RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Achillea millefolium L. (Asteraceae), known as yarrow (milenrama), is a plant used in Mexican traditional medicine for the treatment of hypertension, diabetes, and related diseases. AIM: To determine the vasorelaxant and antihypertensive effect of A. millefollium and to isolate the main bioactive antihypertensive agents. MATERIALS AND METHODS: Organic (hexane, dichloromethane and methanol) and hydro-alcohol (Ethanol-H2O: 70:30) extracts obtained from flowers, leaves and stems were evaluated on isolated aorta rat rings with and without endothelium to determine their vasorelaxant effect. Hexane extract from flowers (HEAmF) was studied to evaluate its antihypertensive effect on spontaneously hypertensive rats (SHR). From HEAmF, bioactive compounds were obtained by bio-guided phytochemical separation through chromatography. RESULTS: Organic extracts showed the best vasorelaxant activity. Hexane extract from flowers was the most potent and efficient ex vivo vasorelaxant agent, showing significant decrease of systolic and diastolic blood pressure in SHR (p < 0.05). Phytochemical separation of HEAmF yielded two epimeric sesquiterpene lactones: leucodin (1) and achillin (2), the major components of the extract. Both 1 and 2 showed similar vasorelaxant action ex vivo (p < 0.05), and their effects where modified by L-NAME (10 µM, nitric oxide synthase inhibitor), by ODQ (1 µM, soluble guanylyl cyclase inhibitor), and also relaxed the contraction induced by KCl (80 mM). Finally, 1 and 2 intragastric administration (50 mg/kg) decreased systolic and diastolic blood pressure in SHR. CONCLUSIONS: Achillea millefolium showed antihypertensive and vasorelaxant effects, due mainly to leucodin and achillin (epimers). Both compounds showed antihypertensive activity by vasorelaxation putatively by endothelium-dependent NO release and cGMP increase, as well as by calcium channels blockade.
Assuntos
Achillea/química , Anti-Hipertensivos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Extratos Vegetais/farmacologia , Sesquiterpenos/farmacologia , Vasodilatadores/farmacologia , Animais , Anti-Hipertensivos/uso terapêutico , Aorta/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio/metabolismo , Simulação por Computador , Frequência Cardíaca/efeitos dos fármacos , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Oxidiazóis/farmacologia , Extratos Vegetais/uso terapêutico , Quinoxalinas/farmacologia , Ratos Endogâmicos SHR , Ratos Wistar , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/uso terapêutico , Vasodilatadores/uso terapêuticoRESUMO
Accumulating evidence demonstrates that microcirculation plays a role in the pathogenesis of hypertension. In the current study, we demonstrated that pancreatic islet microvascular vasomotion of spontaneously hypertensive rats (SHRs) lost the ability to regulate blood flow perfusion and exhibited a lower microvascular blood perfusion pattern which was negative correlated with blood glucose level. SHRs administrated with insulin revealed an improvement of pancreatic islet microvascular vasomotion and blood perfusion pattern. In vitro, the expressions of endothelial nitric oxide synthase (eNOS) and phospho-eNOSser1177 (p-eNOSser1177) were significantly decreased in high glucose exposed islet endothelial cells (iECs), accompanied with a higher ratio of eNOS monomer to eNOS dimer and a significantly increased malondialdehyde and nitrite levels. Meanwhile, barrier function, tube formation and migration capacities of high glucose exposed iECs were significantly inhibited. In contrast, iECs dysfunction induced by glucose toxicity and oxidative stress was attenuated or improved by supplement with insulin, L-arginine and ß-mercaptoethanol. In summary, our findings suggest that functional status of pancreatic islet microvascular vasomotion is impaired in SHRs and provide evidence that treatment with insulin, L-arginine and ß-mercaptoethanol improves endothelium-dependent microvascular vasomotion and meliorates iECs function due to anti-hyperglycemic and anti-oxidative effects, partly through mechanism involving regulation of eNOS and p-eNOSser1177.
Assuntos
Hipertensão/patologia , Ilhotas Pancreáticas/irrigação sanguínea , Microcirculação/fisiologia , Animais , Arginina/farmacologia , Glicemia/análise , Pressão Sanguínea , Movimento Celular/efeitos dos fármacos , Dimerização , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucose/farmacologia , Hipertensão/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Malondialdeído/metabolismo , Mercaptoetanol/farmacologia , Microcirculação/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Endoplasmic reticulum (ER) stress in endothelial cells often leads to endothelial dysfunction which underlies the pathogenesis of cardiovascular diseases. Paeonol, a major phenolic component extracted from Moutan Cortex, possesses various medicinal benefits which have been used extensively in traditional Chinese medicine. The present study investigated the protective mechanism of paeonol against tunicamycin-induced ER stress in isolated mouse aortas and human umbilical vein endothelial cells (HUVECs). Vascular reactivity in aorta was measured using a wire myograph. The effects of paeonol on protein expression of ER stress markers, reactive oxygen species (ROS) production, nitric oxide (NO) bioavailability and peroxisome proliferator-activated receptor δ (PPARδ) activity in the vascular wall were assessed by Western blot, dihydroethidium fluorescence (DHE) or lucigenin enhanced-chemiluminescence, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA) and dual luciferase reporter assay, respectively. Ex vivo treatment with paeonol (0.1µM) for 16h reversed the impaired endothelium-dependent relaxations in C57BJ/6J and PPARδ wild type (WT) mouse aortas following incubation with tunicamycin (0.5µg/mL). Elevated ER stress markers, oxidative stress and reduction of NO bioavailability induced by tunicamycin in HUVECs, C57BJ/6J and PPARδ WT mouse aortas were reversed by paeonol treatment. These beneficial effects of paeonol were diminished in PPARδ knockout (KO) mouse aortas. Paeonol increased the expression of 5' adenosine monophosphate-activated protein kinase (AMPK) and PPARδ expression and activity while restoring the decreased phosphorylation of eNOS. The present study delineates that paeonol protects against tunicamycin-induced vascular endothelial dysfunction by inhibition of ER stress and oxidative stress, thus elevating NO bioavailability via the AMPK/PPARδ signaling pathway.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetofenonas/farmacologia , Antioxidantes/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , PPAR delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Animais , Aorta Torácica , Linhagem Celular , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/agonistas , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , PPAR delta/agonistas , PPAR delta/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnicas de Cultura de TecidosRESUMO
The present research aimed to isolate the non-polar secondary metabolites that produce the vasodilator effects induced by the dichloromethane extract of Prunus serotina (P. serotina) fruits and to determine whether the NO/cGMP and the H2S/KATP channel pathways are involved in their mechanism of action. A bioactivity-directed fractionation of the dichloromethane extract of P. serotina fruits led to the isolation of ursolic acid and uvaol as the main non-polar vasodilator compounds. These compounds showed significant relaxant effect on rat aortic rings in an endothelium- and concentration-dependent manner, which was inhibited by NG-nitro-L-arginine methyl ester (L-NAME), DL-propargylglycine (PAG) and glibenclamide (Gli). Additionally, both triterpenes increased NO and H2S production in aortic tissue. Molecular docking studies showed that ursolic acid and uvaol are able to bind to endothelial NOS and CSE with high affinity for residues that form the oligomeric interface of both enzymes. These results suggest that the vasodilator effect produced by ursolic acid and uvaol contained in P. serotina fruits, involves activation of the NO/cGMP and H2S/KATP channel pathways, possibly through direct activation of NOS and CSE.
Assuntos
Sulfeto de Hidrogênio/agonistas , Óxido Nítrico/agonistas , Prunus avium/química , Triterpenos/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Alcinos/antagonistas & inibidores , Alcinos/farmacologia , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , GMP Cíclico/metabolismo , Cistationina gama-Liase/química , Cistationina gama-Liase/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Frutas/química , Glibureto/antagonistas & inibidores , Glibureto/farmacologia , Glicina/análogos & derivados , Glicina/antagonistas & inibidores , Glicina/farmacologia , Sulfeto de Hidrogênio/metabolismo , Canais KATP/agonistas , Canais KATP/metabolismo , Masculino , Simulação de Acoplamento Molecular , NG-Nitroarginina Metil Éster/antagonistas & inibidores , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Extratos Vegetais/química , Ligação Proteica , Ratos , Triterpenos/isolamento & purificação , Vasodilatadores/isolamento & purificação , Ácido UrsólicoRESUMO
Elevated endothelial arginase activity decreases nitric oxide (NO) production by competing with the substrate l-arginine, previously reported, and reciprocally regulating endothelial nitric oxide synthase (eNOS) activity. Thus, arginase inhibitors may help treat vascular diseases associated with endothelial dysfunction. A screening of metabolites from medicinal plants revealed that (2S)-5,2',5'-trihydroxy-7,8-dimethoxy flavanone (TDF) was a noncompetitive inhibitor of arginase. We investigated whether TDF reciprocally regulated endothelial NO production and its possible mechanism. TDF noncompetitively inhibited arginase I and II activity in a dose-dependent manner. TDF incubation decreased arginase activity and increased NO production in human umbilical vein endothelial cells and isolated mouse aortic vessels and reduced reactive oxygen species (ROS) generation in the endothelium of the latter. These TDF-mediated effects were associated with increased eNOS phosphorylation and dimerization but not with changes in protein content. Endothelium-dependent vasorelaxant responses to acetylcholine (Ach) were significantly increased in TDF-incubated aortic rings and attenuated by incubation with soluble guanylyl cyclase inhibitor. Phenylephrine-induced vasoconstrictor responses were markedly attenuated in TDF-treated vessels from wild-type mice. In atherogenic-prone ApoE(-/-) mice, TDF attenuated the high-cholesterol diet (HCD)-induced increase in arginase activity, which was accompanied by restoration of NO production and reduction of ROS generation. TDF incubation induced eNOS dimerization and phosphorylation at Ser1177. In addition, TDF improved Ach-dependent vasorelaxation responses and attenuated U46619-dependent contractile responses but did not change sodium nitroprusside-induced vasorelaxation or N-NAME-induced vasoconstriction. The findings suggest that TDF may help treat cardiovascular diseases by reducing pathophysiology derived from HCD-mediated endothelial dysfunction.
Assuntos
Apolipoproteínas E/deficiência , Arginase/antagonistas & inibidores , Colesterol na Dieta/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Endotélio Vascular/efeitos dos fármacos , Flavanonas/farmacologia , Scutellaria/química , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/fisiologia , Apolipoproteínas E/genética , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Flavanonas/química , Flavanonas/isolamento & purificação , Flavanonas/uso terapêutico , Deleção de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hiperlipidemias/induzido quimicamente , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/patologia , Hiperlipidemias/fisiopatologia , Masculino , Metanol/química , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The balance of nitric oxide (NO) versus superoxide generation has a major role in the initiation and progression of endothelial dysfunction. Under conditions of high glucose, endothelial nitric oxide synthase (eNOS) functions as a chief source of superoxide rather than NO. In order to improve NO bioavailability within the vessel wall in type-1 diabetes, we investigated treatment strategies that improve eNOS phosphorylation and NO-dependent vasorelaxation. We evaluated methods to increase the eNOS activity by (1) feeding Ins2(Akita) spontaneously diabetic (type-1) mice with l-arginine in the presence of sepiapterin, a precursor of tetrahydrobiopterin; (2) preventing eNOS/NO deregulation by the inclusion of inhibitor kappa B kinase beta (IKKß) inhibitor, salsalate, in the diet regimen in combination with l-arginine and sepiapterin; and (3) independently increasing eNOS expression to improve eNOS activity and associated NO production through generating Ins2(Akita) diabetic mice that overexpress human eNOS predominantly in vascular endothelial cells. Our results clearly demonstrated that diet supplementation with l-arginine, sepiapterin along with salsalate improved phosphorylation of eNOS and enhanced vasorelaxation of thoracic/abdominal aorta in type-1 diabetic mice. More interestingly, despite the overexpression of eNOS, the in-house generated transgenic eNOS-GFP (TgeNOS-GFP)-Ins2(Akita) cross mice showed an unanticipated effect of reduced eNOS phosphorylation and enhanced superoxide production. Our results demonstrate that enhancement of endogenous eNOS activity by nutritional modulation is more beneficial than increasing the endogenous expression of eNOS by gene therapy modalities.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Suplementos Nutricionais , Endotélio Vascular/metabolismo , Hipoglicemiantes/uso terapêutico , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional , Animais , Aorta/citologia , Aorta/metabolismo , Aorta/fisiopatologia , Arginina/metabolismo , Arginina/uso terapêutico , Bovinos , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiopatologia , Feminino , Heterozigoto , Humanos , Hipoglicemiantes/metabolismo , Insulina/genética , Insulina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/genética , Fosforilação , Inibidores de Proteínas Quinases/metabolismo , Pterinas/metabolismo , Pterinas/uso terapêutico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Salicilatos/metabolismo , Salicilatos/uso terapêutico , DesmameRESUMO
This work investigated the blood pressure (BP)-lowering effect of the flavanol (-)-epicatechin in a model of metabolic syndrome. Rats were fed a regular chow diet without (Control) or with 10% (w/v) fructose in the drinking water (high fructose, HF) for 8 weeks. A subgroup of the HF-fed rats was supplemented with (-)-epicatechin 20 mg/kg body weight (HF-EC). Dietary (-)-epicatechin reverted the increase in BP caused by the fructose treatment. In aorta, superoxide anion production and the expression of the NADPH oxidase (NOX) subunits p47(phox) and p22(phox) were enhanced in the HF-fed rats. The increase was prevented by (-)-epicatechin. Similar profile was observed for NOX4 expression. The activity of aorta nitric oxide synthase (NOS) was increased in the HF group and was even higher in the HF-EC rats. These effects were paralleled by increased endothelial NOS phosphorylation at the activation site Ser1177. Among the more relevant mitogen-activated protein kinase pathways in vascular tissue, c-Jun-N-terminal kinase was shown to be activated in the aorta of the HF-fed rats, and (-)-epicatechin supplementation mitigated this activation. Thus, the results suggest that dietary (-)-epicatechin supplementation prevented hypertension in HF-fed rats, decreasing superoxide anion production and elevating NOS activity, favoring an increase in NO bioavailability.
Assuntos
Anti-Hipertensivos/uso terapêutico , Catequina/uso terapêutico , Suplementos Nutricionais , Endotélio Vascular/enzimologia , Hipertensão/prevenção & controle , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/agonistas , Animais , Antioxidantes/uso terapêutico , Aorta Torácica/enzimologia , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Carboidratos da Dieta/efeitos adversos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Frutose/efeitos adversos , Hipertensão/etiologia , Hipertensão/metabolismo , Hipertensão/patologia , Sistema de Sinalização das MAP Quinases , Masculino , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Fosforilação , Processamento de Proteína Pós-Traducional , Distribuição Aleatória , Ratos Sprague-Dawley , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismoRESUMO
The aim of this work was to evaluate the effects of (-)-epicatechin administration in the heart of a rat model with reduced NO production that follows a short-term treatment with L-NAME. Sprague-Dawley rats were treated for 4 d with L-NAME in the absence or presence of (-)-epicatechin in the diet. The redox status in cardiac tissue was improved by (-)-epicatechin administration. L-NAME treatment induced a decrease in NO synthase activity (-62%, p<0.05) and an increase in NADPH-dependent superoxide anion production (+300%, p<0.05) that were totally prevented by (-)-epicatechin administration. These effects of (-)-epicatechin were associated with a higher endothelial NO synthase phosphorylation at an activation site and a reduced expression of the regulatory subunit, p47(phox), suggesting the involvement of posttranslational mechanisms in (-)-epicatechin action. Thus, the (-)-epicatechin treatment would restore NO steady state levels in vivo through effects on both, its synthesis and degradation via the reaction with superoxide anion. The fact that (-)-epicatechin is commonly present in human diet makes this compound a reasonable explanation for the positive cardiovascular effects of a high consumption of fruits and vegetables.
Assuntos
Anti-Hipertensivos/uso terapêutico , Catequina/uso terapêutico , Modelos Animais de Doenças , Ventrículos do Coração/enzimologia , Hipertensão/prevenção & controle , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Vasos Coronários/enzimologia , Vasos Coronários/metabolismo , Suplementos Nutricionais , Ativação Enzimática , Frutas/química , Ventrículos do Coração/metabolismo , Hipertensão/enzimologia , Hipertensão/metabolismo , Masculino , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NG-Nitroarginina Metil Éster , Óxido Nítrico/agonistas , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Fosforilação , Processamento de Proteína Pós-Traducional , Distribuição Aleatória , Ratos Sprague-Dawley , Estereoisomerismo , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismo , Verduras/químicaRESUMO
Diabetic foot ulcer is closely associated with peripheral vascular disease. Enhancement of tissue oxidative stress, reduction of nitric oxide (NO) and angiogenic growth factors, and abnormal matrix metalloproteinase (MMP) activity are pathophysiological factors in post-ischemic neovascularization and diabetic wound healing. Our previous study demonstrated that the Chinese 2-herb formula, NF3, showed significant wound healing effects on diabetic foot ulcer rats. A novel rat diabetic foot ulcer with hindlimb ischemia model was established in order to strengthen our claims on the diabetic wound healing and post-ischemic neovascularization effects of NF3. Our results demonstrate that NF3 can significantly reduce the wound area of the diabetic foot ulcer rat with hindlimb ischemia by 21.6% (p<0.05) compared with the control group. In addition, flow cytometric analysis revealed that NF3 could boost circulating EPC levels for local wound vessel incorporation. Immunohistochemical analysis showed that NF3 could significantly augment blood vessel density, VEGF and eNOS expression, and attenuate tissue oxidative stress of ischemic muscles (p<0.001). NF3 significantly stimulated MMP activity involved in angiogenesis. Our study shows, for the first time, the beneficial effects of NF3 in wound healing and post-ischemic neovascularization in diabetes.
Assuntos
Indutores da Angiogênese/uso terapêutico , Pé Diabético/tratamento farmacológico , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/uso terapêutico , Neovascularização Fisiológica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Pé Diabético/etiologia , Pé Diabético/metabolismo , Pé Diabético/patologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Feminino , Membro Posterior , Isquemia/fisiopatologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular/agonistas , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND AND AIMS: Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are important intestinal neurotransmitters that coexist in the gut enteric nervous system and play an important role in intestinal physiology (e.g., absorption, motility, fluid secretion and smooth muscle relaxation). It is also known that cold exposure alters several aspects of gastrointestinal physiology and induces hyperphagia to meet increased metabolic demands, but there are no data regarding NO and VIP involvement in intestinal response during acclimation to cold. The objective of this study was to determine the influence of long-term L-arginine supplementation on the expression of the three isoforms of nitric oxide synthase (NOS) and VIP in small intestine of rats acclimated to room temperature or cold. METHODS: Animals (six per group) acclimated to room temperature (22 ± 1 °C) and cold (4 ± 1 °C), respectively, were treated with 2.25% L-arginine, a substrate for NOSs, or with 0.01% N(ω)-nitro-L-arginine methyl ester, an inhibitor of NOSs, for 45 days. The topographical distribution of VIP and NOSs expression in small intestine was studied by immunohistochemistry, and ImageJ software was used for semiquantitative densitometric analysis of their immunoexpression. RESULTS: Long-term dietary L-arginine supplementation increases VIP and NOSs immunoexpression at room temperature while at cold increases the endothelial NOS, inducible NOS and VIP but decrease neuronal NOS in rat small intestine. CONCLUSION: Our results demonstrate that long-term dietary L-arginine supplementation modulates NOSs and VIP immunoexpression in rat small intestine with respect to ambient temperature, pointing out the eNOS as a predominant NOS isoform with an immunoexpression pattern similar to VIP.
Assuntos
Arginina/metabolismo , Suplementos Nutricionais , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Regulação para Cima , Peptídeo Intestinal Vasoativo/agonistas , Adaptação Fisiológica/efeitos dos fármacos , Animais , Arginina/antagonistas & inibidores , Temperatura Baixa/efeitos adversos , Cruzamentos Genéticos , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Inibidores Enzimáticos/farmacologia , Imuno-Histoquímica , Células Intersticiais de Cajal/citologia , Células Intersticiais de Cajal/efeitos dos fármacos , Células Intersticiais de Cajal/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/química , Ratos , Regulação para Cima/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
The elevated level of uric acid in the body is associated with increased risk of cardiovascular diseases, which is mediated by endothelial dysfunction. However, its underlying mechanism is not fully understood, although dysregulation of endothelial nitric oxide (NO) production is likely to be involved. Using human umbilical vascular endothelial cells (HUVEC), we explored the molecular mechanism of uric acid on endothelial NO synthase (eNOS) activity and NO production. Although high dose of uric acid (12mg/dl for 24h treatment) significantly decreased eNOS activity and NO production, it did not alter eNOS expression and phosphorylations at eNOS-Ser(1177), eNOS-Thr(495) and eNOS-Ser(114). Under this condition, we also found no alterations in the dimerization and acetylation of eNOS, compared with the control. Furthermore, uric acid did not change the activity of arginase II, an enzyme degrading l-arginine, a substrate of eNOS, and intracellular level of calcium, a cofactor for eNOS activation. We also found that uric acid did not alter xanthine oxidase activity, suggesting no involvement of xanthine oxidase-derived O2(-) production in the observed inhibitory effects. In vitro and in cell coimmunoprecipitation studies, however, revealed that uric acid significantly decreased the interaction between eNOS and calmodulin (CaM), an eNOS activator, although it did not change the intracellular CaM level. Like in HUVEC, uric acid also decreased eNOS-CaM interaction in bovine aortic EC. Finally, uric acid attenuated ionomycin-induced increase in the interaction between eNOS and CaM. This study suggests firstly that uric acid decreased eNOS activity and NO production through reducing the binding between eNOS and CaM in EC. Our result may provide molecular mechanism by which uric acid induces endothelial dysfunction.
Assuntos
Calmodulina/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/biossíntese , Ácido Úrico/farmacologia , Animais , Calmodulina/química , Bovinos , Células Cultivadas , Interações Medicamentosas , Humanos , Ionomicina/farmacologia , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Ligação Proteica , Transdução de SinaisRESUMO
UNLABELLED: Ginsenoside Rb1 and Rg1 are major components of Panax notoginseng (P.N.), an herb with known clinical efficacy in hypertension and myocardial ischemia in Eastern countries. This investigation is to elicit the mechanism of these components in hypertension via their effect on vascular reaction. To assess the ability of P.N. in hypertension, P.N. extracts were injected in spontaneously hypertensive rats (SHR) via the vena caudalis; Low dosages of P.N. extracts significantly lowered blood pressure in SHR. Examination with Rb1 and Rg1 revealed significant vasodilatation using mouse coronary arteries in a dose-dependent manner. Rb1- and Rg1-induced vasodilatation was blocked by pre-incubation with eNOS and PI3K inhibitors. Coronaries of eNOS-/- mice showed attenuated vasodilatation with Rb1 and Rg1. In addition, both Rb1 and Rg1 induce nitric oxide (NO) generation through increasing the phosphorylation of eNOS, activating Na+-independent l-arginine transport, and stimulating cationic amino acid transport (CAT)-1 mRNA expression in cultured endothelial cells. CONCLUSION: Ginsenoside Rb1 and Rg1 increased endothelial-dependent vessel dilatation through the activation of NO by modulating the PI3K/Akt/eNOS pathway and l-arginine transport in endothelial cells. These findings may have important implications for understanding the mechanisms of clinical efficacy of the herb P.N. when used in the regulation of blood pressure.