RESUMO
Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 µM) or Rho kinase (fasudil, 10 µM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.
Assuntos
Retroalimentação Fisiológica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Hipertensão/fisiopatologia , Músculo Liso Vascular/metabolismo , Quinases Associadas a rho/metabolismo , Angiotensina II/metabolismo , Angiotensina II/toxicidade , Animais , Artérias/metabolismo , Western Blotting , Perfilação da Expressão Gênica , Hipertensão/induzido quimicamente , Masculino , Músculo Liso Vascular/fisiopatologia , RNA Interferente Pequeno , Ratos , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
MicroRNAs (miRNAs) are endogenous, small, noncoding RNAs that regulate about 30% of protein-coding genes of the human genome. Thus far, more than 400 miRNAs have been cloned and sequenced in humans. Their biological importance, initially demonstrated in cancer, viral diseases and developmental processes, was more recently investigated in cardiovascular physiology and pathology. MiRNAs expression is tightly controlled in a tissue-specific and developmental stage-specific manner and some of them are highly and specifically expressed in cardiovascular tissues. Through the regulation of the expression of genes involved in cell growth, contractility and electrical conductance, cardiac miRNAs may play a major role in heart development and function. In vascular cells, miRNAs have been linked to vasculoproliferative conditions such as angiogenesis and neointimal lesion formation. Diagnostic use and therapeutic modulation of individual miRNAs or miRNA clusters in cardiovascular diseases will have to be further explored in the future. Molecules specifically regulating cardiovascular miRNAs, either mimicking or antagonizing miRNAs actions, will hopefully normalize dysfunctional gene networks and constitute a new therapy paradigm of cardiovascular diseases.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Fenômenos Fisiológicos Cardiovasculares , MicroRNAs/metabolismo , Animais , Doenças Cardiovasculares/tratamento farmacológico , Sistema Cardiovascular/embriologia , Sistemas de Liberação de Medicamentos , Regulação da Expressão Gênica , HumanosRESUMO
Increasing evidence has accumulated to implicate overactivation of Rho protein as a common component for the pathogenesis of several cardiovascular disorders including hypertension, coronary and cerebral vasospasm, atherosclerosis, and diabetes. Recent advances in Rho protein signaling research indicate that the Rho exchange factors (Rho GEFs) which activate Rho proteins by catalyzing the exchange of GDP for GTP are major regulators of Rho protein activity. In addition, linkage analysis and association studies have recently identified Rho GEFs as susceptibility genes for cardiovascular diseases. All of these data are converging to suggest that as upstream activators of Rho proteins, Rho GEFs expressed in cardiovascular cells are good candidate targets for the treatment of cardiovascular disorders.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Doenças Cardiovasculares/tratamento farmacológico , Fenômenos Fisiológicos Cardiovasculares , Sistemas de Liberação de Medicamentos , Expressão Gênica , Humanos , Polimorfismo Genético , Transdução de SinaisRESUMO
Urotensin II (UII) and UII-related peptide (URP) are paralog neuropeptides whose existence and distribution in mouse have not yet been investigated. In this study, we showed by HPLC/RIA analysis that the UII-immunoreactive molecule in the mouse brain corresponds to a new UII(17) isoform. Moreover, calcium mobilization assays indicated that UII(17) and URP were equally potent in stimulating UII receptor (UT receptor). Quantitative RT-PCR and in situ hybridization analysis revealed that in the CNS UII and URP mRNAs were predominantly expressed in brainstem and spinal motoneurons. Besides, they were differentially expressed in the medial vestibular nucleus, locus coeruleus and the ventral medulla. In periphery, both mRNAs were expressed in skeletal muscle, testis, vagina, stomach, and gall bladder, whereas only URP mRNA could be detected in the seminal vesicle, heart, colon, and thymus. By contrast, the UT receptor mRNA was widely expressed, and notably, very high amounts of transcript occurred in skeletal muscle and prostate. In the biceps femoris muscle, UII-like immunoreactivity was shown to coexist with synaptophysin in muscle motor end plate regions. Altogether these results suggest that (i) UII and URP may have many redundant biological effects, especially at the neuromuscular junction; (ii) URP may more specifically participate to autonomic, cardiovascular and reproductive functions.
Assuntos
Encéfalo/metabolismo , Junção Neuromuscular/metabolismo , Hormônios Peptídicos/metabolismo , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Urotensinas/metabolismo , Animais , Encéfalo/anatomia & histologia , Células CHO , Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Feminino , Masculino , Camundongos , Radioimunoensaio/métodos , Receptores Acoplados a Proteínas G/metabolismo , Sinaptofisina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Urotensinas/químicaRESUMO
Urotensin-II (U-II) and urotensin-II-related peptide (URP) have been identified as the endogenous ligands of the orphan G-protein-coupled receptor GPR14 now renamed UT. The occurrence of U-II and URP in the central nervous system, and the widespread distribution of UT in the brain suggest that U-II and URP may play various behavioral activities. Studies conducted in rodents have shown that central administration of U-II stimulates locomotion, provokes anxiety- and depressive-like states, enhances feeding activity and increases the duration of paradoxical sleep episodes. These observations indicate that, besides the endocrine/paracrine activities of U-II and URP on cardiovascular and kidney functions, these peptides may act as neurotransmitters and/or neuromodulators to regulate various neurobiological activities.
Assuntos
Comportamento Animal/fisiologia , Urotensinas/metabolismo , Animais , Ansiedade/metabolismo , Sistema Nervoso Central/anatomia & histologia , Sistema Nervoso Central/metabolismo , Depressão/metabolismo , Comportamento Alimentar/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Atividade Motora/fisiologia , Hormônios Peptídicos/genética , Hormônios Peptídicos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Urotensinas/genéticaRESUMO
Urotensin II (U-II) and urotensin II-related peptide (URP) are the endogenous ligands for the orphan G-protein-coupled receptor GPR14 now renamed UT. At the periphery, U-II and/or URP exert a wide range of biological effects on cardiovascular tissues, airway smooth muscles, kidney and endocrine glands, while central administration of U-II elicits various behavioral and cardiovascular responses. There is also evidence that U-II and/or URP may be involved in a number of pathological conditions including heart failure, atherosclerosis, renal dysfunction and diabetes. Because of the potential involvement of the urotensinergic system in various physiopathological processes, there is need for the rational design of potent and selective ligands for the UT receptor. Structure-activity relationship studies have shown that the minimal sequence required to retain full biological activity is the conserved U-II(4-11) domain, in particular the Cys5 and Cys10 residues involved in the disulfide bridge, and the Phe6, Lys8 and Tyr9 residues. Free alpha-amino group and C-terminal COOH group are not necessary for the biological activity, and modifications of these radicals may even increase the stability of the analogs. Punctual substitution of native amino acids, notably Phe6 and Trp7, by particular residues generates analogs with antagonistic properties. These studies, which provide crucial information regarding the structural and conformational requirements for ligand-receptor interactions, will be of considerable importance for the design of novel UT ligands with increased selectivity, potency and stability, that may eventually lead to the development of innovative drugs.
Assuntos
Hormônios Peptídicos/química , Hormônios Peptídicos/metabolismo , Relação Estrutura-Atividade , Urotensinas/química , Urotensinas/metabolismo , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Hormônios Peptídicos/genética , Conformação Proteica , Alinhamento de Sequência , Tirosina/química , Tirosina/metabolismo , Urotensinas/genéticaRESUMO
The vasoactive peptide urotensin II (UII) is primarily expressed in motoneurons of the brainstem and spinal cord. Intracerebroventricular injection of UII provokes various behavioral, cardiovascular, motor, and endocrine responses in the rat, but the distribution of the UII receptor in the central nervous system (CNS) has not yet been determined. In the present study, we have investigated the localization of UII receptor (GPR14) mRNA and UII binding sites in the rat CNS. RT-PCR analysis revealed that the highest density of GPR14 mRNA occurred in the pontine nuclei. In situ hybridization histochemistry showed that the GPR14 gene is widely expressed in the brain and spinal cord. In particular, a strong hybridization signal was observed in the olfactory system, hippocampus, olfactory and medial amygdala, hypothalamus, epithalamus, several tegmental nuclei, locus coeruleus, pontine nuclei, motor nuclei, nucleus of the solitary tract, dorsal motor nucleus of the vagus, inferior olive, cerebellum, and spinal cord. Autoradiographic labeling of brain slices with radioiodinated UII showed the presence of UII-binding sites in the lateral septum, bed nucleus of the stria terminalis, medial amygdaloid nucleus, anteroventral thalamus, anterior pretectal nucleus, pedunculopontine tegmental nucleus, pontine nuclei, geniculate nuclei, parabigeminal nucleus, dorsal endopiriform nucleus, and cerebellar cortex. Intense expression of the GPR14 gene in some hypothalamic nuclei (supraoptic, paraventricular, ventromedian, and arcuate nuclei), in limbic structures (amygdala and hippocampus), in medullary nuclei (solitary tract, dorsal motor nucleus of the vagus), and in motor control regions (cerebral and cerebellar cortex, substantia nigra, pontine nuclei) provides the anatomical substrate for the central effects of UII on behavioral, cardiovascular, neuroendocrine, and motor functions. The occurrence of GPR14 mRNA in cranial and spinal motoneurons is consistent with the reported autocrine/paracrine action of UII on motoneurons.
Assuntos
Sistema Nervoso Central/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/genética , Urotensinas/metabolismo , Animais , Ligação Competitiva/fisiologia , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Sistema Nervoso Central/anatomia & histologia , Radioisótopos do Iodo , Masculino , Neurônios Motores/metabolismo , Neurônios/citologia , RNA Mensageiro/metabolismo , Ensaio Radioligante , Ratos , Ratos Wistar , Medula Espinal/anatomia & histologia , Medula Espinal/metabolismoRESUMO
Urotensin II (U-II) is a potent vasoconstrictor peptide which has been identified as the endogenous ligand for the orphan G protein-coupled receptor GPR14 now renamed UT receptor. As the C-terminal cyclic hexapeptide of U-II (U-II(4-11), H-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH) possesses full biological activity, we have synthesized a series of U-II(4-11) analogues and measured their binding affinity on hGPR14-transfected CHO cells and their contractile activity on de-endothelialized rat aortic rings. The data indicate that a free amino group and a functionalized side-chain at the N-terminal extremity of the peptide are not required for biological activity. In addition, the minimal chemical requirement at position 9 of U-II(4-11) is the presence of an aromatic moiety. Most importantly, replacement of the Phe6 residue by cyclohexyl-Ala (Cha) led to an analogue, [Cha6]U-II(4-11), that was devoid of agonistic activity but was able to dose-dependently suppress the vasoconstrictor effect of U-II on rat aortic rings. These new pharmacological data, by providing further information regarding the structure-activity relationships of U-II analogues, should prove useful for the rational design of potent and nonpeptidic UT receptor agonists and antagonists.
Assuntos
Fragmentos de Peptídeos/síntese química , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Urotensinas/antagonistas & inibidores , Urotensinas/síntese química , Vasodilatadores/síntese química , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Técnicas In Vitro , Fragmentos de Peptídeos/farmacologia , Ensaio Radioligante , Ratos , Receptores Acoplados a Proteínas G/genética , Relação Estrutura-Atividade , Urotensinas/farmacologia , Vasodilatadores/farmacologiaRESUMO
BACKGROUND: Both alpha(2)-adrenergic and non--alpha(2)-adrenergic mechanisms seem to be involved in the hypotensive effect of imidazoline-like drugs. This study aimed at investigating how these 2 mechanisms work together to modify blood pressure (BP). METHODS AND RESULTS: LNP 509, which appeared in this study to be devoid of alpha(2A)-adrenergic activity, was administered to anesthetized rabbits and wild-type (WT) mice into the cisterna magna and into the fourth ventricle, respectively. Mean arterial pressure decreased by a maximum of 46 +/- 4% and 16 +/- 2%, respectively. In D79N mice, which lack functional alpha(2A)-adrenergic receptors, LNP 509 also reduced mean arterial pressure by 17 +/- 2%. The hypotension induced by LNP 509 (100 microg/kg intracisternally) was prevented by S23757 (1 mg/kg intracisternally), an antagonist highly selective for I(1)-imidazoline binding sites (I(1)BS). A synergy between LNP 509 and the alpha(2)-adrenergic agonist alpha-methylnoradrenaline (alpha-MNA) was observed in rabbits (cisterna magna injection) and in WT mice (fourth ventricle injection) but not, as expected, in D79N mice. Similar to LNP 509 alone, rilmenidine (fourth ventricle injection), which binds both to alpha(2)-adrenergic receptors and to I(1)BS, decreased BP in D79N mice. In WT animals, rilmenidine had a significantly greater effect. Microinjections performed in rabbits showed that the synergism occurred at least in part in the nucleus reticularis lateralis of the brain stem. CONCLUSIONS: These results demonstrate that a central imidazoline-sensitive, but non--alpha(2)-adrenergic, mechanism can modify BP by itself. This mechanism, which may involve I(1)BS, interacts synergistically with an alpha(2)-adrenergic mechanism to decrease BP.
Assuntos
Pressão Sanguínea/fisiologia , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos/metabolismo , Sistema Nervoso Simpático/fisiologia , Agonistas Adrenérgicos/farmacologia , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/metabolismo , Sistema Cardiovascular/efeitos dos fármacos , Ciclopropanos/farmacologia , Sinergismo Farmacológico , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hipotensão/induzido quimicamente , Hipotensão/prevenção & controle , Imidazóis/farmacologia , Injeções Intraventriculares , Camundongos , Camundongos Transgênicos , Microinjeções , Nordefrin/farmacologia , Oxazóis/farmacologia , Pirróis/farmacologia , Coelhos , Receptores Adrenérgicos alfa 2/deficiência , Receptores Adrenérgicos alfa 2/genética , Rilmenidina , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
Urotensin-II (U-II) receptors are widely distributed in the central nervous system. Intracerebroventricular (i.c.v.) injection of U-II causes hypertension and bradycardia and stimulates prolactin and thyrotropin secretion. However, the behavioral effects of centrally administered U-II have received little attention. In the present study, we tested the effects of i.c.v. injections of U-II on behavioral, metabolic, and endocrine responses in mice. Administration of graded doses of U-II (1-10,000 ng/mouse) provoked: (1) a dose-dependent reduction in the number of head dips in the hole-board test; (2) a dose-dependent reduction in the number of entries in the white chamber in the black-and-white compartment test, and in the number of entries in the central platform and open arms in the plus-maze test; and (3) a dose-dependent increase in the duration of immobility in the forced-swimming test and tail suspension test. Intracerebroventricular injection of U-II also caused an increase in: food intake at doses of 100 and 1,000 ng/mouse, water intake at doses of 100-10,000 ng/mouse, and horizontal locomotion activity at a dose of 10,000 ng/mouse. Whatever was the dose, the central administration of U-II had no effect on body temperature, nociception, apomorphine-induced penile erection and climbing behavior, and stress-induced plasma corticosterone level. Taken together, the present study demonstrates that the central injection of U-II at doses of 1-10,000 ng/mouse induces anxiogenic- and depressant-like effects in mouse. These data suggest that U-II may be involved in some aspects of psychiatric disorders.
Assuntos
Comportamento Animal/efeitos dos fármacos , Urotensinas/farmacologia , Sequência de Aminoácidos , Animais , Temperatura Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Comportamento Exploratório/efeitos dos fármacos , Injeções Intraventriculares , Locomoção/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Fatores de Tempo , Urotensinas/administração & dosagemRESUMO
OBJECTIVE: The objective of this study was to investigate ACE- and chymase-dependent angiotensin I-to-II conversion in human coronary arteries (HCAs). METHODS AND RESULTS: HCA rings were mounted in organ baths, and concentration-response curves to angiotensin II, angiotensin I, and the chymase-specific substrate Pro(11)-D-Ala(12)-angiotensin I (PA-angiotensin I) were constructed. All angiotensins displayed similar efficacy. For a given vasoconstriction, bath (but not interstitial) angiotensin II during angiotensin I and PA-angiotensin I was lower than during angiotensin II, indicating that interstitial (and not bath) angiotensin II determines vasoconstriction. PA-angiotensin I increased interstitial angiotensin II less efficiently than angiotensin I. Separate inhibition of ACE (with captopril) and chymase (with C41 or chymostatin) shifted the angiotensin I concentration-response curve approximately 5-fold to the right, whereas a 10-fold shift occurred during combined ACE and chymase inhibition. Chymostatin, but not captopril and/or C41, reduced bath angiotensin II and abolished PA-Ang I-induced vasoconstriction. Perfused HCA segments, exposed luminally or adventitially to angiotensin I, released angiotensin II into the luminal and adventitial fluid, respectively, and this release was blocked by chymostatin. CONCLUSIONS: Both ACE and chymase contribute to the generation of functionally active angiotensin II in HCAs. However, because angiotensin II loss in the organ bath is chymase-dependent, ACE-mediated conversion occurs more efficiently (ie, closer to AT(1) receptors) than chymase-mediated conversion.
Assuntos
Angiotensina II/metabolismo , Vasos Coronários/enzimologia , Vasos Coronários/metabolismo , Peptidil Dipeptidase A/metabolismo , Serina Endopeptidases/metabolismo , Adolescente , Adulto , Angiotensina I/metabolismo , Angiotensina II/fisiologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Captopril/farmacologia , Quimases , Vasos Coronários/efeitos dos fármacos , Espaço Extracelular/química , Feminino , Humanos , Técnicas In Vitro , Soluções Isotônicas , Masculino , Pessoa de Meia-Idade , Oligopeptídeos/farmacologia , Perfusão , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
2-(4,5-Dihydro-1H-imidazol-2-yl)-3,4-dihydro-2H-1,4-benzoxazine derivatives and tricyclic analogues with a fused additional ring on the nitrogen atom of the benzoxazine moiety have been prepared and evaluated for their cardiovascular effects as potential antihypertensive agents. The imidazoline ring was generated by reaction of the corresponding ethyl ester with ethylenediamine. Affinities for imidazoline binding sites (IBS) I(1) and I(2) and alpha(1) and alpha(2) adrenergic receptors were evaluated as well as the effects on mean arterial blood pressure (MAP) and heart rate (HR) of spontaneously hypertensive rats. With few exceptions the most active compounds on MAP were those with high affinities for IBS and alpha(2) receptor. Among these, compound 4h was the most interesting and is now, together with its enantiomers, under complementary pharmacological evaluation.
Assuntos
Anti-Hipertensivos/síntese química , Imidazóis/síntese química , Oxazinas/síntese química , Medula Suprarrenal/metabolismo , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Sítios de Ligação , Pressão Sanguínea/efeitos dos fármacos , Bovinos , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Imidazóis/química , Imidazóis/farmacologia , Receptores de Imidazolinas , Técnicas In Vitro , Rim/efeitos dos fármacos , Rim/metabolismo , Oxazinas/química , Oxazinas/farmacologia , Coelhos , Ensaio Radioligante , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Melatonin deprival in young rats induces alterations in cerebral arteriolar wall similar to those observed during aging: atrophy and a decrease in distensibility. In this study, we examined the effects of melatonin treatment on cerebral arteriolar structure and distensibility and on the lower limit of cerebral blood flow autoregulation (LLCBF) in old rats. We measured cerebral blood flow (arbitrary unit, laser Doppler, open skull preparation) prior to and during stepwise hypotension (SH) in adult (12/13 months) and old (24/25 months) IcoWI and WAG/Rij male rats. Old rats were untreated or treated for 3 months with melatonin (0.39 (IcoWi) and 0.44 (Wag/Rij) mg kg-1 day-1, drinking water). Stress-strain relationships were determined using cross-sectional area (CSA, microm2, histometry) and values of arteriolar internal diameter (microm) obtained during a second SH following arteriolar deactivation (EDTA, 67 mmol l(-1)). Aging induced (a) atrophy of the arteriolar wall in IcoWI (616+/-20 vs 500+/-27 microm2, P<0.05) but not in WAG/Rij rats (328+/-25 vs 341+/-20 microm2), (b) a decrease in arteriolar wall distensibility and (c) an increase in the LLCBF in both strains (67+/-10 mmHg in 12-month-old vs 95+/-6 mmHg in 24-month-old IcoWi, P<0.05 and 53+/-2 mmHg in 13-month-old vs 67+/-6 mmHg in 25-month-old WAG/Rij). Melatonin treatment induced in IcoWI and WAG/Rij rats (a) hypertrophy of the arteriolar wall (643+/-34 and 435+/-25 microm2, respectively), (b) an increase in arteriolar wall distensibility and (c) a decrease in the LLCBF (64+/-6 and 45+/-4 mmHg, respectively). Melatonin treatment of old rats induced hypertrophy of the arteriolar wall, prevented the age-linked decrease in cerebral arteriolar distensibility and decreased the LLCBF. British Journal of Pharmacology (2004) 141, 399-406. doi:10.1038/sj.bjp.0705629
Assuntos
Envelhecimento/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Melatonina/farmacologia , Envelhecimento/fisiologia , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Encéfalo/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Hipotensão/fisiopatologia , Masculino , Ratos , Especificidade da Espécie , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
(1) The present study was designed to test the hypothesis that melatonin inhibits nitrate tolerance in coronary arteries. (2) Rings of porcine coronary arteries were suspended in organ chambers for isometric tension recording. Nitrate tolerance was induced by incubating the tissues with nitroglycerin (10(-4) M) for 90 min, followed by repeated rinsing for 1 h. Control rings that had not been exposed previously to nitroglycerin, but were otherwise treated identically, were studied simultaneously. The rings were contracted with U46619 (1-3 x 10(-9) M) and concentration-response curves to nitroglycerin (10(-9)-10(-4) M) were obtained. (3) Nitrate tolerance was evident by a 15- to 20-fold rightward shift in the concentration-response curve to nitroglycerin in rings with and without endothelium exposed previously to the drug for 90 min. Addition of melatonin (10(-9)-10(-7) M) to the organ chamber during the 90-min incubation period with nitroglycerin partially inhibited nitrate tolerance in coronary arteries with intact endothelium; however, melatonin had no effect on nitrate tolerance in coronary arteries without endothelium. (4) The effect of melatonin on nitrate tolerance in coronary arteries with endothelium was abolished by the melatonin receptor antagonist, S20928 (10(-6) M). In contrast to melatonin, the selective MT(3)-melatonin receptor agonist, 5-MCA-NAT (10(-8)-10(-7) M), had no effect on nitrate tolerance in coronary arteries. (5) The results demonstrate that melatonin, acting via specific melatonin receptors, inhibits nitrate tolerance in coronary arteries and that this effect is dependent on the presence of the vascular endothelium.
Assuntos
Melatonina/farmacologia , Nitroglicerina/efeitos adversos , Nitroglicerina/farmacologia , Taquifilaxia/fisiologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/antagonistas & inibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Artérias , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiologia , Cromakalim/farmacologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/cirurgia , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Contração Isométrica/fisiologia , Isoproterenol/farmacologia , Melatonina/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Naftalenos/farmacologia , Nitratos/administração & dosagem , Nitratos/efeitos adversos , Nitratos/farmacocinética , Receptores de Melatonina/antagonistas & inibidores , Receptores de Melatonina/efeitos dos fármacos , Suínos , Fatores de Tempo , Triptaminas/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Urotensin II (UII) has been described as the most potent vasoconstrictor peptide and recognized as the endogenous ligand of the orphan G protein-coupled receptor GPR14. Recently, a UII-related peptide (URP) has been isolated from the rat brain and its sequence has been established as H-Ala-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. In order to study the structure-function relationships of URP, we have synthesized a series of URP analogs and measured their binding affinity on hGPR14-transfected cells and their contractile activity in a rat aortic ring bioassay. Alanine substitution of each residue of URP significantly reduced the binding affinity and the contractile activity of the peptides, except for the Ala8-substituted analog that retained biological activity. Most importantly, D-scan of URP revealed that [D-Trp4]URP abrogated and [D-Tyr6]URP partially suppressed the UII-evoked contractile response. [Orn5]URP, which had very low agonistic efficacy, was the most potent antagonist in this series. The solution structure of URP has been determined by 1H NMR spectroscopy and molecular dynamics. URP exhibited a single conformation characterized by an inverse gamma-turn comprising residues Trp-Lys-Tyr which plays a crucial role in the biological activity of URP. These pharmacological and structural data should prove useful for the rational design of non-peptide ligands as potential GPR14 agonists and antagonists.
Assuntos
Hormônios Peptídicos/química , Sequência de Aminoácidos , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Fragmentos de Peptídeos , Conformação ProteicaRESUMO
Hypertension is one of the most frequent pathologies in the industrialized world. Although recognized to be dependent on a combination of genetic and environmental factors, its molecular basis remains elusive. Increased activity of the monomeric G protein RhoA in arteries is a common feature of hypertension. However, how RhoA is activated and whether it has a causative role in hypertension remains unclear. Here we provide evidence that Arhgef1 is the RhoA guanine exchange factor specifically responsible for angiotensin II-induced activation of RhoA signaling in arterial smooth muscle cells. We found that angiotensin II activates Arhgef1 through a previously undescribed mechanism in which Jak2 phosphorylates Tyr738 of Arhgef1. Arhgef1 inactivation in smooth muscle induced resistance to angiotensin II-dependent hypertension in mice, but did not affect normal blood pressure regulation. Our results show that control of RhoA signaling through Arhgef1 is central to the development of angiotensin II-dependent hypertension and identify Arhgef1 as a potential target for the treatment of hypertension.
Assuntos
Angiotensina II/fisiologia , Pressão Sanguínea/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipertensão/etiologia , Hipertensão/fisiopatologia , Janus Quinase 2/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Tirosina/metabolismoRESUMO
In-stent restenosis is a novel pathobiologic process resulting from vascular smooth muscle cell (VSMC) proliferation, migration and excessive matrix production. The present study was designed to assess the activity of RhoA, a major regulator of VSMC proliferation and migration, after stenting and to determine its role in the neointimal formation. Analysis of RhoA activity in an ex vivo organ culture model of human internal mammary arteries demonstrates that stenting induced a time-dependent increase in RhoA activity (4.9 +/- 0.4 vs. 1.2 +/- 0.2 in control at 28 days, n = 4, p < 0.0001) associated with a concomitant decrease in p27 expression. Treatment of stented arteries with the permeant RhoA inhibitor TAT-C3 (10 microg/ml) or Rho-kinase inhibitors (Y-27632, 10 micromol/l; fasudil, 10 micromol/l) inhibited both neointimal formation and decrease in p27 expression. Rapamycin (1 and 10 nmol/l) also inhibited neointimal formation, and induced a loss of RhoA expression. The inhibitory effect of rapamycin on neointimal thickening is prevented by the dominant active form of RhoA. Our study shows that stent implantation induces maintained RhoA activation and demonstrates that the inhibitory action of rapamycin on RhoA expression plays a key role in its antirestenotic effect.
Assuntos
Artéria Torácica Interna/patologia , Sirolimo/farmacologia , Stents/efeitos adversos , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Arteriopatias Oclusivas/prevenção & controle , Proteínas de Ciclo Celular/análise , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p27 , Humanos , Músculo Liso Vascular/patologia , Transdução de Sinais , Proteínas Supressoras de Tumor/análise , Túnica Íntima/patologia , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
The synthesis of new potential inhibitors of human chymase is described. Treatment of dihydroimidazo[1,5-a]indole and [1,5-b]isoquinoline-dione with thioaryl followed by oxidation gave the N-arylsulfonylmethyl of polycyclic hydantoin derivatives 3, 5 and 6.
Assuntos
Inibidores Enzimáticos/síntese química , Hidantoínas/síntese química , Hidantoínas/farmacologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/síntese química , Animais , Células COS , Chlorocebus aethiops , Quimases , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Hidantoínas/química , Indóis/síntese química , Indóis/farmacologia , Cinética , Modelos Moleculares , Conformação Molecular , Proteínas Recombinantes/antagonistas & inibidores , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Tetra-Hidroisoquinolinas/síntese química , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/farmacologia , TransfecçãoRESUMO
In order to specify structure-activity relationships we have synthesized new series of analogues of the Rho-kinase inhibitor (R)-(+)-N-(4-pyridyl)-4-(1-aminoethyl)benzamide (Y-32885). The structural modifications concerned the 1-aminoethyl, the pyridyl and the amide groups which are the main features of this lead compound. Our analogue derivatives were evaluated on GTPgammaS-induced contraction in permeabilized smooth-muscle and on the actin cytoskeleton. All the modifications result in a diminution or a loss of activity showing that interactions of Y-32885 with the catalytic domain of Rho-kinase seem to be particularly definite and sensitive to structural variations. The presence of a pyridine moiety and a basic amine group separated by a spacer bearing an amide function are of utmost importance.
Assuntos
Benzamidas/química , Benzamidas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologia , Actinas/efeitos dos fármacos , Animais , Aorta/citologia , Inibidores Enzimáticos/síntese química , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cobaias , Técnicas In Vitro , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Permeabilidade , Proteínas Serina-Treonina Quinases/química , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Quinases Associadas a rhoRESUMO
With a view to specifying structure-activity relationships we have synthesised a new series of analogues of the Rho-kinase inhibitor 1-(5-isoquinolinesulfonyl)-homopiperazine (Fasudil). The structural modifications concerned the isoquinolinyl heterocycle and the sulfonyl group which are the two main features of this lead compound. These analogues were evaluated on the actin cytoskeleton and on the enzymatic activity of Rho-kinase. Most of the chemical modifications result in a loss of activity showing that interactions of Fasudil with the catalytic domain of Rho-kinase seem to be particularly definite and sensitive to structural variations. The presence of an isoquinolinyl nitrogen and a basic amino group separated by a spacer bearing a sulfonamide function are of utmost importance. Only the tetra-hydroisoquinoline analogue 3 shows the same activity as Fasudil. Moreover, this compound is unable to inhibit PKC biological activity contrary to Fasudil. The loss of the aromatic property could increase the selectivity level in favour of compound 3.