RESUMO
Endothelin (ET)-1 is a likely candidate for a key role in diabetic vascular complications. In the present study, we hypothesized that treatment with pravastatin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) would normalize the ET-1-induced contraction in aortas isolated from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats. Contractile responses were examined by measuring isometric force in endothelium-denuded aortic helical strips from four groups: Long-Evans Tokushima Otsuka (LETO; genetic control), OLETF (type 2 diabetic), pravastatin-treated LETO, and pravastatin-treated OLETF rats. Both immunoblot analysis and immunoprecipitation assays were used to examine Src, protein phosphatase (PP)2A, kinase suppressor of Ras (KSR)1, and ERK signaling pathway protein levels and activities. In endothelium-denuded aortas isolated from OLETF rats at the chronic stage of diabetes (56-60 wk) (vs. those from age-matched LETO rats), we found the following: 1) ET-1-induced contraction was enhanced, 2) ERK1/2 phosphorylation was increased, 3) phosphorylations of KSR1 and PP2A were reduced (i.e., enhancement of the kinase active state), 4) ERK1/2-KSR1 complexes were increased, and 5) Src tyrosine kinase activity was diminished. Endothelium-denuded aortas isolated from OLETF rats treated with pravastatin (10 mg/kg po, daily for 4 wk) exhibited normalized ET-1-induced contractions and suppressed ET-1-stimulated ERK phosphorylation, with the associated phosphorylated KSR1 and phosphorylated PP2A levels being increased toward normal levels. These results suggest that in type 2 diabetic rats, pravastatin normalizes ET-1-induced contraction in aortic smooth muscle via a suppression of PP2A/KSR1/ERK activities after an enhancement of Src kinase activity.
Assuntos
Aorta Torácica/efeitos dos fármacos , Doenças da Aorta/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Endotelina-1/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Pravastatina/farmacologia , Proteínas Quinases/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatologia , Doenças da Aorta/sangue , Doenças da Aorta/enzimologia , Doenças da Aorta/etiologia , Doenças da Aorta/fisiopatologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteína Fosfatase 2/metabolismo , Ratos , Ratos Endogâmicos OLETF , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Quinases da Família src/metabolismoRESUMO
In type 2 diabetes, impaired insulin-induced Akt/endothelial nitric oxide synthase (eNOS) signaling may decrease the vascular relaxation response. Previously, we reported that this response was negatively regulated by G protein-coupled receptor kinase 2 (GRK2). In this study, we investigated whether/how in aortas from ob/ob mice (a model of type 2 diabetes) GRK2 and ß-arrestin 2 might regulate insulin-induced signaling. Endothelium-dependent relaxation was measured in aortic strips. GRK2, ß-arrestin 2, and Akt/eNOS signaling pathway proteins and activities were mainly assayed by Western blotting. In ob/ob (vs. control [Lean]) aortas: 1) insulin-induced relaxation was reduced, and this deficit was prevented by GRK2 inhibitor, anti-GRK2 antibody, and an siRNA specifically targeting GRK2. The Lean aorta relaxation response was reduced to the ob/ob level by pretreatment with an siRNA targeting ß-arrestin 2. 2) Insulin-stimulated Akt and eNOS phosphorylations were decreased. 3) GRK2 expression in membranes was elevated, and, upon insulin stimulation, this expression was further increased, but ß-arrestin 2 was decreased. In ob/ob aortic membranes under insulin stimulation, the phosphorylations of Akt and eNOS were augmented by GRK2 inhibitor. In mouse aorta, GRK2 may be, upon translocation, a key negative regulator of insulin responsiveness and an important regulator of the ß-arrestin 2/Akt/eNOS signaling, which is implicated in diabetic endothelial dysfunction.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Animais , Aorta/metabolismo , Arrestinas/genética , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Insulina/farmacologia , Masculino , Camundongos , Camundongos Obesos , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , beta-Arrestina 2 , beta-ArrestinasRESUMO
Cardiovascular problems are a major cause of morbidity and mortality, mainly due to coronary artery disease and atherosclerosis, in type 2 diabetes mellitus. However, female gender is a protective factor in the development of, for example, atherosclerosis and hypertension. One of the female hormones, 17ß-estradiol (E2), is known to protect against the cardiovascular injury resulting from endothelial dysfunction, but the mechanism by which it does so remains unknown. Our hypothesis was that E2-mediated activation of Akt and mitogen-activated protein kinase (MAPK), and the subsequent endothelial NO synthase (eNOS) phosphorylation, might protect the aorta in diabetic mellitus. The experimental type 2 diabetic model we employed to test that hypothesis (female mice given streptozotocin and nicotinamide) is here termed fDM. In fDM aortas, we examined the E2-induced relaxation response and the associated protein activities. In control (age-matched, nondiabetic) aortas, E2 induced a vascular relaxation response that was mediated via Akt/eNOS and mitogen-activated/ERK-activating kinase (MEK)/eNOS pathways. In fDM aortas (vs. control aortas), (a) the E2-induced relaxation was enhanced, (b) the mediation of the response was different (via Akt/eNOS and p38 MAPK/eNOS pathways), and (c) E2 stimulation increased p38 MAPK and eNOS phosphorylations, decreased MEK phosphorylation, but did not alter estrogen receptor activity. We infer that at least in fDM aortas, E2 has beneficial effects (enhanced vascular relaxation and protection) that are mediated through Akt activation and (compensating for reduced MEK activation) p38 MAPK activation, leading to enhanced eNOS phosphorylation.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Estradiol/farmacologia , Estrogênios/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Diabetes Mellitus Tipo 2/enzimologia , Modelos Animais de Doenças , Feminino , Técnicas In Vitro , Camundongos , Nitratos/análise , Óxido Nítrico Sintase Tipo III/fisiologia , Nitritos/análise , Fosforilação , Vasodilatação/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
In type 2 diabetes, although Akt/endothelial NO synthase (eNOS) activation is known to be negatively regulated by G protein-coupled receptor kinase 2 (GRK2), it is unclear whether the GRK2 inhibitor would have therapeutic effects. Here we examined the hypotensive effect of the GRK2 inhibitor and its efficacy agonist both vascular (aortic) endothelial dysfunction (focusing especially on the Akt/eNOS pathway) and glucose intolerance in two type 2 diabetic models (ob/ob mice and nicotinamide+streptozotocin-induced diabetic mice). Mice were treated with a single injection of the GRK2 inhibitor or vehicle, and the therapeutic effects were compared by examining vascular function and by Western blotting. The GRK2 inhibitor lowered blood pressure in both diabetic models but not in their age-matched controls. The GRK2 inhibitor significantly improved clonidine-induced relaxation only in diabetic (ob/ob and DM) mice, with accompanying attenuations of GRK2 activity and translocation to the plasma membrane. These protective effects of the GRK2 inhibitor may be attributable to the augmented Akt/eNOS pathway activation (as evidenced by increases in Akt phosphorylation at Ser(473) and at Thr(308), and eNOS phosphorylation at Ser(1177)) and to the prevention of the GRK2 translocation and promotion of ß-arrestin 2 translocation to the membrane under clonidine stimulation. Moreover, the GRK2 inhibitor significantly improved the glucose intolerance seen in the ob/ob mice. Our work provides the first evidence that in diabetes, the GRK2 inhibitor ameliorates vascular endothelial dysfunction via the Akt/eNOS pathway by inhibiting GRK2 activity and enhancing ß-arrestin 2 translocation under clonidine stimulation, thereby contributing to a blood pressure-lowering effect. We propose that the GRK2 inhibitor may be a promising therapeutic agent for cardiovascular complications in type 2 diabetes.
Assuntos
Arrestinas/biossíntese , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/genética , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Pressão Sanguínea , Clonidina/farmacologia , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Teste de Tolerância a Glucose , Masculino , Camundongos , Camundongos Obesos , Nitratos/química , Óxido Nítrico Sintase Tipo III/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , beta-Arrestina 2 , beta-ArrestinasRESUMO
In the present sutdy, we have examined the relationship between the CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) pathway and endothelial dysfunction in aortas from GK (Goto-Kakizaki) Type 2 diabetic rats. The ACh (acetylcholine)-induced relaxation and NO production were each attenuated in diabetic aortas (compared with those from age-matched control rats). ACh-stimulated Ser(1177)-eNOS (endothelial NO synthase) phosphorylation was significantly decreased in diabetic aortas (compared with their controls). ACh markedly increased the CaMKII phosphorylation level within endothelial cells only in control aortas (as assessed by immunohistochemistry and Western blotting). ACh-stimulated Thr(286)-CaMKII phosphorylation within endothelial cells was significantly decreased in diabetic aortas (compared with their controls). The ACh-induced relaxations, NO production, eNOS phosphorylation, and CaMKII phosphorylation were inhibited by KN93 and/or by lavendustin C (inhibitors of CaMKII) in control aortas, but not in diabetic ones. Pre-incubation of aortic strips with a PP (protein phosphatase)-1 inhibitor, PPI2 (protein phosphatase inhibitor 2), or with a PP2A inhibitor, CA (cantharidic acid), corrected the above abnormalities in diabetic aortas. The expression of PP2A type A subunit was increased in diabetic aortas. The ACh-stimulated Thr(320)-phosphorylation level of PP1α was lower in diabetic aortas than in their controls, but the total PP1α protein level was not different. These results suggest that the aortic relaxation responses, NO production, and eNOS activity mediated by CaMKII phosphorylation are decreased in this Type 2 diabetic model, and that these impairments of CaMKII signalling may be, at least in part, due to enhancements of PP1α activity and PP2A expression.
Assuntos
Aorta/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Diabetes Mellitus Tipo 2/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Acetilcolina/metabolismo , Acetilcolina/fisiologia , Animais , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Diabetes Mellitus Tipo 2/fisiopatologia , Técnicas In Vitro , Masculino , Óxido Nítrico/biossíntese , Fenóis/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas/metabolismo , Ratos , Ratos Endogâmicos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
Angiotensin II type 2 receptor (AT(2)R) stimulation may cause vasodilation. It could thereby contribute to the antihypertensive effects of angiotensin II type 1 receptor (AT(1)R) antagonists since AT(1)R blockade reportedly increases endogenous levels of Ang II, and this may then bind to the unblocked AT(2)R. Because this is potentially an important consideration in diabetes, we examined whether or not AT(2)R mediates vasorelaxation in db/db diabetic mice. We also examined if AT(2)R-mediated vasorelaxation is preserved after long-term treatment with the AT(1)R antagonist losartan. The effects of AT(2)R stimulation, with either Ang II or the selective agonist CGP-42112A, were studied in aortas from db/db mice (a type 2 diabetic model). CGP-42112A induced a concentration-dependent relaxation in db/db aortas (not in Lean aortas), and this was significantly weakened by the MEK-inhibitor PD98059. CGP-42112A-induced relaxations were increased by Ang II-stimulation (by the organ-culture method) or by AT(1)R blockade (by long-term losartan treatment) only in Lean aortas. Basal AT(2)R expression, and Ang II-stimulated MEK and eNOS phosphorylations were all increased in aortas from db/db (vs. Lean) mice. Long-term losartan treatment increased Ang II-stimulated MEK and eNOS phosphorylations in aortas from Lean, but not db/db, mice. Therefore, this study has provided evidence that AT(2)R-mediated NO production and vasorelaxation through a MEK pathway are enhanced (under basal conditions) in aortas from db/db (vs. Lean) mice. The preservation of such AT(2)R function during AT(1)R blockade needs to be considered in the search for a physiological role for AT(2)R.
Assuntos
Aorta/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Angiopatias Diabéticas/enzimologia , Endotélio Vascular/enzimologia , MAP Quinase Quinase Quinases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Vasodilatação , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/tratamento farmacológico , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Ativação Enzimática , Flavonoides/farmacologia , Losartan/farmacologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Oligopeptídeos/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 2 de Angiotensina/agonistas , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Prostaglandin E(2) (PGE(2)), an important and ubiquitously present vasoactive eicosanoid, may either constrict or dilate systemic vascular beds. However, little is known about the vascular contractile responsiveness to and signaling pathways for PGE(2) at the chronic stage of type 2 diabetes. We hypothesized that PGE(2)-induced arterial contraction is augmented in type 2 diabetic Goto-Kakizaki (GK) rats via the protein kinase Cδ (PKCδ) pathway. Here, we investigated the vasoconstrictor effects of PGE(2) and of sulprostone (EP1-/EP3-receptor agonist) in rings cut from superior mesenteric arteries isolated from GK rats (37-44 weeks old). In arteries from GK rats (vs. those from age-matched Wistar rats), examined in the presence of a nitric oxide synthase inhibitor: 1) the PGE(2)- and sulprostone-induced vasocontractions (which were not blocked by the selective EP1 receptor antagonist sc19220) were enhanced, and these enhancements were suppressed by rottlerin (selective PKCδ inhibitor) but not by Gö6976 (selective PKCα/ß inhibitor); 2) the sulprostone-stimulated phosphorylation of PKCδ (at Thr(505)), which yields an active form, was increased and 3) sulprostone-stimulated caldesmon phosphorylations, which are related to isometric force generation in smooth muscle, were increased. The protein expression of EP3 receptor in superior mesenteric arteries was similar between the two groups of rats. Our data suggest that the diabetes-related enhancement of EP3 receptor-mediated vasocontraction results from activation of the PKCδ pathway. Alterations in EP3 receptor-mediated vasocontraction may be important factors in the pathophysiological influences over arterial tone that are present in diabetic states.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Dinoprostona/análogos & derivados , Dinoprostona/metabolismo , Artérias Mesentéricas/fisiopatologia , Proteína Quinase C-delta/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Vasoconstrição , Animais , Proteínas de Ligação a Calmodulina/metabolismo , Diabetes Mellitus Tipo 2/enzimologia , Dinoprostona/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Fosforilação , Ratos , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP3/agonistas , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
Cardiovascular problems are major causes of morbidity and mortality, the main problems being coronary artery disease and atherosclerosis, in type 2 diabetes mellitus. However, female gender is a protective factor in the development of, for example, atherosclerosis and hypertension. Our aim was to investigate possible gender differences in the activation of Akt/eNOS signaling in aortas from a mouse type 2 diabetic model. Nonfasting plasma glucose was significantly above control in the diabetic mice (both males and females). Plasma insulin was not different between the age-matched controls and the diabetic mice (of either gender). In diabetic males (vs male controls and/or diabetic females): (a) systemic blood pressure was elevated, (b) the clonidine- and insulin-induced Akt-dependent aortic relaxations were impaired, but the ACh-induced Akt-independent and SNP-induced endothelium-independent aortic relaxations were not, (c) Akt and eNOS expression levels were lower, (d) both Akt phosphorylation at Ser(473) and eNOS phosphorylation at Ser(1177) in the aorta were lower under clonidine- or insulin-stimulation, but not under ACh-stimulation. These results suggest that in mice: (i) endothelial functions mediated via the Akt/eNOS pathway are abrogated in type 2 diabetes only in males and (ii) in females (vs males), eNOS expression is elevated and the endothelium resists dysfunction.
Assuntos
Aorta/enzimologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/prevenção & controle , Endotélio Vascular/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Vasodilatação , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/fisiopatologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico/metabolismo , Fosforilação , Fatores Sexuais , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Circulating levels of endothelin (ET)-1 are increased in the diabetic state, as is endogenous ET(A)-receptor-mediated vasoconstriction. However, the responsible mechanisms remain unknown. We hypothesized that ET-1-induced vasoconstriction is augmented in type 2 diabetes with hyperglycemia through an increment in advanced glycation end-products (AGEs). So, we investigated whether treatment with aminoguanidine (AG), an inhibitor of AGEs, would normalize the ET-1-induced contraction induced by ET-1 in strips of thoracic aortas isolated from OLETF rats at the chronic stage of diabetes. In such aortas (vs. those from age-matched genetic control LETO rats): (1) the ET-1-induced contraction was enhanced, (2) the levels of HIF1α/ECE1/plasma ET-1 and plasma CML-AGEs were increased, (3) the ET-1-stimulated ERK phosphorylation mediated by ET(A)-R was increased, (4) the expression level of Jab1-modified ET(A)-R protein was reduced, and (5) the expression level of O-GlcNAcylated ET(A)-R protein was increased. Aortas isolated from such OLETF rats that had been treated with AG (50mg/kg/day for 10 weeks) exhibited reduced ET-1-induced contraction, suppressed ET-1-stimulated ERK phosphorylation accompanied by down-regulation of ET(A)-R, and increased modification of ET(A)-R by Jab1. Such AG-treated rats exhibited normalized plasma ET-1 and CML-AGE levels, and their aortas exhibited decreased HIF1α/ECE1 expression. However, such AG treatment did not alter the elevated levels of plasma glucose or insulin, or systolic blood pressure seen in OLETF rats. These data from the OLETF model suggest that within the timescale studied here, AG normalizes ET-1-induced aortic contraction by suppressing ET(A)-R/ERK activities and/or by normalizing the imbalance between Jab1 and O-GlcNAc in type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Endotelina-1/farmacologia , Guanidinas/farmacologia , Proteínas/metabolismo , Receptor de Endotelina A/metabolismo , Vasoconstrição/efeitos dos fármacos , Acetilglucosamina/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Glicemia/análise , Pressão Sanguínea/efeitos dos fármacos , Complexo do Signalossomo COP9 , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dinoprostona/farmacologia , Endotelina-1/sangue , Endotelina-1/metabolismo , Endotelinas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Técnicas In Vitro , Insulina/sangue , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Fragmentos de Peptídeos/farmacologia , Fenilefrina/farmacologia , Fosforilação , Ratos , Ratos Endogâmicos OLETF , Receptor de Endotelina B/metabolismoRESUMO
In diabetic states, endothelial dysfunction is related to vascular complications. We hypothesized that insulin-induced relaxation and the associated proline-rich tyrosine kinase 2 (Pyk2)/Src/Akt pathway would be abnormal in aortas from the Goto-Kakizaki (GK) type 2 diabetic rat, which exhibits hyperglycemia/insulin resistance, and that losartan treatment of such rats (25 mg·kg(-1)·day(-1) for 2 wk) would correct these abnormalities. Endothelium-dependent relaxation was by measuring isometric force in helical strips of aortas from four groups, each of 30 rats: normal Wistar (control), GK (diabetic), losartan-treated normal, and losartan-treated GK. Pyk2, Src, and Akt/endothelial nitric oxide synthase (eNOS) signaling-pathway protein levels and activities were assayed mainly by Western blotting and partly by immunohistochemistry. In GK (vs. age-matched control) aortas, various insulin-stimulated levels [nitric oxide production and the phosphorylations of eNOS at Ser(1177), of Akt at Thr(308), of phosphoinositide-dependent kinase-1 (PDK1) at Ser(241), of Src at Tyr(416), and of Pyk2 at Tyr(579)] were all significantly decreased and unaffected by either Src inhibitor (PP2) or Pyk2 inhibitor (AG17), while the insulin-stimulated levels of insulin receptor substrate (IRS)-1 phosphorylation at Ser(307), total-eNOS, and total-Akt were significantly increased. Losartan treatment normalized these altered levels. The insulin-stimulated phosphorylation levels of Src/PDK1/Akt/eNOS, but not of Pyk2, were decreased by PP2 in control and losartan-treated GK, but not in GK, aortas. These results suggest that in the GK diabetic aorta increased phospho-IRS-1 (at Ser(307)) and decreased Pyk2/Src activity inhibit insulin-induced stimulation of the PDK/Akt/eNOS pathway. The observed increase in phospho-IRS-1 (at Ser(307)) may result from increased angiotensin II activity.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Aorta/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Quinase 2 de Adesão Focal/metabolismo , Losartan/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasodilatação/efeitos dos fármacos , Quinases da Família src/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Angiotensina II/metabolismo , Animais , Aorta/enzimologia , Aorta/fisiopatologia , Western Blotting , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Quinase 2 de Adesão Focal/antagonistas & inibidores , Imuno-Histoquímica , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Vasodilatadores/farmacologia , Quinases da Família src/antagonistas & inibidoresRESUMO
Little is known about the vascular contractile responsiveness to, and signaling pathways for, extracellular nucleotides in the chronic stage of type 2 diabetes or whether the ANG II type 1 receptor blocker losartan might alter such responses. We hypothesized that nucleotide-induced arterial contractions are augmented in diabetic Goto-Kakizaki (GK) rats and that treatment with losartan would normalize the contractions. Here, we investigated the vasoconstrictor effects of ATP/UTP in superior mesenteric arteries isolated from GK rats (37-42 wk old) that had or had not received 2 wk of losartan (25 mg·kg(-1)·day(-1)). In arteries from GK rats (vs. those from Wistar rats), 1) ATP- and UTP-induced contractions, which were blocked by the nonselective P2 antagonist suramin, were enhanced, and these enhancements were suppressed by endothelial denudation, by cyclooxygenase (COX) inhibitors, or by a cytosolic phospholipase A(2) (cPLA(2)) inhibitor; 2) both nucleotides induced increased release of PGE(2) and PGF(2α); 3) nucleotide-stimulated cPLA(2) phosphorylations were increased; 4) COX-1 and COX-2 expressions were increased; and 5) neither P2Y2 nor P2Y6 receptor expression differed, but P2Y4 receptor expression was decreased. Mesenteric arteries from GK rats treated with losartan exhibited (vs. untreated GK) 1) reduced nucleotide-induced contractions, 2) suppressed UTP-induced release of PGE(2) and PGF(2α), 3) suppressed UTP-stimulated cPLA(2) phosphorylation, 4) normalized expressions of COX-2 and P2Y4 receptors, and 5) reduced superoxide generation. Our data suggest that the diabetes-related enhancement of ATP-mediated vasoconstriction was due to P2Y receptor-mediated activation of the cPLA(2)/COX pathway and, moreover, that losartan normalizes such contractions by a suppressing action within this pathway.
Assuntos
Trifosfato de Adenosina/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Losartan/farmacologia , Artéria Mesentérica Superior/efeitos dos fármacos , Uridina Trifosfato/metabolismo , Vasoconstrição/efeitos dos fármacos , Animais , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/fisiopatologia , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Artéria Mesentérica Superior/metabolismo , Artéria Mesentérica Superior/fisiopatologia , Fosforilação , Antagonistas do Receptor Purinérgico P2/farmacologia , Ratos , Ratos Wistar , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
In diabetic states, hyperinsulinemia may negatively regulate Akt/endothelial nitric oxide synthase (eNOS) activation. Our main aim was to investigate whether and how insulin might negatively regulate Akt/eNOS activities via G protein-coupled receptor kinase 2 (GRK2) in aortas from ob/ob mice. Endothelium-dependent relaxation was measured in aortic rings from ob/ob mice (a type 2 diabetes model). GRK2, ß-arrestin2, and Akt/eNOS signaling-pathway protein levels and activities were mainly assayed by Western blotting. Plasma insulin was significantly elevated in ob/ob mice. Insulin-induced relaxation was significantly decreased in the ob/ob aortas [vs. age-matched control (lean) ones]. The response in ob/ob aortas was enhanced by PKC inhibitor or GRK2 inhibitor. Akt (at Thr(308)) phosphorylation and eNOS (at Ser(1177)) phosphorylation, and also the ß-arrestin2 protein level, were markedly decreased in the membrane fraction of insulin-stimulated ob/ob aortas (vs. insulin-stimulated lean ones). These membrane-fraction expressions were enhanced by GRK2 inhibitor and by PKC inhibitor in the ob/ob group but not in the lean group. PKC activity was much greater in ob/ob than in lean aortas. GRK2 protein and activity levels were increased in ob/ob and were greatly reduced by GRK2 inhibitor or PKC inhibitor pretreatment. These results suggest that in the aorta in diabetic mice with hyperinsulinemia an upregulation of GRK2 and a decrease in ß-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.
Assuntos
Aorta/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Endotélio Vascular/enzimologia , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Insulina/sangue , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasodilatação , Análise de Variância , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Arrestinas/metabolismo , Glicemia/metabolismo , Western Blotting , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Ativação Enzimática , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Hiperinsulinismo/enzimologia , Hiperinsulinismo/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , beta-ArrestinasRESUMO
Nitric oxide (NO) production and endothelial function are mediated via the Akt/eNOS pathway. We investigated the reductions of these mechanism(s) in type 2 diabetes. Diabetic model (nicotinamide+streptozotocin-induced) mice were fed for 4 weeks on a normal diet either containing or not containing losartan, an AT1 R antagonist. Relaxations and NO productions were measured in isolated aortas. G-protein coupled receptor kinase 2 (GRK2) protein levels and activities in the Akt/eNOS signaling-pathway were mainly assayed by Western blotting. Clonidine- and insulin-induced relaxations and NO productions, all of which were significantly decreased in aortas isolated from the diabetics, were normalized by 4 weeks' losartan administration. Plasma angiotensin II (Ang II) and GRK2 protein levels were increased in diabetes, and each was normalized by 4 week's losartan administration. Additionally, there was a direct correlation between the plasma Ang II and aortic GRK2 protein levels. In the diabetics, the clonidine-induced responses (but not the insulin-induced ones) were enhanced by GRK2-inhibitor. Akt phosphorylation was markedly below control in the clonidine-stimulated diabetes. The phosphorylation of Akt at Thr³°8 was significantly normalized and the phosphorylation of eNOS at Ser¹¹77 tended to be increased by GRK2-inhibitor in the clonidine-stimulated diabetics. Our data suggest that (a) the Akt/eNOS pathway is downstream of GRK2, and that GRK2 inhibits Akt/eNOS activities, and (b) this pathway underlies the impaired NO production seen in type 2 diabetes, in which there are defective phosphorylations of Akt and eNOS that may be caused by an upregulation of GRK2 secondary to a high plasma Ang II level. Inhibitors of GRK2 warrant further investigation as potential new therapeutic agents in diabetes.
Assuntos
Angiotensina II/metabolismo , Aorta/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Endotélio Vascular/fisiopatologia , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Angiotensina II/sangue , Animais , Aorta/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/sangue , Masculino , Camundongos , Óxido Nítrico/metabolismoRESUMO
In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.
Assuntos
Células Endoteliais/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais , Animais , Gorduras na Dieta , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Camundongos Knockout , Camundongos Obesos , Óxido Nítrico Sintase Tipo III/metabolismo , FosforilaçãoRESUMO
The aim of the present study was to assess gender differences in diabetes-related vascular reactivity in murine aortas. Diabetes is a risk factor for ischemic heart disease, cerebral ischemia, and atherosclerosis, conditions in which endothelial dysfunction plays a pathogenetic role. We examined vascular responses in aortas isolated from streptozotocin (STZ)-induced type 1 diabetic mice and age-matched control mice, and looked for gender differences in the diabetes-induced changes in these responses. For each gender, the plasma adiponectin levels were lower in diabetic mice than in the controls, and they were significantly higher in females than in males. The acetylcholine (ACh)-induced endothelium-dependent relaxation of aortic rings was impaired (vs. that in the age-matched controls) in diabetic male mice, but not in diabetic female mice. The sodium nitroprusside-induced endothelium-independent aortic relaxation was not altered by diabetes in either male or female mice. The norepinephrine-induced aortic contraction was enhanced (vs. that in the control group) in diabetic female mice, but not in diabetic male mice, whereas in the presence of N(G)-nitro-L-arginine neither gender exhibited a significant diabetes-induced change in this contraction. The clonidine-induced and insulin-induced endothelium-dependent aortic relaxations were impaired only in the diabetic female group (vs. the age-matched controls). These results suggest that: a) in male diabetic mice, which exhibited low adiponectin levels, these were impairments of both the aortic relaxation and nitric oxide (NO) production induced by ACh, whereas b) in female diabetic mice, there were impairments of the aortic relaxations induced by both insulin and clonidine.
Assuntos
Adiponectina/sangue , Aorta/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Endotélio Vascular/fisiopatologia , Fatores Sexuais , Vasoconstrição/fisiologia , Vasodilatação/fisiologia , Acetilcolina , Animais , Arginina , Clonidina , Feminino , Insulina , Masculino , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico/biossíntese , Nitroprussiato , NorepinefrinaRESUMO
We investigated the vasorelaxant activity of the methanolic extracts of Sappan Lignum (CSE) and its constituents, brazilin, sappanchalcone, and protosappanins A-E, on rat aorta and mesenteric artery. By comparing the vasorelaxant activity of CSE and brazilin on both blood vessels, we found that CSE contained active constituents other than brazilin. When added to brazilin, sappanchalcone and protosappanin D showed vasorelaxant activity on both blood vessels precontracted with phenylephrine. We clarified that the vasorelaxant activity of brazilin was endothelium-independent, while that of sappanchalcone was endothelium-dependent, on both blood vessels. On the other hand, the vasorelaxant activity of protosappanin D was independent of the endothelium of the aorta and dependent on the endothelium of the mesenteric artery. Experiments on sappanchalcone and protosappanin D using NG-nitro-L-arginine and indomethacin revealed the involvement of nitric oxide and prostaglandin as endothelium-derived relaxation factors (EDRFs). The anti-oketsu effect of Sappan Lignum might be attributable to the interaction of those compounds. We could partly evaluate the anti-oketsu activity of Sappan Lignum using both the aorta and the mesenteric artery. Through this study, we showed the importance of comparing the effects on the aorta and the mesenteric artery as we found that natural compounds showed different mechanisms of action on the two blood vessels.
Assuntos
Aorta Torácica/efeitos dos fármacos , Caesalpinia , Medicamentos de Ervas Chinesas/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Vasodilatadores/farmacologia , Animais , Aorta Torácica/fisiologia , Benzopiranos/isolamento & purificação , Benzopiranos/farmacologia , Chalconas/isolamento & purificação , Chalconas/farmacologia , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Masculino , Artérias Mesentéricas/fisiologia , Fenóis/isolamento & purificação , Fenóis/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Ratos , Ratos Wistar , Vasodilatadores/química , Vasodilatadores/isolamento & purificaçãoRESUMO
BACKGROUND AND PURPOSE: Arteries from hypertensive subjects are reportedly hyperresponsive to 5-hydroxytryptamine (5-HT), but it remains unclear whether this is true in chronic type 2 diabetes. We have assessed responses to 5-HT shown by mesenteric arteries from type 2 diabetic ob/ob mice (27-32 weeks old) and have identified the molecular mechanisms involved. EXPERIMENTAL APPROACH: Contractions of mesenteric rings to 5-HT were examined in vitro. Activation of mesenteric RhoA, Rho kinase and Src was measured by Western blotting or by modified enzyme-linked immunosorbent assay. KEY RESULTS: Concentration-dependent contractions to 5-HT were greater in mesenteric rings from the ob/ob than in those from the age-matched control ('Lean') group. In each group, there was no significant change in the 5-HT-induced contractions after inhibition of nitric oxide synthase (with N(G)-nitro-L-arginine), of cyclooxygenase (with indomethacin) or of protein kinase C (with chelerythrine). However inhibition of the MEK/ERK pathway (with PD98059) decreased the response to 5-HT. Although the diabetes-related enhancement of the 5-HT response was preserved with each of these inhibitors, enhancement was abolished by a Rho kinase inhibitor (Y27632) and by Src kinase inhibitors (PP1 analogue or Src kinase inhibitor I). 5-HT-induced activation of RhoA, Rho kinase and Src kinase in mesenteric arteries was greater in the ob/ob than in the Lean group, but the expression of RhoA, Rho kinase isoforms and Src did not differ between these groups. CONCLUSIONS AND IMPLICATIONS: These results suggest that the enhancement of 5-HT-induced contraction in mesenteric arteries from ob/ob mice may be attributable to increased activation of RhoA/Rho kinase and Src kinase.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Artérias Mesentéricas/fisiopatologia , Contração Muscular/fisiologia , Serotonina/farmacologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/fisiologia , Quinases da Família src/fisiologia , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Contração Muscular/efeitos dos fármacos , Óxido Nítrico Sintase/antagonistas & inibidores , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/fisiologia , Quinases da Família src/antagonistas & inibidoresRESUMO
The aim of the present study was to investigate the relationship among superoxide anion, peroxisome proliferator-activated receptors (PPARs), and endothelium-dependent relaxation in murine aortas organ-cultured in a high-glucose condition. Aortas organ-cultured with a high concentration of glucose (40 mmol/L, 20 h; HG group) exhibited the following characteristics (versus aortas cultured in serum-free medium): (i) significantly weaker relaxation to acetylcholine, but unchanged relaxation to SNP and unchanged contractions to norepinephrine and isotonic K+, (ii) significantly greater superoxide generation (indicated by the amount of nitroblue tetrazolium reduced, (iii) significantly higher protein expression levels of gp91phox, NAD(P)H oxidase subunits, and endothelial NO synthase, (iv) significantly lower protein expression level of Mn-superoxide dismutase (SOD), and (v) markedly greater reduction in the protein expression of PPARgamma than in that of PPARalpha. The HG-induced impairment of endothelium-dependent relaxation was prevented by cotreatment with tempol (a SOD mimetic). These results suggest that in the mouse aorta, exposure to high glucose levels may lead to an excessive generation of superoxide via increased gp91phox and decreased Mn-SOD protein expression and that this may in turn trigger an impairment of endothelium-dependent relaxation. Moreover, such protein changes in gp91phox and Mn-SOD may be secondary to a decreased expression of PPARgamma protein.
Assuntos
Aorta/fisiologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Superóxidos/metabolismo , Vasodilatação/fisiologia , Animais , Aorta/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos ICR , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Técnicas de Cultura de Órgãos , PPAR alfa/metabolismo , PPAR gama/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Superóxido Dismutase/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
The aim of this study was to investigate the vasorelaxant effect induced by cassiarin A, a novel antiplasmodial alkaloid from Cassia siamea, in rings cut from rat superior mesenteric arteries. In rings precontracted with phenylephrine, cassiarin A induced a concentration-dependent relaxation. This relaxation was attenuated: 1) after removal of the endothelium or after pretreatment of rings with 100 microM of N(G)-nitro-L-arginine (nitric oxide synthase inhibitor) or 10 microM of 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (guanylyl cyclase inhibitor), but not after pretreatment with 10 microM of indomethacin (cyclooxygenase inhibitor); and 2) after pretreatment of preparations with either a nonselective or selective inhibitor of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels [1 mM of tetraethylammonium or 100 nM of iberiotoxin, respectively]. The cassiarin A-induced relaxation was also attenuated by these BK(Ca) inhibitors in endothelium-denuded preparations. The cassiarin A-induced relaxation was not altered by treatment with the ATP-sensitive K(+)-channel inhibitor glibenclamide (10 microM) or with the voltage-dependent K(+)-channel inhibitor 4-aminopyridine (1 mM). In isolated mesenteric artery rings, cassiarin A tended to increase nitric oxide (NO) levels. These results suggest that in the rat mesenteric artery, cassiarin A-induced relaxation may be mediated by endothelial NO and may occur partly via BK(Ca)-channel activation.
Assuntos
Cassia/química , Endotélio Vascular/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasodilatadores/farmacologia , Alcaloides/farmacologia , Animais , Antimaláricos/farmacologia , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , Artéria Mesentérica Superior/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Fenilefrina , Fitoterapia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar , VasoconstritoresRESUMO
It is well known that type 2 diabetes mellitus is frequently associated with vascular dysfunction and an elevated systemic blood pressure, yet the underlying mechanisms are not completely understood. We previously reported that in mesenteric arteries from established type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats, which exhibit endothelial dysfunction, there is an imbalance between endothelium-derived vasodilators [namely, nitric oxide (NO) and hyperpolarizing factor (EDHF)] and vasoconstrictors [contracting factors (EDCFs) such as cyclooxygenase (COX)-derived prostanoids]. Here, we investigated whether the angiotensin II receptor antagonist losartan might improve endothelial dysfunction in OLETF rats at the established stage of diabetes. In mesenteric arteries isolated from OLETF rats [vs. those from age-matched control Long-Evans Tokushima Otsuka (LETO) rats]: (1) the acetylcholine (ACh)-induced relaxation was impaired, (2) the NO- and EDHF-mediated relaxations were reduced, (3) the ACh-induced EDCF-mediated contraction and the production of prostanoids were increased, and (4) superoxide generation was increased. After such OLETF rats had received losartan (25 mg/kg/day p.o. for 4 weeks), their isolated mesenteric arteries exhibited: (1) improvements in ACh-induced NO- and EDHF-mediated relaxations, (2) reduced EDCF- and arachidonic acid-induced contractions, (3) suppressed production of prostanoids, (4) reduced PGE(2)-mediated contraction, and (5) reduced superoxide generation. Within the timescale studied here, losartan did not change the protein expressions of endothelial NO synthase, COX1, or COX2 in mesenteric arteries from either OLETF or LETO rats. Losartan thus normalizes vascular dysfunction in this type 2 diabetic model, and the above effects may contribute to the reduction of adverse cardiovascular events seen in diabetic patients treated with angiotensin II receptor blockers.