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1.
Nat Commun ; 13(1): 6941, 2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396957

RESUMO

Chronic obstructive airway diseases are a global medical burden that is expected to increase in the near future. However, the underlying mechanistic processes are poorly understood so far. Herein, we show that the endocannabinoid anandamide (AEA) induces prominent airway relaxation in vitro and in vivo. In contrast to 2-arachidonlyglycerol-induced airway relaxation, this is mediated by fatty acid amide hydrolase (FAAH)-dependent metabolites. In particular, we identify mouse and also human epithelial and airway smooth muscle cells as source of AEA-induced prostaglandin E2 production and cAMP as direct mediator of AEA-dependent airway relaxation. Mass spectrometry experiments demonstrate reduced levels of endocannabinoid-like compounds in lungs of ovalbumin-sensitized mice indicating a pathophysiological relevance of endocannabinoid signalling in obstructive airway disease. Importantly, AEA inhalation protects against airway hyper-reactivity after ovalbumin sensitization. Thus, this work highlights the AEA/FAAH axis as a critical regulator of airway tone that could provide therapeutic targets for airway relaxation.


Assuntos
Ácidos Araquidônicos , Endocanabinoides , Animais , Camundongos , Humanos , Endocanabinoides/metabolismo , Ovalbumina , Ácidos Araquidônicos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo
2.
Sci Transl Med ; 14(638): eabl6328, 2022 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-35353541

RESUMO

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fosfatidilinositol 3-Quinase , Animais , Classe Ib de Fosfatidilinositol 3-Quinase , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Inflamação , Camundongos , Peptídeos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo
3.
Arterioscler Thromb Vasc Biol ; 41(12): 2974-2989, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34615374

RESUMO

OBJECTIVE: Pathological angiogenesis is a hallmark of various diseases characterized by local hypoxia and inflammation. These disorders can be treated with inhibitors of angiogenesis, but current compounds display a variety of side effects and lose efficacy over time. This makes the identification of novel signaling pathways and pharmacological targets involved in angiogenesis a top priority. Approach and Results: Here, we show that inactivation of FAAH (fatty acid amide hydrolase), the enzyme responsible for degradation of the endocannabinoid anandamide, strongly impairs angiogenesis in vitro and in vivo. Both, the pharmacological FAAH inhibitor URB597 and anandamide induce downregulation of gene sets for cell cycle progression and DNA replication in endothelial cells. This is underscored by cell biological experiments, in which both compounds inhibit proliferation and migration and evoke cell cycle exit of endothelial cells. This prominent antiangiogenic effect is also of pathophysiological relevance in vivo, as laser-induced choroidal neovascularization in the eye of FAAH-/- mice is strongly reduced. CONCLUSIONS: Thus, elevation of endogenous anandamide levels by FAAH inhibition represents a novel antiangiogenic mechanism.


Assuntos
Amidoidrolases/farmacocinética , Ácidos Araquidônicos/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Endocanabinoides/farmacologia , Endotélio Vascular/crescimento & desenvolvimento , Músculo Liso Vascular/efeitos dos fármacos , Alcamidas Poli-Insaturadas/farmacologia , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/patologia , Agonistas de Receptores de Canabinoides/farmacologia , Bovinos , Linhagem Celular , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Humanos , Camundongos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Neovascularização Patológica
4.
ACS Pharmacol Transl Sci ; 4(2): 888-897, 2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33860209

RESUMO

Guanine nucleotide-binding proteins (G proteins) transduce extracellular signals received by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. While GPCRs represent the largest class of drug targets, G protein inhibition has only recently been recognized as a novel strategy for treating complex diseases such as asthma, inflammation, and cancer. The structurally similar macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent selective inhibitors of the Gq subfamily of G proteins. FR and YM differ in two positions, FR being more lipophilic than YM. Both compounds are utilized as pharmacological tools to block Gq proteins in vitro and in vivo. However, no detailed characterization of FR and YM has been performed, which is a prerequisite for the compounds' translation into clinical application. Here, we performed a thorough study of both compounds' physicochemical, pharmacokinetic, and pharmacological properties. Chemical stability was high across a large range of pH values, with FR being somewhat more stable than YM. Oral bioavailability and brain penetration of both depsipeptides were low. FR showed lower plasma protein binding and was metabolized significantly faster than YM by human and mouse liver microsomes. FR accumulated in lung after chronic intratracheal or intraperitoneal application, while YM was more distributed to other organs. Most strikingly, the previously observed longer residence time of FR resulted in a significantly prolonged pharmacologic effect as compared to YM in a methacholine-induced bronchoconstriction mouse model. These results prove that changes within a molecule which seem marginal compared to its structural complexity can lead to crucial pharmacological differences.

5.
Mol Cell ; 80(6): 940-954.e6, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33202251

RESUMO

Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Fosfolipase C beta/genética , Cálcio/metabolismo , Sinalização do Cálcio/genética , Citosol/metabolismo , Células HEK293 , Humanos , Ligação Proteica/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética
6.
Front Chem ; 8: 833, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33173765

RESUMO

The cyclic depsipeptide FR900359 (FR) isolated from the plant Ardisia crenata and produced by endosymbiotic bacteria acts as a selective Gq protein inhibitor. It is a powerful tool to study G protein-coupled receptor signaling, and has potential as a novel drug for the treatment of pulmonary diseases and cancer. For pharmacokinetic studies, sensitive quantitative measurements of drug levels are required. In the present study we established an LC-MS/MS method to detect nanomolar concentrations of FR and the structurally related natural product YM-254890 (YM) in biological samples. HPLC separation coupled to ESI-QTOF-MS and UV-VIS detection was applied. For identification and quantification, the extract ion chromatogram (EIC) of M+1 was evaluated. Limits of detection (LOD) of 0.53-0.55 nM and limits of quantification (LOQ) of 1.6-1.7 nM were achieved for both FR and YM. This protocol was subsequently applied to determine FR concentrations in mouse organs and tissues after peroral application of the drug. A three-step liquid-liquid extraction protocol was established, which resulted in adequate recovery rates of typically around 50%. The results indicated low peroral absorption of FR. Besides the gut, highest concentrations were determined in eye and kidney. The developed analytical method will be useful for preclinical studies to evaluate these potent Gq protein inhibitors, which may have potential as future drugs for complex diseases.

7.
Br J Pharmacol ; 177(8): 1898-1916, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31881095

RESUMO

BACKGROUND AND PURPOSE: G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The Gq protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up-regulated in cancer and inflammatory diseases. Gq inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for Gq proteins are lacking. EXPERIMENTAL APPROACH: We have now developed Gq -specific, cell-permeable 3 H-labelled high-affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM). The tracers served to specifically label and quantify Gq proteins in their native conformation in cells and tissues with high accuracy. KEY RESULTS: FR and YM displayed low nanomolar affinity for Gαq , Gα11 and Gα14 expressed in CRISPR/Cas9 Gαq -knockout cells, but not for Gα15 . The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a "dowel" effect of the pseudoirreversibly binding FR. A high-throughput binding assay led to the discovery of novel Gq inhibitors, which inhibited Gq signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation. CONCLUSIONS AND IMPLICATIONS: The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Transdução de Sinais , Animais , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Cinética , Camundongos
8.
FASEB J ; 32(5): 2519-2530, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29295855

RESUMO

Pulmonary hypertension is a severe, incurable disease with a poor prognosis. Although treatment regimens have improved during the last 2 decades, current pharmacologic strategies are limited and focus on the modulation of only a few pathways related to endothelin, NO, and prostacyclin signaling. Therefore, the identification of novel molecular targets is urgently needed. We found that the ß2 adrenoceptor (AR) agonists terbutaline (TER) and salbutamol induced a dose-dependent vasorelaxation in large pulmonary arteries but not aortas of mouse. This effect was found to be independent of ß ARs and the endothelium but was determined by the type of the preconstrictor. Vasodilation by ß2 AR agonists occurred after pretreatment of pulmonary arteries with phenylephrine and serotonin, both agonists of α1 ARs, but was absent after preconstriction with the thromboxane analog U46619. These data indicated α-adrenolytic activity of ß2 AR agonists, which was confirmed by a right shift of the phenylephrine dose-response curve by TER. This effect was physiologically relevant because TER also relaxed small intrapulmonary arteries in lung slices and diminished pulmonary arterial pressure in an isolated perfused lung model under normoxia and hypoxia. Finally, TER applied as an aerosol also selectively decreased pulmonary arterial pressure without effects on systemic blood pressure and heart rate in mouse in vivo. Thus, ß2 AR agonists display α-adrenolytic activity in pulmonary arteries ex vivo and in vivo, and may provide a novel option to reduce pulmonary arterial pressure in pulmonary hypertension.-Neumann, V., Knies, R., Seidinger, A., Simon, A., Lorenz, K., Matthey, M., Breuer, J., Wenzel, D. The ß2 agonist terbutaline specifically decreases pulmonary arterial pressure under normoxia and hypoxia via α adrenoceptor antagonism.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Pulmão , Artéria Pulmonar/fisiopatologia , Receptores Adrenérgicos alfa 1/metabolismo , Terbutalina/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Pulmão/irrigação sanguínea , Pulmão/fisiopatologia , Camundongos , Fenilefrina/farmacologia , Artéria Pulmonar/metabolismo
9.
Sci Transl Med ; 9(407)2017 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-28904224

RESUMO

Obstructive lung diseases are common causes of disability and death worldwide. A hallmark feature is aberrant activation of Gq protein-dependent signaling cascades. Currently, drugs targeting single G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) are used to reduce airway tone. However, therapeutic efficacy is often limited, because various GPCRs contribute to bronchoconstriction, and chronic exposure to receptor-activating medications results in desensitization. We therefore hypothesized that pharmacological Gq inhibition could serve as a central mechanism to achieve efficient therapeutic bronchorelaxation. We found that the compound FR900359 (FR), a membrane-permeable inhibitor of Gq, was effective in silencing Gq signaling in murine and human airway smooth muscle cells. Moreover, FR both prevented bronchoconstrictor responses and triggered sustained airway relaxation in mouse, pig, and human airway tissue ex vivo. Inhalation of FR in healthy wild-type mice resulted in high local concentrations of the compound in the lungs and prevented airway constriction without acute effects on blood pressure and heart rate. FR administration also protected against airway hyperreactivity in murine models of allergen sensitization using ovalbumin and house dust mite as allergens. Our findings establish FR as a selective Gq inhibitor when applied locally to the airways of mice in vivo and suggest that pharmacological blockade of Gq proteins may be a useful therapeutic strategy to achieve bronchorelaxation in asthmatic lung disease.


Assuntos
Asma/metabolismo , Asma/fisiopatologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Pulmão/metabolismo , Pulmão/fisiopatologia , Relaxamento Muscular , Transdução de Sinais , Animais , Asma/tratamento farmacológico , Asma/parasitologia , Broncoconstrição , Depsipeptídeos/farmacologia , Depsipeptídeos/uso terapêutico , Modelos Animais de Doenças , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Relaxamento Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ovalbumina , Pneumonia/complicações , Pneumonia/tratamento farmacológico , Pneumonia/patologia , Pyroglyphidae , Sus scrofa
10.
Basic Res Cardiol ; 109(4): 425, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24980781

RESUMO

Ischemic heart disease is associated with inflammation, interstitial fibrosis and ventricular dysfunction prior to the development of heart failure. Endocannabinoids and the cannabinoid receptor CB2 have been claimed to be involved, but their potential role in cardioprotection is not well understood. We therefore explored the role of the cannabinoid receptor CB2 during the initial phase of ischemic cardiomyopathy development prior to the onset of ventricular dysfunction or infarction. Wild type and CB2-deficient mice underwent daily brief, repetitive ischemia and reperfusion (I/R) episodes leading to ischemic cardiomyopathy. The relevance of the endocannabinoid-CB2 receptor axis was underscored by the finding that CB2 was upregulated in ischemic wild type cardiomyocytes and that anandamide level was transiently increased during I/R. CB2-deficient mice showed an increased rate of apoptosis, irreversible loss of cardiomyocytes and persistent left ventricular dysfunction 60 days after the injury, whereas wild type mice presented neither morphological nor functional defects. These defects were due to lack of cardiomyocyte protection mechanisms, as CB2-deficient hearts were in contrast to controls unable to induce switch in myosin heavy chain isoforms, antioxidative enzymes and chemokine CCL2 during repetitive I/R. In addition, a prolonged inflammatory response and adverse myocardial remodeling were found in CB2-deficient hearts because of postponed activation of the M2a macrophage subpopulation. Therefore, the endocannabinoid-CB2 receptor axis plays a key role in cardioprotection during the initial phase of ischemic cardiomyopathy development.


Assuntos
Cardiomiopatias/prevenção & controle , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Transdução de Sinais , Animais , Apoptose , Ácidos Araquidônicos/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Endocanabinoides/metabolismo , Feminino , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/patologia , Alcamidas Poli-Insaturadas/metabolismo , Receptor CB2 de Canabinoide/deficiência , Receptor CB2 de Canabinoide/genética , Fatores de Tempo , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Disfunção Ventricular Esquerda/prevenção & controle , Função Ventricular Esquerda , Remodelação Ventricular
11.
FASEB J ; 28(5): 2281-92, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24500921

RESUMO

Novel relaxants of pulmonary arteries and airways are of special interest to obtain insights into pulmonary signaling pathways and to develop treatment strategies for lung diseases. Herein, we demonstrate that Arg-Gly-Asp (RGD) peptides induce a dose-dependent relaxation of pulmonary arteries and airways in mouse. The relaxing effect was specific because it was strongly reduced using the control peptides RGE or RAD (P<0.001). Longer peptide sequences containing RGD and its flanking amino acids found in fibronectin showed a similar effect even at a 10-fold lower concentration. The relevance of RGD-induced pulmonary vasorelaxation was demonstrated in isometric force measurements, lung slices, and the isolated perfused lung model under normoxia and hypoxia and in vivo. As cell surface receptor we identified ß3- but not ß1-integrin subunits. Moreover, vasorelaxation by RGD peptides was strongly diminished after removal of the endothelium in endothelial nitric oxide synthase-deficient (eNOS(-/-) mice; P<0.01) and after pharmacological inhibition of the NO/sGC pathway (P<0.05). Additionally, several potassium channels like Kv, Kir, and KATP played a role. In airways the response was mediated by Kv and KCa channels. Thus, RGD peptides are relaxants of pulmonary arteries and airways. These findings may help to establish novel therapeutic approaches for pulmonary hypertension and obstructive lung disease.


Assuntos
Regulação da Expressão Gênica , Integrina beta3/metabolismo , Pulmão/metabolismo , Oligopeptídeos/química , Artéria Pulmonar/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Ventrículos do Coração/patologia , Hipóxia , Técnicas In Vitro , Camundongos , Óxido Nítrico Sintase Tipo III/genética , Perfusão , Canais de Potássio/metabolismo , Transdução de Sinais , Vasodilatação
12.
Proc Natl Acad Sci U S A ; 110(46): 18710-5, 2013 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-24167249

RESUMO

Endocannabinoids are important regulators of organ homeostasis. Although their role in systemic vasculature has been extensively studied, their impact on pulmonary vessels remains less clear. Herein, we show that the endocannabinoid anandamide (AEA) is a key mediator of hypoxic pulmonary vasoconstriction (HPV) via fatty acid amide hydrolase (FAAH)-dependent metabolites. This is underscored by the prominent vasoconstrictive effect of AEA on pulmonary arteries and strongly reduced HPV in FAAH(-/-) mice and wild-type mice upon pharmacological treatment with FAAH inhibitor URB597. In addition, mass spectrometry measurements revealed a clear increase of AEA and the FAAH-dependent metabolite arachidonic acid in hypoxic lungs of wild-type mice. We have identified pulmonary vascular smooth muscle cells as the source responsible for hypoxia-induced AEA generation. Moreover, either FAAH(-/-) mice or wild-type mice treated with FAAH inhibitor URB597 are protected against hypoxia-induced pulmonary hypertension and the concomitant vascular remodeling in the lung. Thus, the AEA/FAAH pathway is an important mediator of HPV and is involved in the generation of pulmonary hypertension.


Assuntos
Ácidos Araquidônicos/metabolismo , Endocanabinoides/metabolismo , Hipertensão Pulmonar/prevenção & controle , Hipóxia/fisiopatologia , Pulmão/fisiopatologia , Alcamidas Poli-Insaturadas/metabolismo , Transdução de Sinais/fisiologia , Vasoconstrição/fisiologia , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Amidoidrolases/metabolismo , Análise de Variância , Animais , Benzamidas/farmacologia , Western Blotting , Carbamatos/farmacologia , Cromatografia Líquida , Primers do DNA/genética , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Hipóxia/complicações , Hipóxia/metabolismo , Imuno-Histoquímica , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Hypertension ; 59(6): 1256-62, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22547445

RESUMO

Some small molecular weight peptides possess potent vasoactive properties. Herein we have identified the laminin nonapeptide (LNP) CDPGYIGSR as a novel vasoconstrictive agent. Isometric force measurements revealed that LNP induced vasoconstriction in small and large murine arteries in a dose-dependent fashion; LNP also increased vascular tone in human mammary arteries. The vasoactive response was specific for the nonapeptide, because neither scrambled nor very similar peptide sequences modulated vascular tone. As an underlying mechanism we found in [Ca(2+)](i) imaging experiments that the nonapeptide induced transmembrane [Ca(2+)](i) influx in vascular smooth muscle cells. Patch clamp experiments showed that LNP activated nonselective cation channels, causing depolarization of the membrane potential and opening of L-type Ca(2+) channels. The functional effect of LNP was also assessed with catheter measurements in mice in vivo and confirmed vasoconstriction. This effect was restricted to the systemic circulation, because measurements with the perfused lung system demonstrated that LNP did not alter vascular tone in pulmonary arteries. Thus, LNP is a vasoconstrictor in mouse and human arteries, and its vasoactivity is restricted to the systemic vasculature.


Assuntos
Circulação Sanguínea/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Oligopeptídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Sequência de Aminoácidos , Animais , Artérias/efeitos dos fármacos , Artérias/fisiologia , Circulação Sanguínea/fisiologia , Pressão Sanguínea/fisiologia , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/fisiologia , Feminino , Humanos , Técnicas In Vitro , Laminina/química , Laminina/farmacologia , Masculino , Artéria Torácica Interna/efeitos dos fármacos , Artéria Torácica Interna/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Oligopeptídeos/química , Técnicas de Patch-Clamp , Cauda/irrigação sanguínea , Vasoconstrição/fisiologia
14.
PLoS One ; 5(12): e14240, 2010 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-21151899

RESUMO

BACKGROUND: Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. METHODOLOGY/PRINCIPAL FINDINGS: High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCßII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCßII, significantly reducing atherosclerosis. CONCLUSIONS: High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.


Assuntos
Apolipoproteínas E/genética , Aterosclerose/metabolismo , Cisteína Endopeptidases/genética , Diabetes Mellitus Experimental/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ubiquitina/química , Animais , Cisteína Endopeptidases/metabolismo , Glicosilação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos de Músculo Liso/citologia , NF-kappa B/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa
15.
Hypertension ; 54(1): 157-63, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19470879

RESUMO

beta(2)-adrenoceptors are important modulators of vascular tone, particularly in the pulmonary circulation. Because neurohormonal activation occurs in pulmonary arterial hypertension, we have investigated the effect of different adrenergic vasoactive substances on tone regulation in large and small pulmonary arteries, as well as in systemic vessels of mice. We found that the beta(2)-adrenoceptor antagonist ICI 118,551 (ICI) evoked a decrease of vascular tone in large pulmonary arteries and reduced the sensitivity of pulmonary arteries toward different contracting agents, eg, norepinephrine, serotonin, or endothelin. ICI proved to act specifically on pulmonary vessels, because it shifted the dose-response curve of norepinephrine to the right in pulmonary arteries, whereas there was no effect in the aorta. Pharmacological experiments proved that the right shift of the norepinephrine dose-response curve by ICI was mediated via a beta(2)-adrenoceptor/G(i/o) protein-dependent pathway enhancing NO production in the endothelium; these results were corroborated in beta-adrenoceptor and endothelial NO synthase knockout mice where ICI had no effect. ICI increased vascular lumen diameter in lung sections and reduced pulmonary arterial pressure under normoxia and under hypoxia in the isolated perfused lung model. These effects were found to be physiologically relevant, because ICI specifically decreased pulmonary but not systemic blood pressure in vivo. Thus, the beta(2)-adrenoceptor antagonist ICI is a pulmonary arterial-specific vasorelaxant and, therefore, a potentially interesting novel therapeutic agent for the treatment of pulmonary arterial hypertension.


Assuntos
Antagonistas de Receptores Adrenérgicos beta 2 , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Óxido Nítrico/metabolismo , Propanolaminas/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Animais , Western Blotting , Relação Dose-Resposta a Droga , Feminino , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Norepinefrina/farmacologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiologia , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/fisiologia , Transdução de Sinais/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia
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