapsigargina (3,9±0,25mg.s−1), enquanto o bloqueador mais seletivo para TRPC3, Pyr10 (1 a 3μM), não apresentou efeito. ML204 (1 a 3μM), bloqueador seletivo de TRPC4, descrito há pouco tempo, reverteu os relaxamentos induzidos por acetilcolina de forma completa, mas afetou minimamente aqueles produzidos por tapsigargina. Os derivados pirazólicos ou ML204 não afetaram os relaxamentos estimulados com GSK1016790A (TRPV4-agonista). Ainda, após pré-incubação com Pyr2 e Pyr6, acetilcolina e tapsigargina provocaram relaxamentos transitórios semelhantes em magnitude e cinética àqueles observados na ausência de cálcio extracelular. Os relaxamentos do nitroprussiato de sódio e as contrações induzidas pela fenilefrina (aorta sem endotélio) não foram afetados pelos compostos pirazólicos (1 a 3μM). Conclusão: Essas observações revelaram uma complexidade desconhecida das vias de influxo de cálcio no endotélio da aorta de rato, que resultam na produção e na liberação de óxido nítrico. Vias distinguíveis farmacologicamente medeiam o influxo estimulado por acetilcolina (ORAI TRPC, diferentes de TRPC3 TRPC4) e tapsigargina (TRPC4 não requerido).

Effect of tobacco smoke on hydrogen sulfide-induced rat thoracic aorta relaxation

Levels of hydrogen sulfide (H2S), a gaseous signaling molecule, are reduced in the serum of individuals who smoke. We hypothesized that tobacco smoke influenced smooth muscle relaxation by decreasing H2S levels and this effect could also influence expression of cystathionine γ-lyase (CSE) and sulfonylurea receptor-2 (SUR-2). The aim of this study was to explore the effect of tobacco smoke on H2S-mediated rat thoracic aorta relaxation and its possible mechanism. Thirty-two Sprague-Dawley rats were divided into four groups: control (C) group, short-term smoker (SS) group, mid-term smoker (MS) group, and long-term smoker (LS) group. H2S concentrations in serum, action of H2S on rat aortic vascular relaxation, and expression of CSE and SUR-2 in thoracic aortic smooth muscle were measured. Although there was no significant difference in H2S between the C and the SS groups, concentration of H2S was significantly reduced in both the LS and MS groups compared to control (P<0.01). Furthermore, H2S was significantly lower in the LS than in the MS group (P<0.05). Rat aortic vascular relaxation was lower in all three treatment groups compared to the control, with the most significant decrease observed in the LS group (P<0.05 compared to the MS group). Expression of CSE and SUR-2 was reduced in the LS and MS groups compared to control (P<0.05), with the lowest levels observed in the LS group (P<0.05). Therefore, tobacco smoke reduced expression of CSE and SUR-2 in rat thoracic aorta, which may inhibit H2S production and vascular dilation.

Possible mechanisms involved in the vasorelaxant effect produced by clobenzorex in aortic segments of rats

Clobenzorex is a metabolic precursor of amphetamine indicated for the treatment of obesity. Amphetamines have been involved with cardiovascular side effects such as hypertension and pulmonary arterial hypertension. The aim of the present study was to investigate whether the direct application of 10-9-10-5 M clobenzorex on isolated phenylephrine-precontracted rat aortic rings produces vascular effects, and if so, what mechanisms may be involved. Clobenzorex produced an immediate concentration-dependent vasorelaxant effect at the higher concentrations (10-7.5-10-5 M). The present outcome was not modified by 10-6 M atropine (an antagonist of muscarinic acetylcholine receptors), 3.1×10-7 M glibenclamide (an ATP-sensitive K+ channel blocker), 10-3 M 4-aminopyridine (4-AP; a voltage-activated K+ channel blocker), 10-5 M indomethacin (a prostaglandin synthesis inhibitor), 10-5 M clotrimazole (a cytochrome P450 inhibitor) or 10-5 M cycloheximide (a general protein synthesis inhibitor). Contrarily, the clobenzorex-induced vasorelaxation was significantly attenuated (P<0.05) by 10-5 M L-NAME (a direct inhibitor of nitric oxide synthase), 10-7 M ODQ (an inhibitor of nitric oxide-sensitive guanylyl cyclase), 10-6 M KT 5823 (an inhibitor of protein kinase G), 10-2 M TEA (a Ca2+-activated K+ channel blocker and non-specific voltage-activated K+ channel blocker) and 10-7 M apamin plus 10-7 M charybdotoxin (blockers of small- and large-conductance Ca2+-activated K+ channels, respectively), and was blocked by 8×10-2 M potassium (a high concentration) and removal of the vascular endothelium. These results suggest that the direct vasorelaxant effect by clobenzorex on phenylephrine-precontracted rat aortic rings involved stimulation of the NO/cGMP/PKG/Ca2+-activated K+ channel pathway.

A spleen tyrosine kinase inhibitor attenuates the proliferation and migration of vascular smooth muscle cells

BACKGROUND: Pathologic vascular smooth muscle cell (VSMC) proliferation and migration after vascular injury promotes the development of occlusive vascular disease. Therefore, an effective chemical agent to suppress aberrant proliferation and migration of VSMCs can be a potential therapeutic modality for occlusive vascular disease such as atherosclerosis and restenosis. To find an anti-proliferative chemical agent for VSMCs, we screened an in-house small molecule library, and the selected small molecule was further validated for its anti-proliferative effect on VSMCs using multiple approaches, such as cell proliferation assays, wound healing assays, transwell migration assays, and ex vivo aortic ring assay. RESULTS: Among 43 initially screened small molecule inhibitors of kinases that have no known anti-proliferative effect on VSMCs, a spleen tyrosine kinase (Syk) inhibitor (BAY61-3606) showed significant anti-proliferative effect on VSMCs. Further experiments indicated that BAY61 attenuated the VSMC proliferation in both concentration- and time-dependent manner, and it also significantly suppressed the migration of VSMCs as assessed by both wound healing assays and transwell assays. Additionally, BAY61 suppressed the sprouting of VSMCs from endothelium-removed aortic rings. CONCLUSION: The present study identified a Syk kinase inhibitor as a potent VSMC proliferation and migration inhibitor and warrants further studies to elucidate its underlying molecular mechanisms, such as its primary target, and to validate its in vivo efficacy as a therapeutic agent for restenosis and atherosclerosis.

Jabuticaba-Induced Endothelium-Independent Vasodilating Effect on Isolated Arteries / Efeito Vasodilatador Independente do Endotélio Induzido pela Jabuticaba (M. cauliflora) em Artérias Isoladas

Abstract Background: Despite the important biological effects of jabuticaba, its actions on the cardiovascular system have not been clarified. Objectives: To determine the effects of jabuticaba hydroalcoholic extract (JHE) on vascular smooth muscle (VSM) of isolated arteries. Methods: Endothelium-denuded aortic rings of rats were mounted in isolated organ bath to record isometric tension. The relaxant effect of JHE and the influence of K+ channels and Ca2+ intra- and extracellular sources on JHE-stimulated response were assessed. Results: Arteries pre-contracted with phenylephrine showed concentration-dependent relaxation (0.380 to 1.92 mg/mL). Treatment with K+ channel blockers (tetraethyl-ammonium, glibenclamide, 4-aminopyridine) hindered relaxation due to JHE. In addition, phenylephrine-stimulated contraction was hindered by previous treatment with JHE. Inhibition of sarcoplasmic reticulum Ca2+ ATPase did not change relaxation due to JHE. In addition, JHE inhibited the contraction caused by Ca2+ influx stimulated by phenylephrine and KCl (75 mM). Conclusion: JHE induces endothelium-independent vasodilation. Activation of K+ channels and inhibition of Ca2+ influx through the membrane are involved in the JHE relaxant effect.

Resumo Fundamentos: Embora a jabuticaba apresente importantes efeitos biológicos, suas ações sobre o sistema cardiovascular ainda não foram esclarecidas. Objetivos: Determinar os efeitos do extrato de jabuticaba (EHJ) sobre o músculo liso vascular (MLV) em artérias isoladas. Métodos: Aortas (sem endotélio) de ratos foram montadas em banho de órgãos isolados para registro de tensão isométrica. Foram verificados o efeito relaxante, a influência dos canais de K+ e das fontes de Ca2+ intra- e extracelular sob a resposta estimulada pelo EHJ. Resultados: Artérias pré-contraídas com fenilefrina apresentaram relaxamento concentração-dependente (0,380 a 1,92 mg/mL). O tratamento com bloqueadores de canais de K+ (tetraetilamônio, glibenclamida, 4-aminopiridina) prejudicaram o relaxamento pelo EHJ. A contração estimulada com fenilefrina também foi prejudicada pelo tratamento prévio com EHJ. A inibição da Ca2+ATPase do reticulo sarcoplasmático não alterou o relaxamento pelo EHJ. Além disso, o EHJ inibiu a contração causada pelo influxo de Ca2+ estimulado por fenilefrina e KCl (75 mM). Conclusão: O EHJ induz vasodilatação independente do endotélio. Ativação dos canais de K+ e inibição do influxo de Ca2+ através da membrana estão envolvidas no efeito relaxante do EHJ.

Treatment of patients with aortic atherosclerotic disease with paclitaxel-associated lipid nanoparticles

OBJECTIVE: The toxicity of anti-cancer chemotherapeutic agents can be reduced by associating these compounds, such as the anti-proliferative agent paclitaxel, with a cholesterol-rich nanoemulsion (LDE) that mimics the lipid composition of low-density lipoprotein (LDL). When injected into circulation, the LDE concentrates the carried drugs in neoplastic tissues and atherosclerotic lesions. In rabbits, atherosclerotic lesion size was reduced by 65% following LDE-paclitaxel treatment. The current study aimed to test the effectiveness of LDE-paclitaxel on inpatients with aortic atherosclerosis. METHODS: This study tested a 175 mg/m2 body surface area dose of LDE-paclitaxel (intravenous administration, 3/3 weeks for 6 cycles) in patients with aortic atherosclerosis who were aged between 69 and 86 yrs. A control group of 9 untreated patients with aortic atherosclerosis (72-83 yrs) was also observed. RESULTS: The LDE-paclitaxel treatment elicited no important clinical or laboratory toxicities. Images were acquired via multiple detector computer tomography angiography (64-slice scanner) before treatment and at 1-2 months after treatment. The images showed that the mean plaque volume in the aortic artery wall was reduced in 4 of the 8 patients, while in 3 patients it remained unchanged and in one patient it increased. In the control group, images were acquired twice with an interval of 6-8 months. None of the patients in this group exhibited a reduction in plaque volume; in contrast, the plaque volume increased in three patients and remained stable in four patients. During the study period, one death unrelated to the treatment occurred in the LDE-paclitaxel group and one death occurred in the control group. CONCLUSION: Treatment with LDE-paclitaxel was tolerated by patients with cardiovascular disease and showed the potential to reduce atherosclerotic lesion size.

The Labdane Ent-3-Acetoxy-Labda-8(17), 13-Dien-15-Oic Decreases Blood Pressure In Hypertensive Rats / O Labdano Ácido Ent-3-Acetóxi-Labda-8(17), 13-Dieno-15-Óico Reduz Pressão Arterial Em Ratos Hipertensos

Abstract 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.

Resumo Fundamento: Diterpenos do tipo labdano induzem uma queda da pressão arterial por meio do relaxamento do músculo liso vascular; todavia, não há estudos que descrevam os efeitos de labdanos em ratos hipertensos. Objetivo: O presente estudo foi desenvolvido para investigar as ações cardiovasculares do labdano ácido ent-3-acetóxi-labda-8(17),13-dieno-15-óico (labda-15-óico) na hipertensão renal dois rins-1 clipe (2R-1C). Métodos: Foram feitos experimentos de reatividade vascular em anéis aórticos isolados de ratos machos 2R-1C e normotensos (2R). A medição de Nitrato/Nitrito (NOx) foi feita nas aortas por meio de ensaio colorimétrico. As medidas de pressão arterial foram feitas em ratos conscientes. Resultados: O ácido labda-15-óico (0,1 - 300 µmol/l) e a forscolina (0,1 nmol/l - 1 µmol/l) relaxaram as aortas com endotélio intacto e as aortas sem endotélio dos ratos 2R-1C e 2R. O labda-15-óico mostrou-se mais eficaz na indução do relaxamento em aortas com endotélio intacto de 2R pré-contraídas com fenilefrina em comparação àquelas sem endotélio. A forscolina mostrou-se mais potente do que o ácido labda-15-óico na indução do relaxamento vascular nas artérias tanto de ratos 2R-1C quanto de ratos 2R. O aumento dos níveis de NOx induzido pelo ácido labda-15-óico foi menor nas artérias de ratos 2R-1C em comparação a ratos 2R. A administração intravenosa de ácido labda-15-óico (0,3-3 mg/kg) ou forscolina (0,1-1 mg/kg) induziu hipertensão em ratos 2R-1C e 2R conscientes. Conclusão: Os presentes resultados mostram que o labda-15-óico induz relaxamento vascular e hipotensão em ratos hipertensos.

Role of adenylyl cyclase in reduced ß-adrenoceptor-mediated vasorelaxation during maturation

Beta-adrenergic receptor (βAR)-dependent blood vessel relaxation is impaired in older animals and G protein activation has been suggested as the causative mechanism. Here, we investigated the role of βAR subtypes (β1AR, β2AR, and β3AR) and cAMP in maturation-dependent vasorelaxation impairment. Aortic rings from 15 Sprague-Dawley male rats (3 or 9 weeks old) were harvested and left intact or denuded of the endothelium. Vascular relaxation in aortic rings from younger and older groups was compared in the presence of βAR subtype agonists and antagonists along with cAMP and cGMP antagonists. Isolated aortic rings were used to evaluate relaxation responses, protein expression was evaluated by western blot or real time PCR, and metabolites were measured by ELISA. Expression of βAR subtypes and adenylyl cyclase was assessed, and cAMP activity was measured in vascular tissue from both groups. Isoproterenol- and BRL744-dependent relaxation in aortic rings with and without endothelium from 9-week-old rats was impaired compared with younger rats. The β1AR antagonist CGP20712A (10-7 M) did not affect isoproterenol or BRL744-dependent relaxation in arteries from either group. The β2AR antagonist ICI-118,551 (10-7 M) inhibited isoproterenol-dependent aortic relaxation in both groups. The β3AR antagonist SR59230A (10-7 M) inhibited isoproterenol- and BRL744-dependent aortic ring relaxation in younger but not in older rats. All βAR subtypes were expressed in both groups, although β3AR expression was lower in the older group. Adenylyl cyclase (SQ 22536) or protein kinase A (H89) inhibitors prevented isoproterenol-induced relaxation in younger but not in older rats. Production of cAMP was reduced in the older group. Adenylyl cyclase III and RyR3 protein expression was higher in the younger group. In conclusion, altered expression of β3AR and adenylyl cyclase III may be responsible for reduced cAMP production in the older group.

Pharmacological study of the mechanisms involved in the vasodilator effect produced by the acute application of triiodothyronine to rat aortic rings

A relationship between thyroid hormones and the cardiovascular system has been well established in the literature. The present in vitro study aimed to investigate the mechanisms involved in the vasodilator effect produced by the acute application of 10-8–10-4 M triiodothyronine (T3) to isolated rat aortic rings. Thoracic aortic rings from 80 adult male Wistar rats were isolated and mounted in tissue chambers filled with Krebs-Henseleit bicarbonate buffer in order to analyze the influence of endothelial tissue, inhibitors and blockers on the vascular effect produced by T3. T3 induced a vasorelaxant response in phenylephrine-precontracted rat aortic rings at higher concentrations (10-4.5–10-4.0 M). This outcome was unaffected by 3.1×10-7 M glibenclamide, 10-3 M 4-aminopyridine (4-AP), 10-5 M indomethacin, or 10-5 M cycloheximide. Contrarily, vasorelaxant responses to T3 were significantly (P<0.05) attenuated by endothelium removal or the application of 10-6 M atropine, 10-5 M L-NG-nitroarginine methyl ester (L-NAME), 10-7 M 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10-6 M (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i](1,6)benzodiazocine-10-carboxylic acid, methyl ester KT 5823, 10-2 M tetraethylammonium (TEA), or 10-7 M apamin plus 10-7 M charybdotoxin. The results suggest the involvement of endothelial mechanisms in the vasodilator effect produced by the acute in vitro application of T3 to rat aortic rings. Possible mechanisms include the stimulation of muscarinic receptors, activation of the NO-cGMP-PKG pathway, and opening of Ca2+-activated K+ channels.

Long-lasting endothelium-dependent relaxation of isolated arteries caused by an extract from the bark of Combretum leprosum / Extrato das cascas de Combretum leprosum causa relaxamento dependente de endotélio de longa duração em artérias isoladas

Objective To describe and to characterize the relaxing effect of an extract of the bark of Combretum leprosum on isolated arterial rings from different animals.Methods Rings (3 to 4mm) from rabbit, rat, or porcine arteries rings were suspended in an organ bath (Krebs, 37°C, 95%O2/5%CO2) to record isometric contractions. After the stabilization period (2 to 3 hours) contractions were induced by the addition of phenylephrine (0.1 to 0.3µM) or U46619 (10 to 100nM), and Combretum leprosum extract was added on the plateau of the contractions. Experiments were performed to determine the potency, duration, reversibility, and to get insights on the potential mechanism involved in extract-induced relaxations.Results In all rings tested, Combretumleprosum extract (1.5μg/mL) was able to cause relaxations, which were strictly endothelium-dependent. In rabbit or rat thoracic aorta rings, the relaxations were reversed by vitamin B12a or L-NG-nitroarginine. In porcine right coronary arteries and rabbit abdominal aorta, extract caused both L-NG-nitroarginine-sensitive and L-NG-nitroarginine-resistant relaxations. In rabbit thoracic aorta, the extract was relatively potent (EC50=0.20µg/mL) and caused relaxations; intriguingly the endothelium continued to produce relaxing factors for a long period after removing the extract. The magnitude of extract-induced relaxations was significantly reduced in the absence of extracellular Ca2+; in addition, the TRPs channels blocker ruthenium red (10µM) was able to revert extract-induced relaxations. Phytochemical analyses indicated that the extract was rich in polyphenol-like reacting substances.ConclusionsCombretum leprosum extract contains bioactive compounds capable of promoting Ca2+-dependent stimulation of endothelial cells which results in a prolonged production of relaxing factors.

Objetivo Descrever e caracterizar os relaxamentos induzidos por um extrato das cascas de Combretum leprosum em anéis de artérias de diferentes espécies de animais.Métodos Anéis (3 a 4mm) de artérias de coelho, rato e porco foram montados em cubas para órgão isolado (Krebs, 37°C, 95%O2/5%CO2) para registro das contrações isométricas. Após um período de estabilização (2 a 3 horas), as contrações foram induzidas com fenilefrina (0,1 a 0,3µM) ou U46619 (10 a 100nM); no platô dessas contrações, adicionamos o extrato Combretum leprosum. Diferentes protocolos foram realizados para determinar potência, duração, reversibilidade e mecanismo dos relaxamentos induzidos pelo extrato.Resultados Em todas as preparações testadas, o extrato de Combretum leprosum (1,5µg/mL) provocou relaxamentos dependentes de endotélio. Em aorta torácica de coelho ou rato, os relaxamentos foram revertidos pela vitamina B12a ou L-NG-nitro-arginina. Em anéis de aorta abdominal de coelho e de artérias coronárias de porco, o extrato causou relaxamentos sensíveis e resistentes à L-NG-nitro-arginina. Em aorta torácica de coelho, o extrato foi relativamente muito potente (EC50=0,20μg/mL) e quando causou relaxamentos; intrigantemente o endotélio continuou a produzir fatores relaxantes por um longo período após remoção do extrato. A magnitude dos relaxamentos induzidos pelo extrato foi significativamente reduzida em ausência Ca2+ extracelular; ademais, o vermelho de rutênio (10μM), um bloqueador de canais TRPs, foi capaz de reverter os relaxamentos induzidos pelo extrato. Análises preliminares indicaram que o extrato continha compostos com reatividade química semelhante à polifenóis.Conclusão O extrato de Combretum leprosum contem compostos bioativos capazes de promover estimulação dependente de Ca2+ das células endoteliais a qual resulta numa produção prolongada de fatores relaxantes.

Mechanisms involved in the vasorelaxant effects produced by the acute application of amfepramone in vitro to rat aortic rings

Amfepramone (diethylpropion) is an appetite-suppressant drug used for the treatment of overweight and obesity. It has been suggested that the systemic and central activity of amfepramone produces cardiovascular effects such as transient ischemic attacks and primary pulmonary hypertension. However, it is not known whether amfepramone produces immediate vascular effects when applied in vitro to rat aortic rings and, if so, what mechanisms may be involved. We analyzed the effect of amfepramone on phenylephrine-precontracted rat aortic rings with or without endothelium and the influence of inhibitors or blockers on this effect. Amfepramone produced a concentration-dependent vasorelaxation in phenylephrine-precontracted rat aortic rings that was not affected by the vehicle, atropine, 4-AP, glibenclamide, indomethacin, clotrimazole, or cycloheximide. The vasorelaxant effect of amfepramone was significantly attenuated by NG-nitro-L-arginine methyl ester (L-NAME) and tetraethylammonium (TEA), and was blocked by removal of the vascular endothelium. These results suggest that amfepramone had a direct vasorelaxant effect on phenylephrine-precontracted rat aortic rings, and that inhibition of endothelial nitric oxide synthase and the opening of Ca2+-activated K+ channels were involved in this effect.

Effect of Pitavastatin on Vascular Reactivity in Hypercholesterolemic Rabbits / O Efeito da Pitavastatina na Reatividade Vascular em Coelhos Hipercolesterolêmicos

Background: Pitavastatin is the newest statin available in Brazil and likely the one with fewer side effects. Thus, pitavastatin was evaluated in hypercholesterolemic rabbits in relation to its action on vascular reactivity. Objective: To assess the lowest dose of pitavastatin necessary to reduce plasma lipids, cholesterol and tissue lipid peroxidation, as well as endothelial function in hypercholesterolemic rabbits. Methods: Thirty rabbits divided into six groups (n = 5): G1 - standard chow diet; G2 - hypercholesterolemic diet for 30 days; G3 - hypercholesterolemic diet and after the 16th day, diet supplemented with pitavastatin (0.1 mg); G4 - hypercholesterolemic diet supplemented with pitavastatin (0.25 mg); G5 - hypercholesterolemic diet supplemented with pitavastatin (0.5 mg); G6 - hypercholesterolemic diet supplemented with pitavastatin (1.0 mg). After 30 days, total cholesterol, HDL, triglycerides, glucose, creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT) were measured and LDL was calculated. In-depth anesthesia was performed with sodium thiopental and aortic segments were removed to study endothelial function, cholesterol and tissue lipid peroxidation. The significance level for statistical tests was 5%. Results: Total cholesterol and LDL were significantly elevated in relation to G1. HDL was significantly reduced in G4, G5 and G6 when compared to G2. Triglycerides, CK, AST, ALT, cholesterol and tissue lipid peroxidation showed no statistical difference between G2 and G3-G6. Significantly endothelial dysfunction reversion was observed in G5 and G6 when compared to G2. Conclusion: Pitavastatin starting at a 0.5 mg dose was effective in reverting endothelial dysfunction in hypercholesterolemic rabbits. .

Fundamento: A pitavastatina é a mais nova estatina disponível no Brasil e com prováveis menores efeitos colaterais. Assim, a pitavastatina foi avaliada em coelhos hipercolesterolêmicos em relação à ação na reatividade vascular. Objetivo: Investigar a menor dose de pitavastatina na redução dos lípides plasmáticos, colesterol e peroxidação lipídica tecidual e função endotelial em coelhos hipercolesterolêmicos. Métodos: Trinta coelhos divididos em seis grupos (n=5): G1 - dieta ração padrão; G2 - dieta hipercolesterolêmica por 30 dias; G3 - dieta hipercolesterolêmica e a partir do 16º dia suplementada com pitavastatina (0,1 mg); G4 - dieta hipercolesterolêmica e suplementada com pitavastatina (0.25 mg); G5 - dieta hipercolesterolêmica e suplementada com pitavastatina (0,5 mg); G6 - dieta hipercolesterolêmica e suplementada com pitavastatina (1,0 mg). Após 30 dias foram dosados o colesterol total, HDL, triglicérides, glicose, creatinoquinase, aspartato aminotransferase, alanina aminotransferase e o LDL calculado. Aprofundada a anestesia com tiopental sódico e retirado segmentos de aorta para estudo da função endotelial, colesterol e peroxidação lipídica tecidual. O nível de significância adotado para os testes estatísticos foi 5%. Resultados: Colesterol total e LDL elevados significativamente em relação ao G1. HDL reduzida significativamente em G4, G5 e G6 em relação ao G2. Triglicérides, creatinoquinase, aspartato aminotransferase, alanina aminotransferase, colesterol e peroxidação lipídica tecidual sem diferença estatística entre G3-G6 e G2. Disfunção endotelial revertida significativamente em G5 e G6 em relação ao G2. Conclusão: A pitavastatina, a partir da ...

Estresse Isolado ou Associado ao Etanol Libera Prostanóides em Aorta de Ratos via ?2-Adrenoceptores / Stress Alone or associated with Ethanol Induces Prostanoid Release in Rat Aorta via ?2-Adrenoceptor

Fundamento: Estresse e etanol são ambos, independentemente, importantes fatores de risco cardiovascular. Objetivo: avaliar o risco cardiovascular do consumo de etanol e exposição ao estresse, isolados e em associação, em ratos machos adultos. Métodos: Os ratos foram separados em quatro grupos: controle, etanol (20% na água de beber durante seis semanas), estresse (imobilização 1h dia/5 dias por semana/ 6 semanas) e estresse/etanol. As curvas de concentração-resposta à noradrenalina - na ausência e na presença de ioimbina, L-NAME ou indometacina - ou fenilefrina foram determinadas em aortas torácicas com e sem endotélio. EC50 e resposta máxima (n = 8-12) foram comparadas através de ANOVA de dois fatores (two-way) / método de Bonferroni. Resultados: Estresse ou estresse em associação com o consumo de etanol aumentaram as respostas máximas de noradrenalina em aortas intactas. Essa hiper-reatividade foi eliminada pela remoção do endotélio, ou pela presença da indometacina ou ioimbina, mas não foi alterada pela presença de L-NAME. Enquanto isso, o consumo de etanol não alterou a reatividade à noradrenalina. As respostas da fenilefrina em aortas com e sem endotélio também permaneceram inalteradas independentemente do protocolo. Conclusão: O estresse crônico aumentou as respostas aórticas dos ratos à noradrenalina. Esse efeito é dependente do endotélio vascular e envolve a liberação de prostanóides vasoconstritores através da estimulação de α-2 adrenoceptores endoteliais. Além disso, o consumo crônico de etanol pareceu não influenciar as respostas de noradrenalina em aorta de rato, nem modificar o aumento de tais respostas observadas em consequência da exposição ao estresse. .

Background: Stress and ethanol are both, independently, important cardiovascular risk factors. Objective: To evaluate the cardiovascular risk of ethanol consumption and stress exposure, isolated and in association, in male adult rats. Methods: Rats were separated into 4 groups: Control, ethanol (20% in drinking water for 6 weeks), stress (immobilization 1h day/5 days a week for 6 weeks) and stress/ethanol. Concentration-responses curves to noradrenaline - in the absence and presence of yohimbine, L-NAME or indomethacin - or to phenylephrine were determined in thoracic aortas with and without endothelium. EC50 and maximum response (n=8-12) were compared using two-way ANOVA/Bonferroni method. Results: Either stress or stress in association with ethanol consumption increased the noradrenaline maximum responses in intact aortas. This hyper-reactivity was eliminated by endothelium removal or by the presence of either indomethacin or yohimbine, but was not altered by the presence of L-NAME. Meanwhile, ethanol consumption did not alter the reactivity to noradrenaline. The phenylephrine responses in aortas both with and without endothelium also remained unaffected regardless of protocol. Conclusion: Chronic stress increased rat aortic responses to noradrenaline. This effect is dependent upon the vascular endothelium and involves the release of vasoconstrictor prostanoids via stimulation of endothelial alpha-2 adrenoceptors. Moreover, chronic ethanol consumption appeared to neither influence noradrenaline responses in rat thoracic aorta, nor did it modify the increase of such responses observed as a consequence of stress exposure. .

Vascular adaptive responses to physical exercise and to stress are affected differently by nandrolone administration

Androgenic anabolic steroid, physical exercise and stress induce cardiovascular adaptations including increased endothelial function. The present study investigated the effects of these conditions alone and in combination on the vascular responses of male Wistar rats. Exercise was started at 8 weeks of life (60-min swimming sessions 5 days per week for 8 weeks, while carrying a 5 percent body-weight load). One group received nandrolone (5 mg/kg, twice per week for 8 weeks, im). Acute immobilization stress (2 h) was induced immediately before the experimental protocol. Curves for noradrenaline were obtained for thoracic aorta, with and without endothelium from sedentary and trained rats, submitted or not to stress, treated or not with nandrolone. None of the procedures altered the vascular reactivity to noradrenaline in denuded aorta. In intact aorta, stress and exercise produced vascular adaptive responses characterized by endothelium-dependent hyporeactivity to noradrenaline. These conditions in combination did not potentiate the vascular adaptive response. Exercise-induced vascular adaptive response was abolished by nandrolone. In contrast, the aortal reactivity to noradrenaline of sedentary rats and the vascular adaptive response to stress of sedentary and trained rats were not affected by nandrolone. Maximum response for 7-10 rats/group (g): sedentary 3.8 ± 0.2 vs trained 3.0 ± 0.2*; sedentary/stress 2.7 ± 0.2 vs trained/stress 3.1 ± 0.1*; sedentary/nandrolone 3.6 ± 0.1 vs trained/nandrolone 3.8 ± 0.1; sedentary/stress/nandrolone 3.2 ± 0.1 vs trained/stress/nandrolone 2.5 ± 0.1*; *P < 0.05 compared to its respective control. Stress and physical exercise determine similar vascular adaptive response involving distinct mechanisms as indicated by the observation that only the physical exercise-induced adaptive response was abolished by nandrolone.

The effects of heated vegetable oils on blood pressure in rats

OBJECTIVES: The goal of this study was to determine the possible mechanism that is involved in the blood pressureraising effect of heated vegetable oils. METHODS: Adult male Sprague-Dawley rats were divided into 11 groups; the control group was fed with rat chow, and the other groups were fed with chow that was mixed with 15 percent weight/weight palm or soy oils, which were either in a fresh form or heated once, twice, five, or ten times. Blood pressures were measured at the baseline and throughout the 24-week study. Plasma nitric oxide levels were assessed prior to treatment and at the end of the study. Following 24 weeks, the rats were sacrificed to investigate their vascular reactivity using the thoracic aorta. RESULTS: Palm and soy oils had no detrimental effects on blood pressure, and they significantly elevated the nitric oxide contents and reduced the contractile responses to phenylephrine. However, trials using palm and soy oils that were repeatedly heated showed an increase in blood pressure, enhanced phenylephrine-induced contractions, reduced acetylcholine- and sodium nitroprusside-induced relaxations relative to the control and rats that were fed fresh vegetable oils. CONCLUSIONS: The blood pressure-raising effect of the heated vegetable cooking oils is associated with increased vascular reactivity and a reduction in nitric oxide levels. The chronic consumption of heated vegetable oils leads to disturbances in endogenous vascular regulatory substances, such as nitric oxide. The thermal oxidation of the cooking oils promotes the generation of free radicals and may play an important contributory role in the pathogenesis of hypertension in rats.

Oxidative stress is not associated with vascular dysfunction in a model of alloxan-induced diabetic rats / Estresse oxidativo não está associado à disfunção vascular em um modelo de diabetes induzida por aloxana em ratos

OBJECTIVES: To verify if an experimental model of alloxan-diabetic rats promotes oxidative stress, reduces nitric oxide bioavailability and causes vascular dysfunction, and to evaluate the effect of N-acetylcysteine (NAC) on these parameters. METHODS: Alloxan-diabetic rats were treated or not with NAC for four weeks. Plasmatic levels of malondialdehyde (MDA) and nitrite/nitrate (NOx), the endothelial and inducible nitric oxide synthase (eNOS and iNOS) immunostaining and the vascular reactivity of aorta were compared among diabetic (D), treated diabetic (TD) and control (C) rats. RESULTS: MDA levels increased in D and TD. NOx levels did not differ among groups. Endothelial eNOS immunostaining reduced and adventitial iNOS increased in D and TD. The responsiveness of rings to acetylcholine, sodium nitroprusside, and phenylephrine did not differ among groups. CONCLUSIONS: NAC had no effect on the evaluated parameters and this experimental model did not promote vascular dysfunction despite the development of oxidative stress.

OBJETIVOS: Verificar se um modelo experimental de diabetes por aloxana promove estresse oxidativo, reduz a disponibilidade de óxido nítrico e causa disfunção vascular, e avaliar o efeito da N-acetilcisteína (NAC) nesses parâmetros. MÉTODOS: Ratos diabéticos por aloxana foram tratados com NAC por quatro semanas. Níveis plasmáticos de malondialdeído (MDA) e nitrito/nitrato (NOx), imunomarcação para óxido nítrico sintase endotelial e induzida (eNOS e iNOS) e reatividade vascular da aorta foram comparados entre ratos diabéticos (D), diabéticos tratados (TD) e controles (C). RESULTADOS: O MDA aumentou nos grupos D e TD. O NOx não diferiu entre os grupos. A marcação da eNOS no endotélio reduziu e a da iNOS na adventícia aumentou nos grupos D e TD. A responsividade dos anéis vasculares à acetilcolina, nitroprussiato de sódio e fenilefrina não diferiu entre os grupos. CONCLUSÕES: A NAC não teve efeito sobre os parâmetros avaliados. Esse modelo experimental não promoveu disfunção vascular apesar do desenvolvimento de estresse oxidativo.

Kinetics of cardiac and vascular remodeling by spontaneously hypertensive rats after discontinuation of long-term captopril treatment

Angiotensin-converting enzyme inhibitors reduce blood pressure and attenuate cardiac and vascular remodeling in hypertension. However, the kinetics of remodeling after discontinuation of the long-term use of these drugs are unknown. Our objective was to investigate the temporal changes occurring in blood pressure and vascular structure of spontaneously hypertensive rats (SHR). Captopril treatment was started in the pre-hypertensive state. Rats (4 weeks) were assigned to three groups: SHR-Cap (N = 51) treated with captopril (1 g/L) in drinking water from the 4th to the 14th week; SHR-C (N = 48) untreated SHR; Wistar (N = 47) control rats. Subgroups of animals were studied at 2, 4, and 8 weeks after discontinuation of captopril. Direct blood pressure was recorded in freely moving animals after femoral artery catheterism. The animals were then killed to determine left ventricular hypertrophy (LVH) and the aorta fixed at the same pressure measured in vivo. Captopril prevented hypertension (105 ± 3 vs 136 ± 5 mmHg), LVH (2.17 ± 0.05 vs 2.97 ± 0.14 mg/g body weight) and the increase in cross-sectional area to luminal area ratio of the aorta (0.21 ± 0.01 vs 0.26 ± 0.02 ìm²) (SHR-Cap vs SHR-C). However, these parameters increased progressively after discontinuation of captopril (22nd week: 141 ± 2 mmHg, 2.50 ± 0.06 mg/g, 0.27 ± 0.02 ìm²). Prevention of the development of hypertension in SHR by using captopril during the prehypertensive period prevents the development of cardiac and vascular remodeling. Recovery of these processes follows the kinetic of hypertension development after discontinuation of captopril.

Cardiovascular activity of the n-butanol fraction of the methanol extract of Loranthus ferrugineus Roxb

We investigated the vascular responses and the blood pressure reducing effects of different fractions obtained from the methanol extract of Loranthus ferrugineus Roxb. (F. Loranthaceae). By means of solvent-solvent extraction, L. ferrugineus methanol extract (LFME) was successively fractionated with chloroform, ethyl acetate and n-butanol. The ability of these LFME fractions to relax vascular smooth muscle against phenylephrine (PE)- and KCl-induced contractions in isolated rat aortic rings was determined. In another set of experiments, LFME fractions were tested for blood pressure lowering activity in anesthetized adult male Sprague-Dawley rats (250-300 g, 14-18 weeks). The n-butanol fraction of LFME (NBF-LFME) produced a significant concentration-dependent inhibition of PE- and KCl-induced aortic ring contractions compared to other fractions. Moreover, NBF-LFME had a significantly higher relaxant effect against PE- than against high K+-induced contractions. In anesthetized Sprague-Dawley rats, NBF-LFME significantly lowered blood pressure in a dose-dependent manner and with a relatively longer duration of action compared to the other fractions. HPLC, UV and IR spectra suggested the presence of terpenoid constituents in both LFME and NBF-LFME. Accordingly, we conclude that NBF-LFME is the most potent fraction producing a concentration-dependent relaxation in vascular smooth muscle in vitro and a dose-dependent blood pressure lowering activity in vivo. The cardiovascular effects of NBF-LFME are most likely attributable to its terpenoid content.

[ effect of tribulus terrestris on thoracic aorta contractile response in diabetic rats]

Considering the higher incidence of cardiovascular disorders in diabetes mellitus and some evidence on antioxidant and antidiabetic potential of Tribulus terrestris [TT], this study was conducted to evaluate the beneficial effect of 6-week oral administration of TT on contractile reactivity of isolated thoracic aorta in diabetic rats. Female Wistar rats were divided into control, TT-treated control, diabetic, glibenclamide-treated, and TT-treated diabetic groups. For induction of diabetes, streptozotcin [STZ] was administered [60 mg/Kg]. Meanwhile, TT-treated groups received TT-mixed with standard pelleted food at a weight ratio of 6.25% for 6 weeks. Serum glucose level was measured at weeks 3 and 6. Finally, contractile reactivity of thoracic aortic rings to KCl and phenylephrine [PE] was determined. Serum glucose level at weeks 3 and 6 showed a significant decrease in TT-treated diabetic group [P<0.01 and P<0.005 respectively] compared to diabetics. In addition, TT-treated diabetic group showed a significant lower contraction to PE [P<0.05] as compared to diabetic group and such significant reduction was also observed for KCl [P<0.05]. Meanwhile, there was no significant difference between control and TT-treated control groups regarding their contractile reactivity to KCl and PE. Oral administration of TT for 6 weeks could exert a hypoglycemic effect and also attenuates the contractile responsiveness of the vascular system and this may prevent the development of hypertension in diabetic rats

Fresh soy oil protects against vascular changes in an estrogen-deficient rat model: an electron microscopy study

OBJECTIVE: To observe the effects of consuming repeatedly heated soy oil on the aortic tissues of estrogen-deficient rats. METHODS: Thirty female Sprague Dawley rats (200- 250 g) were divided equally into five groups. One group served as the normal control (NC) group. The four treated groups were ovariectomized and were fed as follows: 2 percent cholesterol diet (OVXC); 2 percent cholesterol diet + fresh soy oil (FSO); 2 percent cholesterol diet + once-heated soy oil (1HSO); and 2 percent cholesterol diet + five-times-heated soy oil (5HSO). After four months, the rats were sacrificed, and the aortic tissues were obtained for histological studies. RESULTS: After four months of feeding, the NC, FSO and 1HSO groups had a lower body weight gain compared to the OVXC and 5HSO groups. The tunica intima/media ratio in the 5HSO group was significantly thicker (p < 0.05) compared to the NC, OVXC and FSO groups. Electron microscopy showed that endothelial cells were normally shaped in the FSO and NC groups but irregular in the 1HSO and 5HSO groups. A greater number of collagen fibers and vacuoles were observed in the 5HSO group compared to the other treatment groups. CONCLUSIONS: Fresh soy oil offered protection in the estrogen-deficient state, as these rats had similar features to those of the NC group. The damage to the tunica intima and the increase in the ratio of tunica intima/media thickness showed the deleterious effect of consuming repeatedly heated soy oil in castrated female rats.