RESUMO
The present study was initiated to characterize vascular dysregulations (contraction and relaxation) associated with metabolic defects in Merions shawi, a rodent from the gerbillidae family, submitted to 12 weeks high-calorie diet. This diet induces a type 2 diabetes/metabolic syndrome phenotype with hypertension. In diabetic meriones, body weight increase was associated with hyperglycemia, increased insulinemia, and insulin resistance. Compared to lean meriones, diabetic meriones showed decreased aorta contraction to noradrenaline, which was normalized after NOS inhibition. Endothelium-dependent relaxation to carbachol was enhanced, while relaxing effects of the NO donor SNAP and of diazoxide were unchanged. Insulin-evoked relaxation was depressed in aorta from diabetic meriones, and L-arginine relaxed contracted arteries from diabetic meriones, but not from lean meriones. Urine NOX level and iNOS mRNA muscle expression were significantly higher in diabetic meriones compared to lean animals. These data strongly suggest that iNOS may have a pathogenic role in vascular dysfunction observed in diet-induced diabetic meriones.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Ingestão de Energia , Hipertensão/fisiopatologia , Síndrome Metabólica/fisiopatologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta/fisiopatologia , Arginina/farmacologia , Carbacol/farmacologia , Diazóxido/farmacologia , Endotélio Vascular/fisiopatologia , Gerbillinae , Insulina/farmacologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Óxido Nítrico/urina , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Norepinefrina/farmacologia , RNA Mensageiro/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacologiaRESUMO
Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca(2+) signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.
Assuntos
Movimento Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Adesões Focais/fisiologia , Proteínas de Membrana/fisiologia , Proteínas dos Microfilamentos/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Adesões Focais/efeitos dos fármacos , Masculino , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Pseudópodes/fisiologia , Ratos , Ratos WistarRESUMO
Calycotome villosa subsp. intermedia is used in traditional medicine for the prevention and self-treatment of a variety of illnesses, including diabetes mellitus, obesity, and hypertension. The present study aims to investigate the in vivo, ex vivo, and in vitro hypoglycemic and hypotensive effects of the lyophilized aqueous extract of Calycotome villosa subsp. intermedia seeds (CV) on Meriones shawi submitted to hypercaloric diet and physical inactivity (HCD/PI) for 12 weeks. This diet induces a type 2 diabetes/metabolic syndrome phenotype with hypertension. Furthermore, HCD/PI decreased aorta contraction due to noradrenaline, enhanced L-arginine, and depressed insulin-evoked relaxation, while the relaxing effects of the NO donor SNAP and of diazoxide were unchanged. In vivo experiments showed that the oral administration of the CV extract (50 mg/kg b.wt) for 3 consecutive weeks significantly attenuated the development of type 2 diabetes, obesity, dyslipidemia, and hypertension. These effects may involve the improvement of lipid metabolism, insulin sensitivity, systolic arterial pressure, and urine output. Additionally, ex vivo and in vitro investigations revealed that CV treatment improved vascular contraction to noradrenaline, induced a slight aorta relaxation in response to carbachol, increased the vasorelaxation effect evoked by insulin, and depressed the L-arginine evoked relaxation. However, CV did not change the endothelium-independent vasorelaxation response evoked by SNAP or diazoxide. Hence, the present study provides useful information and supports the traditional use of CV in the prevention and self-treatment of numerous ailments. Overall, it can be concluded that Calycotome villosa subsp. intermedia seed extracts might be useful in the management of type 2 diabetes and hypertension.
RESUMO
In addition to its role in the regulation of artery contraction, Rho kinase (ROCK) was reported to be involved in the cytosolic calcium response to vasoconstrictor agonists in rat aorta and superior mesenteric artery (SMA). However, it remains to be determined whether ROCK also contributes to calcium signaling in resistance arteries, which play a major role in blood pressure regulation. The investigation of the effect of ROCK inhibition on the calcium and contractile responses of rat resistance mesenteric artery (RMA), in comparison with aorta and SMA, indicated that the calcium response to noradrenaline was inhibited by the ROCK inhibitor Y-27632 in aorta and SMA but not in RMA. The effect of Y-27632 on the calcium signal was unaffected by cytochalasin-D. ROCK activation in noradrenaline-stimulated arteries was confirmed by the inhibition of myosin light chain phosphorylation by Y-27632. Moreover, noradrenaline-induced calcium signaling was similarly inhibited by nimodipine in aorta, SMA and RMA, but nimodipine sensitivity of the contraction increased from the aorta to the RMA, suggesting that the contraction was controlled by different sources of calcium. In pressurized RMA, Y-27632 and H-1152 depressed pressure-induced calcium responses and abolished myogenic contraction. These results stress the important differences in calcium signaling between conductance and resistance arteries.
Assuntos
Amidas/farmacologia , Aorta/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Aorta/metabolismo , Masculino , Artéria Mesentérica Superior/efeitos dos fármacos , Norepinefrina/farmacologia , Ratos , Ratos Wistar , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
The Liver Toxicity Biomarker Study is a systems toxicology approach to discover biomarkers that are indicative of a drug's potential to cause human idiosyncratic drug-induced liver injury. In phase I, the molecular effects in rat liver and blood plasma induced by tolcapone (a "toxic" drug) were compared with the molecular effects in the same tissues by dosing with entacapone (a "clean" drug, similar to tolcapone in chemical structure and primary pharmacological mechanism). Two durations of drug exposure, 3 and 28 days, were employed. Comprehensive molecular analysis of rat liver and plasma samples yielded marker analytes for various drug-vehicle or drug-drug comparisons. An important finding was that the marker analytes associated with tolcapone only partially overlapped with marker analytes associated with entacapone, despite the fact that both drugs have similar chemical structures and the same primary pharmacological mechanism of action. This result indicates that the molecular analyses employed in the study are detecting substantial "off-target" markers for the two drugs. An additional interesting finding was the modest overlap of the marker data sets for 3-day exposure and 28-day exposure, indicating that the molecular changes in liver and plasma caused by short- and long-term drug treatments do not share common characteristics.
Assuntos
Benzofenonas/toxicidade , Catecóis/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Nitrilas/toxicidade , Nitrofenóis/toxicidade , Animais , Biomarcadores/análise , Proteínas Sanguíneas/análise , Doença Hepática Induzida por Substâncias e Drogas/sangue , Feminino , Perfilação da Expressão Gênica , Fígado/química , Fígado/metabolismo , Masculino , Metaboloma/efeitos dos fármacos , Metabolômica , Proteoma/análise , Proteoma/efeitos dos fármacos , Proteômica , Ratos , Projetos de Pesquisa , Tolcapona , Testes de Toxicidade Aguda/métodos , Testes de Toxicidade Crônica/métodosRESUMO
Ezrin, Radixin, Moesin binding phosphoprotein 50 (EBP50) is a scaffold protein that possesses two PDZ interacting domains. We have shown that, in isolated artery stimulated with noradrenaline, EBP50 interacts with several elements of the cytoskeleton. However, the contribution of EBP50 to the organization of the cytoskeleton is unknown. We have used primary cultured vascular smooth muscle cells to investigate the involvement of EBP50 in the regulation of cell architecture, motility and cell cycle, and to identify its target proteins and subsequent action mechanism. The results showed that depletion of EBP50 by siRNA transfection induced changes in cell architecture and increased cell migration. The same phenotype was induced by inhibition of myosin IIa and this effect was not additive in cells depleted for EBP50. Moreover, a larger proportion of binucleated cells was observed after EBP50 depletion, indicating a defect in cytokinesis. The identification, after co-immunoprecipitation, of a direct interaction of EBP50 with both tubulin and myosin IIa suggested that EBP50 could regulate cell migration and cytokinesis by linking myosin IIa fibers and microtubule network. Indeed, depletion of EBP50 also dismantled myosin IIa fibers and induced the formation of stable microtubules in lamellae expansions and Rac1 activation. This signaling cascade leads to the formation of lamellipodia, trailing tails and decrease of focal adhesion formation, triggering cell migration.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Citocinese , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Fosfoproteínas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Aorta/citologia , Proteínas de Transporte/genética , Forma Celular , Células Cultivadas , Masculino , Microscopia de Fluorescência , Microtúbulos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Fosforilação , Cultura Primária de Células , Interferência de RNA , Ratos , Ratos Wistar , Trocadores de Sódio-HidrogênioRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Ajuga iva (L.) Schreb. (Labiatae) (AI) is used in folk medicine for a variety of ailments, including diabetes mellitus and hypertension. AIM OF THE STUDY: In this work, we aimed to investigate the antihypertensive and vasorelaxant effects of AI aqueous extract in stroke prone spontaneously hypertensive rats (SHR-SP). MATERIAL AND METHODS: Male SHR-SP rats were orally force-fed AI aqueous extract (500 mg/kg body weight) daily for one week. Systolic blood pressure and urine output were recorded in vivo by non-invasive methods. AI vasoactive effects on noradrenaline contractile response and acetylcholine-evoked relaxation were assessed ex vivo on aorta rings of treated and untreated SHR-SP rats. AI extract was then subjected to bio-guided fractionation using solvents of increasing polarity. For each fraction, in vitro vasorelaxation assay was performed on noradrenaline-precontracted aorta of Wistar rats, in the absence/presence of N-nitro-L-arginine (L-NNA). HPLC analysis of AI total extract, and the most in vitro active AI residual aqueous extract fraction (A1) was performed using naringin, naringenin, apigenin, apigenin 7-O-glucoside as marker compounds. RESULTS: AI aqueous extract (500 mg/kg) significantly (P < 0.05) decreased systolic blood pressure (SBP) in SHR-SP rats, while not affecting the urine output. In ex vivo experiments, the total extract decreased contractile response to noradrenaline of aortic rings isolated from AI-treated SHR-SP rats with or without addition of N-nitro-L-arginine, but endothelium dependent relaxation evoked by acetylcholine in noradrenaline-contracted aortic rings was not affected by the extract treatment. In vitro experiments on AI aqueous extract fractions showed that its polar fraction was the only one affecting in vitro noradrenaline induced contractions, but only in an endothelium dependent manner. This fraction was shown by HPLC-UV to contain flavonoid glycosides among other polar compounds whose activity and mode of action may be modified in vivo by metabolization. CONCLUSION: These results support the use of AI as antihypertensive treatment in folk medicine. The systolic blood pressure decrease may be attributed at least in part to vasorelaxant glycosylated/polar phenolic compounds as flavonoids and/or their metabolites.
Assuntos
Ajuga/química , Anti-Hipertensivos/farmacologia , Hipertensão/tratamento farmacológico , Extratos Vegetais/farmacologia , Vasodilatadores/farmacologia , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/uso terapêutico , Aorta/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Fracionamento Químico , Flavonoides/química , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Masculino , Medicina Tradicional , Marrocos , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Ratos Endogâmicos SHR , Ratos Wistar , Solventes/química , Micção/efeitos dos fármacos , Vasodilatadores/química , Vasodilatadores/uso terapêutico , Água/químicaRESUMO
Synchronous calcium oscillations are observed in primary cultures of rat cortical neurons when mature networks are formed. This spontaneous neuronal activity needs an accurate control of calcium homeostasis. Alteration of intraneuronal calcium concentration is described in many neurodegenerative disorders, including Alzheimer disease (AD). Although processing of amyloid precursor protein (APP) that generates Abeta peptide has critical implications for AD pathogenesis, the neuronal function of APP remains unclear. Here, we report that expression of human APP (hAPP) in rat cortical neurons increases L-type calcium currents, which stimulate SK channels, calcium-dependent K(+) channels responsible for medium afterhyperpolarization (mAHP). In a neuronal network, increased mAHP in some neurons expressing hAPP leads to inhibition of calcium oscillations in all the cells of the network. This inhibition is independent of production and secretion of Abeta and other APP metabolites. In a neuronal network, reduction of endogenous APP expression using shRNA increases the frequency and reduces the amplitude of calcium oscillations. Altogether, these data support a key role for APP in the control of neuronal excitability.
Assuntos
Precursor de Proteína beta-Amiloide/biossíntese , Sinalização do Cálcio/fisiologia , Cálcio/antagonistas & inibidores , Córtex Cerebral/fisiologia , Regulação para Baixo/fisiologia , Neurônios/fisiologia , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/genética , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/biossíntese , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Humanos , Neurônios/citologia , Neurônios/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos WistarRESUMO
Ezrin, radixin, and moesin (ERM) proteins are known to be substrates of Rho kinase (ROCK), a key player in vascular smooth muscle regulation. Their function in arteries remains to be elucidated. The objective of the present study was to investigate ERM phosphorylation and function in rat aorta and mesenteric artery and the influence of ERM-binding phosphoprotein 50 (EBP50), a scaffold partner of ERM proteins in several cell types. In isolated arteries, ERM proteins are phosphorylated by PKC and ROCK with different kinetics after either agonist stimulation or KCl-induced depolarization. Immunoprecipitation of EBP50 in noradrenaline-stimulated arteries allowed identification of its interaction with moesin and several other proteins involved in cytoskeleton regulation. This interaction was inhibited by Y27632, a ROCK inhibitor. Moesin or EBP50 depletion after small interfering RNA transfection by reverse permeabilization in intact mesenteric arteries both potentiated the contractility in response to agonist stimulation without any effect on contractile response induced by high KCl. This effect was preserved in ionomycin-permeabilized arteries. These results indicate that, in agonist-stimulated arteries, the activation of ROCK leads to the binding of moesin to EBP50, which interacts with several components of the cytoskeleton, resulting in a decrease in the contractile response.
Assuntos
Proteínas de Transporte/metabolismo , Artérias Mesentéricas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Norepinefrina/metabolismo , Fosfoproteínas/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Animais , Anti-Hipertensivos/metabolismo , Aorta/efeitos dos fármacos , Aorta/metabolismo , Ionomicina/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Cloreto de Potássio/farmacologia , Ligação Proteica , Proteína Quinase C/análise , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Piridinas/farmacologia , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Trocadores de Sódio-Hidrogênio , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
In this study, we determined the vasorelaxant activity of essential oils of different samples of CROTON ZAMBESICUS collected in the same area in Benin at different periods and analysed their compositions by GC-FID and GC-MS. 68 compounds were identified among which 20 have not been described previously in this plant's essential oils. We observed quantitative differences among essential oils but all possess significant vasorelaxant activity on intact rat aortae contracted by KCl (IC (50) 5.6-11.8 µg/mL). This activity may, at least in part, be explained by the presence of vasorelaxant diterpenes such as ENT-18-hydroxy-trachyloban-3-one, isopimara-7,15-dien-3ß-ol, and ENT-18-hydroxy-isopimar-7,15-dien-3ß-ol, previously isolated from the dichloromethane extract of the leaves, but also to linalool (IC (50) 43.4 µg/mL) and particularly to caryophyllene oxide (IC (50) 2.5 µg/mL).
Assuntos
Aorta/efeitos dos fármacos , Croton/química , Óleos Voláteis/farmacologia , Vasodilatadores/farmacologia , Animais , Diterpenos/química , Diterpenos/isolamento & purificação , Diterpenos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Técnicas In Vitro , Óleos Voláteis/química , Óleos Voláteis/isolamento & purificação , RatosRESUMO
The aim of this study was to explore the changes evoked by organ culture in the signalling pathways activated by noradrenaline in rat resistance mesenteric artery. Contractile responses and calcium signalling were significantly more sensitive to noradrenaline in arteries cultured for 48-72 h in the absence of growth factors compared to fresh arteries. Both calcium release activated by noradrenaline in calcium-free solution and calcium entry measured after the addition of external calcium were higher in cultured arteries than in fresh tissue. Blockers of non-selective cation channels (SKF-96365, flufenamic acid, Gd(3+)) more potently inhibited noradrenaline contraction in cultured arteries than in fresh ones. The src kinase inhibitors genistein or PP2 normalised the increased contraction and the increased calcium release evoked by noradrenaline in cultured arteries. In cultured arteries, trpc1 (transient receptor potential canonical 1) mRNA expression was decreased by 47 +/- 8% (n = 5, p < 0.05), while trpc6 mRNA expression was increased by 92 +/- 24% (n = 5, p < 0.05) in comparison with non-cultured arteries. Immunofluorescence analysis of protein expression confirmed the up-regulation of TRPC6 protein after culture. These results indicate that mesenteric artery culture results in src kinase-dependent increase in the responses to noradrenaline and in a change in cation channel activity, which could contribute to the increased contraction.
Assuntos
Sinalização do Cálcio , Artérias Mesentéricas/metabolismo , Norepinefrina/metabolismo , Canais de Cátion TRPC/metabolismo , Vasoconstrição , Compostos de Alumínio/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Fluoretos/farmacologia , Proteínas de Ligação ao GTP/agonistas , Proteínas de Ligação ao GTP/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/enzimologia , Técnicas de Cultura de Órgãos , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Ratos , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Drug-induced liver injury (DILI) is the primary adverse event that results in withdrawal of drugs from the market and a frequent reason for the failure of drug candidates in development. The Liver Toxicity Biomarker Study (LTBS) is an innovative approach to investigate DILI because it compares molecular events produced in vivo by compound pairs that (a) are similar in structure and mechanism of action, (b) are associated with few or no signs of liver toxicity in preclinical studies, and (c) show marked differences in hepatotoxic potential. The LTBS is a collaborative preclinical research effort in molecular systems toxicology between the National Center for Toxicological Research and BG Medicine, Inc., and is supported by seven pharmaceutical companies and three technology providers. In phase I of the LTBS, entacapone and tolcapone were studied in rats to provide results and information that will form the foundation for the design and implementation of phase II. Molecular analysis of the rat liver and plasma samples combined with statistical analyses of the resulting datasets yielded marker analytes, illustrating the value of the broad-spectrum, molecular systems analysis approach to studying pharmacological or toxicological effects.
Assuntos
Antiparkinsonianos/toxicidade , Benzofenonas/toxicidade , Biomarcadores/metabolismo , Catecóis/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fígado/metabolismo , Nitrilas/toxicidade , Nitrofenóis/toxicidade , Animais , Antiparkinsonianos/farmacocinética , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Metabolômica , Análise de Sequência com Séries de Oligonucleotídeos , Proteômica , Ratos , Ratos Sprague-Dawley , TolcaponaRESUMO
The present study was designed to further evaluate the biological effects and tissue selectivity of new 3-alkylaminobenzo- and 3-alkylaminopyridothiadiazine 1,1-dioxides bearing identical branched alkylamino chains at the 3-position. These original compounds were compared with their parent molecules; namely the K(ATP) channel openers diazoxide and pinacidil. All tested 3-alkylamino-substituted derivatives provoked a marked, concentration-dependent inhibition of the glucose-induced insulin secretion from rat pancreatic islets. The 3-alkylaminopyridothiadiazine 1,1-dioxides evoked a weak vasorelaxant activity whilst their 7-halo-substituted benzothiadiazine counterparts elicited pronounced, concentration-dependent, relaxations of rat aortic rings. The myorelaxant effect of the different drugs was tightly correlated with their capacity to increase (86)Rb outflow ((42)K substitute) from prelabelled and perifused rat aortic rings. EC(50)/IC(50) ratios (vascular/pancreatic) revealed a pronounced selectivity of the 3-alkylaminopyridothiadiazine 1,1-dioxides towards the pancreatic endocrine tissue. Structure-activity relationships suggest that, besides the requirement of an electronegative pole at the 7-position of the heterocycle, a minimal steric hindrance confers an optimal insulin-secreting cell selectivity. Lastly, radioisotopic, electrophysiological and pharmacological investigations indicate that the marked vasorelaxant properties of the 3-alkylaminobenzothiadiazine 1,1-dioxides are related to the activation of smooth muscle K(ATP) channels.
Assuntos
Canais KATP/efeitos dos fármacos , Tiadiazinas/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Glibureto/farmacologia , Técnicas In Vitro , Insulina/metabolismo , Secreção de Insulina , Potenciais da Membrana/efeitos dos fármacos , Especificidade de Órgãos , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Vasoconstrição/efeitos dos fármacosRESUMO
Antihypertensive peptides received much interest over the last decade. These peptides are known to be angiotensin converting enzyme (ACE) inhibitors in vitro, but the actual antihypertensive mechanisms in vivo are still unclear. In this research, we used rat aortic rings in organ bath experiments to investigate five potential vascular antihypertensive mechanisms of the dipeptide Val-Tyr. Only one significant effect was observed, namely preincubation of the aorta with Val-Tyr led to a significant shift of the concentration-response curve evoked by angiotensin I (Ang I). Val-Tyr had no effect on the angiotensin II receptor or the alpha-adrenergic receptor. Furthermore, it did not interact with voltage-operated Ca2+ channels, or with nitric oxide production/availability. In conclusion, our results show that Val-Tyr specifically inhibits Ang I-evoked contraction through ACE inhibition and that four other main mechanisms of vascular tone regulation are not affected.
Assuntos
Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Dipeptídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Acetilcolina/farmacologia , Angiotensina I/farmacologia , Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anti-Hipertensivos/química , Aorta/fisiologia , Dipeptídeos/química , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Técnicas In Vitro , Lisinopril/química , Lisinopril/farmacologia , Masculino , Norepinefrina/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos WistarRESUMO
Transient receptor potential canonical (TRPC) proteins have been proposed to function as plasma membrane Ca2+ channels activated by store depletion and/or by receptor stimulation. However, their role in the increase in cytosolic Ca2+ activated by contractile agonists in vascular smooth muscle is not yet elucidated. The present study was designed to investigate the functional and molecular properties of the Ca2+ entry pathway activated by endothelin-1 in primary cultured aortic smooth muscle cells. Measurement of the Ca2+ signal in fura-2-loaded cells allowed to characterize endothelin-1-evoked Ca2+ entry, which was resistant to dihydropyridine, and was blocked by 2-aminoethoxydiphenylborate (2-APB) and micromolar concentration of Gd3+. It was not activated by store depletion, but was inhibited by the endothelin ETA receptor antagonist BQ-123, and by heparin. On the opposite, thapsigargin-induced store depletion activated a Ca2+ entry pathway that was not affected by 2-APB, BQ-123 or heparin, and was less sensitive to Gd3+ than was endothelin-1-evoked Ca2+ entry. Investigation of the gene expression of TRPC isoforms by real-time RT-PCR revealed that TRPC1 was the most abundant. In cells transfected with TRPC1 small interfering RNA sequence, TRPC1 mRNA and protein expression were decreased by 72+/-3% and 86+/-2%, respectively, while TRPC6 expression was unaffected. In TRPC1 knockdown cells, both endothelin-1-evoked Ca2+ entry and store-operated Ca2+ entry evoked by thapsigargin were blunted. These results indicate that in aortic smooth muscle cells, TRPC1 is not only involved in Ca2+ entry activated by store depletion but also in receptor-operated Ca2+ entry, which requires inositol (1,4,5) triphosphate receptor activation.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Canais de Cátion TRPC/agonistas , Animais , Anticoagulantes/administração & dosagem , Anticoagulantes/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Endotelina-1/farmacologia , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Heparina/administração & dosagem , Heparina/farmacologia , Masculino , Microinjeções , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPC/genética , Transfecção , Vasoconstritores/farmacologiaRESUMO
AIM OF THE STUDY: Tanacetum vulgare L. (Asteraceae/Compositae) is principally used in traditional Moroccan medicine as antihypertensive remedy. The aim of the present study was to investigate the in vitro vascular activity of the aqueous extract of Tanacetum vulgare L. MATERIALS AND METHODS: The activity of Tanacetum vulgare L. extract was tested on contractile response of Wistar rat aorta to high KCl and noradrenaline and on endothelium-dependent relaxation evoked by acetylcholine. RESULTS: The addition of Tanacetum extract during the plateau phase of noradrenaline-evoked contraction produced a rapid relaxation that reached a maximum of 30% of the contraction and was suppressed by the NO synthase inhibitor N(G)-nitro-l-arginine. At higher extract concentrations this rapid relaxation was followed by a slowly developing, N(G)-nitro-l-arginine-resistant, relaxing effect. Tanacetum extract also depressed KCl-evoked contraction by 30% at maximum. This effect was abolished in the presence of N(G)-nitro-l-arginine. The endothelium-dependent relaxation induced by acetylcholine was depressed in the presence of Tanacetum extract in the bathing solution. CONCLUSION: This study indicates that the aqueous extract of Tanacetum possesses NO-mediated and NO-independent vasorelaxing properties in vitro.
Assuntos
Extratos Vegetais/farmacologia , Tanacetum/química , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Acetilcolina , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Técnicas In Vitro , Medicina Tradicional , Marrocos , Óxido Nítrico/metabolismo , Norepinefrina , Extratos Vegetais/administração & dosagem , Folhas de Planta , Cloreto de Potássio , Ratos , Ratos Wistar , Vasodilatadores/administração & dosagem , Vasodilatadores/isolamento & purificaçãoRESUMO
OBJECTIVE: The present investigation was carried out to evaluate the safety of an aqueous extract of the seeds of Calycotome villosa (Poiret) Link (subsp. intermedia) by determining its cytotoxicity and potential toxicity after acute and sub-chronic administration in rodents. MATERIALS AND METHODS: Cytotoxic activity was tested in cancer and non-cancer cell lines HeLa, Mel-5, HL-60 and 3T3. Acute toxicity tests were carried out in mice by a single oral administration of Calycotome seed-extract (0 - 12 g/kg) as well as intraperitoneal doses of 0 - 5 g/kg. Sub-chronic studies were conducted in Wistar rats by administration of oral daily doses for up to 90 days. Changes in body and vital organ weights, mortality, haematology, clinical biochemistry and histologic morphology were evaluated. RESULTS: The lyophilized aqueous extract of C. villosa exhibited a low cytotoxicity in all cell lines tested with an IC50 > 100 µg/ml. In the acute study in mice, intra-peritoneal administration caused dose-dependent adverse effects and mortality with an LD50 of 4.06 ± 0.01 g/kg. In the chronic tests, neither mortality nor visible signs of lethality was seen in rats. Even AST and ALT were not affected while a significant decrease in serum glucose levels, at 300 and 600 mg/kg was detected. Histopathological examination of the kidney and liver did not show any alteration or inflammation at the end of treatment. CONCLUSION: In conclusion, the aqueous extract of C. villosa seed appeared to be non-toxic and did not produce mortality or clinically significant changes in the haematological and biochemical parameters in rats.
RESUMO
The diacylglycerol lipase inhibitor 1,6-bis(cyclohexyloximinocarbonylamino) hexane (RHC-80267) was tested for its effect on acetylcholine-evoked relaxation in rat mesenteric artery. In artery contracted with either noradrenaline or KCl, RHC-80267 (0.1-10 muM) potentiated the relaxation evoked by acetylcholine. The effect of RHC-80267 was not affected by nitric oxide synthase inhibition or by inhibitors of protein kinase C or of phospholipase A(2). The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol did not change the relaxation to acetylcholine. RHC-80267 did not affect the relaxation evoked by carbachol, by the nitric oxide donor SNAP (S-nitroso-N-acetylpenicillamine) or by the K(+) channel opener cromakalim. Neostigmine, a cholinesterase inhibitor, produced the same effect as RHC-80267 on acetylcholine-evoked relaxation. When tested on cholinesterase in brain homogenate, RHC-80267 concentration-dependently inhibited cholinesterase activity with an IC(50) of 4 muM. These results indicate that the potentiation of acetylcholine-evoked responses by RHC-80267 in rat mesenteric artery is caused by the inhibition of the cholinesterase activity in the vascular wall.
Assuntos
Acetilcolina/farmacologia , Inibidores da Colinesterase/farmacologia , Cicloexanonas/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Animais , Fatores Biológicos/fisiologia , Colinesterases/metabolismo , Cromakalim/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Lipase Lipoproteica/antagonistas & inibidores , Masculino , Artérias Mesentéricas/fisiologia , Naftalenos/farmacologia , Neostigmina/farmacologia , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroarginina/farmacologia , Norepinefrina/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Fosfolipases A/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Endogâmicos WKY , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Integrative (or systems biology) is a new approach to analyzing biological entities as integrated systems of genetic, genomic, protein, metabolite, cellular, and pathway events that are in flux and interdependent. Here, we demonstrate the application of intregrative biological analysis to a mammalian disease model, the apolipoprotein E3-Leiden (APO*E3) transgenic mouse. Mice selected for the study were fed a normal chow diet and sacrificed at 9 weeks of age-conditions under which they develop only mild type I and II atherosclerotic lesions. Hepatic mRNA expression analysis showed a 25% decrease in APO A1 and a 43% increase in liver fatty acid binding protein expression between transgenic and wild type control mice, while there was no change in PPAR-alpha expression. On-line high performance liquid chromatography-mass spectrometry quantitative profiling of tryptic digests of soluble liver proteins and liver lipids, coupled with principle component analysis, enabled rapid identification of early protein and metabolite markers of disease pathology. These included a 44% increase in L-FABP in transgenic animals compared to controls, as well as an increase in triglycerides and select bioactive lysophosphatidylcholine species. A correlation analysis of identified genes, proteins, and lipids was used to construct an interaction network. Taken together, these results indicate that integrative biology is a powerful tool for rapid identification of early markers and key components of pathophysiologic processes, and constitute the first application of this approach to a mammalian system.
Assuntos
Apolipoproteínas E/genética , Apolipoproteínas E/fisiologia , Camundongos Transgênicos , Animais , Apolipoproteína E3 , Arteriosclerose/metabolismo , Proteínas de Transporte/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Proteínas de Ligação a Ácido Graxo , Genoma , Humanos , Metabolismo dos Lipídeos , Fígado/metabolismo , Lisofosfatidilcolinas/metabolismo , Espectrometria de Massas , Camundongos , Modelos Biológicos , Análise Multivariada , Análise de Sequência com Séries de Oligonucleotídeos , RNA/metabolismo , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Tripsina/metabolismo , Tripsina/farmacologiaRESUMO
Multitiered quantitative analysis of biological systems is rapidly becoming the desired approach to study hierarchical functional interactions between proteins and metabolites. We describe here a novel systematic approach to analyze organisms with complex metabolic regulatory networks. By using precise analytical methods to measure biochemical constituents and their relative abundance in whole plasma of transgenic ApoE*3-Leiden mice and an isogenic wild-type control group, simultaneous snapshots of metabolic and protein states were obtained. Novel data processing and multivariate analysis tools such as Impurity Resolution Software (IMPRESS) and Windows-based linear fit program (WINLIN) were used to compare protein and metabolic profiles in parallel. Canonical correlations of the resulting data show quantitative relationships between heterogeneous components in the TG animals. These results, obtained solely from whole plasma analysis allowed us, in a rapid manner, to corroborate previous findings as well as find new events pertaining to dominant and peripheral events in lipoprotein metabolism of a genetically modified mammalian organism in relation to ApoE3, a key mediator of lipoprotein metabolism.