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1.
Genes Dev ; 33(17-18): 1136-1158, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31481537

RESUMO

Circadian rhythms are driven by a transcription-translation feedback loop that separates anabolic and catabolic processes across the Earth's 24-h light-dark cycle. Central pacemaker neurons that perceive light entrain a distributed clock network and are closely juxtaposed with hypothalamic neurons involved in regulation of sleep/wake and fast/feeding states. Gaps remain in identifying how pacemaker and extrapacemaker neurons communicate with energy-sensing neurons and the distinct role of circuit interactions versus transcriptionally driven cell-autonomous clocks in the timing of organismal bioenergetics. In this review, we discuss the reciprocal relationship through which the central clock drives appetitive behavior and metabolic homeostasis and the pathways through which nutrient state and sleep/wake behavior affect central clock function.


Assuntos
Relógios Circadianos/fisiologia , Metabolismo Energético/genética , Hipotálamo/metabolismo , Neurônios/fisiologia , Animais , Ritmo Circadiano/fisiologia , AMP Cíclico/metabolismo , Comportamento Alimentar/fisiologia , Humanos , Transdução de Sinais
2.
Gene ; 715: 144028, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31374326

RESUMO

BACKGROUND: Type 2 diabetes (T2D) is a complex polygenic disease with unclear mechanism. In an attempt to identify novel genes involved in ß-cell function, we harness a bioinformatics method called Loss-of-function tool (LoFtool) gene score. METHODS: RNA-sequencing data from human islets were used to cross-reference genes within the 1st quartile of most intolerant LoFtool score with the 100th most expressed genes in human islets. Out of these genes, GNAS and EEF1A1 genes were selected for further investigation in diabetic islets, metabolic tissues along with their correlation with diabetic phenotypes. The influence of GNAS and EEF1A1 on insulin secretion and ß-cell function were validated in INS-1 cells. RESULTS: A comparatively higher expression level of GNAS and EEF1A1 was observed in human islets than fat, liver and muscle tissues. Furthermore, diabetic islets displayed a reduced expression of GNAS, but not of EEF1A, compared to non-diabetic islets. The expression of GNAS was positively correlated with insulin secretory index, GLP1R, GIPR and inversely correlated with HbA1c. Diabetic human islets displayed a reduced cAMP generation and insulin secretory capacity in response to glucose. Moreover, siRNA silencing of GNAS in INS-1 cells reduced insulin secretion, insulin content, and cAMP production. In addition, the expression of Insulin, PDX1, and MAFA was significantly down-regulated in GNAS-silenced cells. However, cell viability and apoptosis rate were unaffected. CONCLUSION: LoFtool is a powerful tool to identify genes associated with pancreatic islets dysfunction. GNAS is a crucial gene for the ß-cell insulin secretory capacity.


Assuntos
Cromograninas/biossíntese , Subunidades alfa Gs de Proteínas de Ligação ao GTP/biossíntese , Regulação da Expressão Gênica , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Idoso , Animais , Linhagem Celular , Cromograninas/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Células Secretoras de Insulina/citologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Ratos
3.
Environ Toxicol ; 34(9): 1052-1062, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31161640

RESUMO

Manganese (Mn) plays a critical role in individual growth and development, yet excessive exposure can result in neurotoxicity, especially cognitive impairment. Neuronal apoptosis is considered as one of the mechanisms of Mn-induced neurotoxicity. Recent evidence suggests that cAMP-PKA-CREB signaling regulates apoptosis and is associated with cognitive function. However, whether this pathway participates in Mn-induced neurotoxicity is not completely understood. To fill this gap, in vitro cultures of PC12 cells were exposed to 0, 400, 500, and 600 µmol/L Mn for 24 hours, respectively. Another group of cells were pretreated with 10.0 µmol/L rolipram (a phosphodiesterase-4 [PDE4] inhibitor) for 1 hour followed by 500 µmol/L Mn exposure for 24 hours. Flow cytometry, immunofluorescence staining, enzyme-linked immunosorbent assay, and Western blot analysis were used to detect the apoptosis rate, protein levels of PDE4, cAMP signaling, and apoptosis-associated proteins, respectively. We found that Mn exposure significantly inhibited cAMP signaling and protein expression of Bcl-2, while increasing apoptosis rate, protein levels of PDE4, Bax, activated caspase-3, and activated caspase-8 in PC12 cells. Pretreatment of rolipram ameliorated Mn-induced deficits in cAMP signaling and apoptosis. These findings demonstrate that cAMP-PKA-CREB signaling pathway-induced apoptosis is involved in Mn-induced neurotoxicity.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Poluentes Ambientais/toxicidade , Manganês/toxicidade , Neurônios/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neurônios/metabolismo , Neurônios/patologia , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Células PC12 , Ratos , Transdução de Sinais/efeitos dos fármacos
4.
Nat Commun ; 10(1): 2572, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189917

RESUMO

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities.


Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Retroalimentação Fisiológica/fisiologia , Chaperonas Moleculares/metabolismo , Ataxias Espinocerebelares/patologia , Animais , Retroalimentação Fisiológica/efeitos dos fármacos , Fibroblastos , Células HEK293 , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Hipocampo/patologia , Holoenzimas/metabolismo , Humanos , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Nucleosídeos de Purina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ataxias Espinocerebelares/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia
5.
Bioelectrochemistry ; 129: 100-105, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31153124

RESUMO

Electrochemically active bacteria (EAB) are capable of electrochemical interactions with electrodes via extracellular electron transfer (EET) pathways and serve as essential components in bioelectrochemical systems. Previous studies have suggested that EAB, such as Shewanella oneidensis MR-1, use cyclic AMP (cAMP) receptor proteins to coordinately regulate the expression of catabolic and EET-related genes, prompting us to hypothesize that the intracellular cAMP concentration is an important factor determining the electrochemical activities of EAB. The present study constructed an MR-1 mutant, cyaC-OE, that overexpressed cyaC, a gene encoding a membrane-bound class III adenylate cyclase, and examined its electrochemical and transcriptomic characteristics. We show that the intracellular cAMP concentration in cyaC-OE is more than five times that in wild-type MR-1, and that cya-OE generates approximately two-fold higher current in BES than the wild-type strain. In addition, the expression of genes involved in EET and anaerobic carbon catabolism is up-regulated in cya-OE compared to that in the wild-type strain. These results suggest that increasing the intracellular cAMP level is a promising approach for constructing EAB with high catabolic and electrochemical activities.


Assuntos
Adenilil Ciclases/genética , Proteínas de Bactérias/genética , Fontes de Energia Bioelétrica/microbiologia , Shewanella/genética , Regulação para Cima , Adenilil Ciclases/metabolismo , Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Eletricidade , Genes Bacterianos , Shewanella/fisiologia
6.
Eur J Med Chem ; 176: 61-67, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096119

RESUMO

The present study was designed to synthesize and evaluate ursolic acid hybrid compounds against glioma cells. Initial screening revealed that most of the synthesized compounds displayed better inhibitory effect on glioma cell proliferation compared to parent ursolic acid. The mechanism of inhibitory effect of the most potent compound 6d on glioma cells was investigated in detail. Treatment with compound 6d significantly (p < 0.001) reduced U251 and C6 cell proliferation at 48 h. The growth of U251 and C6 glioma cells was reduced to minimum level (17 and 21%) on treatment with 10 µM concentration of compound 6d. Treatment of the U251 cells with 10 µM concentration of compound 6d caused a significant (p < 0.05) inhibition of cAMP level. In U251 cell cultures treatment with compound 6d at 10 µM concentration enhanced proportion of apoptotic cells to 69.32% compared to 2.34% in the control cultures. The compound 6d treatment of U251 cells for 48 h caused arrest of cell cycle in the G0/G1 phase with consequent decrease of cell population in G2/M and S phases. The results from TEM showed that compound 6d treatment of U251 cells for 48 h caused blebbing of the cell membranes, chromatin condensation, appearance of foamy cytoplasmic material and autophagic vacuoles. The results from SEM revealed that compound 6d treatment of U251 cells caused a marked inhibition of microvilli and extensions on the cell surfaces. Thus present study demonstrates that compound 6d inhibits glioma cell growth, induces apoptosis and arrest cell cycle through metabolic pathway down-regulation. Therefore, compound 6d can be evaluated further for the treatment of glioma.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , AMP Cíclico/metabolismo , Glioma/tratamento farmacológico , Triterpenos/farmacologia , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Glioma/metabolismo , Glioma/patologia , Humanos , Ratos , Transdução de Sinais/efeitos dos fármacos , Triterpenos/síntese química
7.
Plant Sci ; 283: 1-10, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128679

RESUMO

Colletotrichum higginsianum causes anthracnose disease in a wide range of cruciferous crops and has been used as a model system to study plant-pathogen interactions and pathogenicity of hemibiotrophic plant pathogens. Conidiation, hyphae growth, appressorial development and appressorial penetration are significant steps during the infection process of C. higginsianum. However, the mechanisms of these important steps during infection remain incompletely understood. To further investigate the mechanisms of the plant-C. higginsianum interactions during infection progress, we characterized Cyclase-Associated Protein (ChCAP) gene. Deletion of the ChCAP gene resulted in reduction in conidiation and hyphal growth rate. The pathogenicity of ΔChCAP mutants was significantly reduced with much smaller lesion on the infected leaves compared to that of wild type strain with typically water-soaked and dark necrotic lesions on Arabidopsis leaves. Further study demonstrated that the appressorial formation rate, turgor pressure, penetration ability and switch from biotrophic to necrotrophic phases decreased obviously in ΔChCAP mutants, indicating that the attenuated pathogenicity of ΔChCAP mutants was due to these defective phenotypes. In addition, the ΔChCAP mutants sectored on PDA with abnormal, dark color, vesicle-like colony morphology and hyphae tip. Moreover, the ΔChCAP mutants had a reduced intracellular cAMP levels and exogenous cAMP can partially rescue the defects of ΔChCAP mutants in appressorial formation and penetration rate, but not in colony morphology, conidial shape and virulence, indicating that ChCAP is a key component in cAMP signaling pathway and likely play other roles in biology of C. higginsianum. In summary, our findings support the role of ChCAP in regulating conidiation, intracellular cAMP level, hyphal growth, appressorial formation, penetration ability and pathogenicity of this hemibiotrophic fungus.


Assuntos
Colletotrichum/crescimento & desenvolvimento , AMP Cíclico/metabolismo , Proteínas Fúngicas/fisiologia , Hifas/crescimento & desenvolvimento , Esporos Fúngicos/crescimento & desenvolvimento , Arabidopsis/microbiologia , Colletotrichum/metabolismo , Colletotrichum/patogenicidade , Colletotrichum/fisiologia , Proteínas Fúngicas/metabolismo , Hifas/fisiologia , Filogenia , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Esporos Fúngicos/fisiologia , Estresse Fisiológico
8.
Nat Commun ; 10(1): 1971, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036819

RESUMO

ORAI1 constitutes the store-operated Ca2+ release-activated Ca2+ (CRAC) channel crucial for life. Whereas ORAI1 activation by Ca2+-sensing STIM proteins is known, still obscure is how ORAI1 is turned off through Ca2+-dependent inactivation (CDI), protecting against Ca2+ toxicity. Here we identify a spatially-restricted Ca2+/cAMP signaling crosstalk critical for mediating CDI. Binding of Ca2+-activated adenylyl cyclase 8 (AC8) to the N-terminus of ORAI1 positions AC8 near the mouth of ORAI1 for sensing Ca2+. Ca2+ permeating ORAI1 activates AC8 to generate cAMP and activate PKA. PKA, positioned by AKAP79 near ORAI1, phosphorylates serine-34 in ORAI1 pore extension to induce CDI whereas recruitment of the phosphatase calcineurin antagonizes the effect of PKA. Notably, CDI shapes ORAI1 cytosolic Ca2+ signature to determine the isoform and degree of NFAT activation. Thus, we uncover a mechanism of ORAI1 inactivation, and reveal a hitherto unappreciated role for inactivation in shaping cellular Ca2+ signals and NFAT activation.


Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Proteína ORAI1/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Western Blotting , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Fosforilação , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/genética , Molécula 2 de Interação Estromal/metabolismo
9.
Zhongguo Zhong Yao Za Zhi ; 44(8): 1654-1659, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-31090331

RESUMO

To explore the anti-platelet aggregation and anti-thrombotic mechanisms of Trichosanthis Fructus combined with aspirin based on network pharmacology and the validation of arteriovenous by pass model in rats. The databases of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Drug Repositioning and Adverse Drug Reaction Chemical-Protein Interactome(DRAR-CPI),Universal Protein Resource(Uniprot) and the Database for Annotation,Visualization,and Integrated Discovery(DAVID) were used to predict protein targets and analyze biological pathway and signal pathway in the combination of Trichosanthis Fructus with aspirin. The effects of pretreatment with Trichosanthis Fructus pellets,aspirin pellets and their combination on thromboxane B2(TXB2),6-keto prostaglandin F1α(6-keto-PGF1α) and cyclic adenosine monophosphate(c AMP) in rat thrombotic model were studied. Through the study of network pharmacology,12 components of aspirin and Trichosanthis Fructus,including hydroxygenkwanin,quercetin and adenosine,were found to show the anti-platelet aggregation and anti-thrombosis mechanisms through9 common protein targets,such as SRC,RAC1,MAPK14,MAPK1,AKT1,and 14 common signaling pathways,such as VEGF signaling pathway. After the intervention with Trichosanthis Fructus pellets combined with aspirin pellets,the vascular endothslia growth factor(VEGF) signaling pathway can be activated to inhibit platelet aggregation and improve vascular endothelial function,and show the anti-platelet aggregation and anti-thrombosis mechanisms,which verify the results of the network pharmacology,and explain the anti-platelet aggregation and anti-thrombotic mechanisms of the combination of Trichosanthis Fructus pellets with aspirin pellets.


Assuntos
Aspirina/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Trombose/tratamento farmacológico , Trichosanthes/química , 6-Cetoprostaglandina F1 alfa/metabolismo , Animais , AMP Cíclico/metabolismo , Frutas/química , Ratos , Transdução de Sinais , Tromboxano B2/metabolismo
10.
Zoolog Sci ; 36(2): 105-111, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31120644

RESUMO

Mammalian T cell death-associated gene 8 (TDAG8)s are activated by extracellular protons. In the present study, we examined whether the TDAG8 homologs of other species are activated by protons as they are in mammals. We found that Xenopus TDAG8 also stimulated cAMP response element (CRE)-driven promoter activities reflecting the activation of Gs/cAMP signaling pathways when they are stimulated by protons. On the other hand, the activities of chicken and zebrafish TDAG8s are hardly affected by protons. Results using chimeric receptors of human and zebrafish TDAG8s indicate that the specificity of the proton-induced activation lies in the extracellular region. These results suggest that protons are not an evolutionarily conserved agonist of TDAG8.


Assuntos
Prótons , Receptores Acoplados a Proteínas-G/genética , Animais , Galinhas , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Receptores Acoplados a Proteínas-G/metabolismo , Xenopus , Peixe-Zebra
11.
Molecules ; 24(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035694

RESUMO

The antispasmodic effect of drugs is used for the symptomatic treatment of cramping and discomfort affecting smooth muscles from the gastrointestinal, billiary or genitourinary tract in a variety of clinical situations.The existing synthetic antispasmodic drugs may cause a series of unpleasant side effects, and therefore the discovery of new molecules of natural origin is an important goal for the pharmaceutical industry. This review describes a series of recent studies investigating the antispasmodic effect of essential oils from 39 plant species belonging to 12 families. The pharmacological models used in the studies together with the mechanistic discussions and the chemical composition of the essential oils are also detailed. The data clearly demonstrate the antispasmodic effect of the essential oils from the aromatic plant species studied. Further research is needed in order to ascertain the therapeutic importance of these findings.


Assuntos
Óleos Voláteis/química , Óleos Voláteis/farmacologia , Parassimpatolíticos/química , Parassimpatolíticos/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Estudos Clínicos como Assunto , AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Estrutura Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Óleos Voláteis/análise , Óleos Voláteis/uso terapêutico , Parassimpatolíticos/uso terapêutico , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/análise , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Relação Estrutura-Atividade , Resultado do Tratamento
12.
Life Sci ; 230: 150-161, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31125564

RESUMO

Despite novel technologies, colon cancer remains undiagnosed and 25% of patients are diagnosed with metastatic colon cancer. Resistant to chemotherapeutic agents is one of the major problems associated with treating colon cancer which creates the need to develop novel agents targeting towards newer targets. A phosphodiesterase is a group of isoenzyme, which, hydrolyze cyclic nucleotides and thereby lowers intracellular levels of cAMP and cGMP leading to tumorigenic effects. Many in vitro and in vivo studies have confirmed increased PDE expression in different types of cancers including colon cancer. cAMP-specific PDE inhibitors increase intracellular cAMP that leads to activation of effector molecules-cAMP-dependent protein kinase A, exchange protein activated by cAMP and cAMP gated ion channels. These molecules regulate cellular responses and exert its anticancer role through different mechanisms including apoptosis, inhibition of angiogenesis, upregulating tumor suppressor genes and suppressing oncogenes. On the other hand, cGMP specific PDE inhibitors exhibit anticancer effects through cGMP dependent protein kinase and cGMP dependent cation channels. Elevation in cGMP works through activation of caspases, suppression of Wnt/b-catenin pathway and TCF transcription leading to inhibition of CDK and survivin. These studies point out towards the fact that PDE inhibition is associated with anti-proliferative, anti-apoptotic and anti-angiogenic pathways involved in its anticancer effects in colon cancer. Thus, inhibition of PDE enzymes can be used as a novel approach to treat colon cancer. This review will focus on cAMP and cGMP signaling pathways leading to tumorigenesis and the use of PDE inhibitors in colon cancer.


Assuntos
Neoplasias do Colo/terapia , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Humanos , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Cell Physiol Biochem ; 52(6): 1339-1360, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050282

RESUMO

BACKGROUND/AIMS: Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration. METHODS: We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²âº mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis. RESULTS: We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²âº signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes. CONCLUSION: Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries.


Assuntos
Receptor Tipo 1 de Melanocortina/metabolismo , Aorta/citologia , Sinalização do Cálcio/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Melanocortina/genética , alfa-MSH/farmacologia
14.
Int J Mol Sci ; 20(9)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035588

RESUMO

Melanogenesis is the sequential process of melanin production by melanocytes in order to protect the skin from harmful stimuli. Melanogenesis is disrupted by radiation exposure, which results in the differentiation of melanocytes into melanoma. Recently, some methods have been developed to maintain the instability of melanogenesis in melanoma by activating cellular autophagy. However, there is still a lack of knowledge about how autophagy is involved in the regulation of melanogenesis in melanoma cells. Here, we used rottlerin as an autophagy inducer to investigate the role of the cyclic adenosine monophosphate (cAMP)/cAMP response element binding (CREB) signaling pathway in melanogenesis. We found that rottlerin can inhibit melanin production by targeting cAMP, which is initially activated by alpha-melanocyte stimulating hormone (α-MSH). Our findings suggest that rottlerin has a pivotal role as an autophagy inducer in the regulation of melanogenesis by targeting the cAMP/CREB signaling pathway.


Assuntos
Acetofenonas/farmacologia , Autofagia/efeitos dos fármacos , Benzopiranos/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Melaninas/metabolismo , Acetofenonas/química , Animais , Benzopiranos/química , Linhagem Celular Tumoral , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanoma Experimental , Camundongos , Transdução de Sinais
15.
Methods Mol Biol ; 1947: 361-376, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969428

RESUMO

Engineered G protein-coupled receptors (DREADDs, designer receptors exclusively activated by designer drugs) are convenient tools for specific activation of GPCR signaling in many cell types. DREADDs have been utilized as research tools to study numerous cellular and physiologic processes, including regulation of neuronal activity, behavior, and metabolism. Mice with random insertion transgenes and adeno-associated viruses have been widely used to express DREADDs in individual cell types. We recently created and characterized ROSA26-GsDREADD knock-in mice to allow Cre recombinase-dependent expression of a Gαs-coupled DREADD (GsD) fused to GFP in distinct cell populations in vivo. These animals also harbor a CREB-activated luciferase reporter gene for analysis of CREB activity by in vivo imaging, ex vivo imaging, or biochemical reporter assays. In this chapter, we provide detailed methods for breeding GsD animals, inducing GsD expression, stimulating GsD activity, and measuring basal and stimulated CREB reporter bioluminescence in tissues in vivo, ex vivo, and in vitro. These animals are available from our laboratory for non-profit research.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Genes Reporter , Processamento de Imagem Assistida por Computador/métodos , Medições Luminescentes/métodos , Moduladores de Transporte de Membrana/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Clozapina/análogos & derivados , Clozapina/farmacologia , Integrases/metabolismo , Camundongos , Especificidade de Órgãos , Receptores Acoplados a Proteínas-G/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais
16.
Infect Immun ; 87(6)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936159

RESUMO

Cyclic di-AMP (c-di-AMP) is a recently discovered second messenger in bacteria. The cellular level of c-di-AMP in Streptococcus pyogenes is predicted to be controlled by the synthase DacA and two putative phosphodiesterases, GdpP and Pde2. To investigate the role of c-di-AMP in S. pyogenes, we generated null mutants in each of these proteins by gene deletion. Unlike those in other Gram-positive pathogens such as Staphylococcus aureus and Listeria monocytogenes, DacA in S. pyogenes was not essential for growth in rich media. The DacA null mutant presented a growth defect that manifested through an increased lag time, produced no detectable biofilm, and displayed increased susceptibility toward environmental stressors such as high salt, low pH, reactive oxygen radicals, and cell wall-targeting antibiotics, suggesting that c-di-AMP plays significant roles in crucial cellular processes involved in stress management. The Pde2 null mutant exhibited a lower growth rate and increased biofilm formation, and interestingly, these phenotypes were distinct from those of the null mutant of GdpP, suggesting that Pde2 and GdpP play distinctive roles in c-di-AMP signaling. DacA and Pde2 were critical to the production of the virulence factor SpeB and to the overall virulence of S. pyogenes, as both DacA and Pde2 null mutants were highly attenuated in a mouse model of subcutaneous infection. Collectively, these results show that c-di-AMP is an important global regulator and is required for a proper response to stress and for virulence in S. pyogenes, suggesting that its signaling pathway could be an attractive antivirulence drug target against S. pyogenes infections.


Assuntos
Proteínas de Bactérias/genética , Biofilmes , Parede Celular/metabolismo , AMP Cíclico/metabolismo , Exotoxinas/genética , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/fisiologia , Streptococcus pyogenes/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Exotoxinas/metabolismo , Feminino , Regulação Bacteriana da Expressão Gênica , Homeostase , Humanos , Masculino , Camundongos , Camundongos Pelados , Sistemas do Segundo Mensageiro , Streptococcus pyogenes/genética , Virulência
17.
Cell Commun Signal ; 17(1): 34, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987657

RESUMO

BACKGROUNDS/AIM: Male and female hearts have many structural and functional differences. Here, we investigated the role of estrogen (E2) in the mechanisms of sex differences in contraction through the cAMP-L-type Ca2+channel pathway in adult mice left ventricular (LV) apical myocytes at basal and stress state. METHODS: Isolated LV apical myocytes from male, female (Sham) and ovariectomised mice (OVX) were used to investigate contractility, Ca2+ transients and L-type Ca2+ channel (LTCC) function. The levels of ß2AR, intracellular cAMP, phosphodiesterase (PDE 3 and PDE 4), RyR2, PLB, SLN, and SERCA2a were compared among the experimental groups. RESULTS: We found that (1) intracellular cAMP, ICaL density, contraction and Ca2+ transient amplitudes were larger in Sham and OVX + E2 myocytes compared to male and OVX. (2) The mRNA expression of PDE 3 and 4 were lower in Sham and OVX + E2 groups compared with male and OVX groups. Treatment of myocytes with IBMX (100 µM) increased contraction and Ca2+ transient amplitude in both sexes and canceled differences between them. (3) ß2AR-mediated stress decreased cAMP concentration and peak contraction and Ca2+ transient amplitude only in male and OVX groups but not in Sham or OVX + E2 groups suggesting a cardioprotective role of E2 in female mice. (4) Pretreatment of OVX myocytes with GPR30 antagonist G15 (100 nM) abolished the effects of E2, but ERα and ERß antagonist ICI 182,780 (1 µM) did not. Moreover, activation of GPR30 with G1 (100 nM) replicated the effects of E2 on cAMP, contraction and Ca2+ transient amplitudes suggesting that the acute effects of E2 were mediated by GPR30 via non-genomic signaling. (5) mRNA expression of RyR2 was higher in myocytes from Sham than those of male while PLB and SLN were higher in male than Sham but no sex differences were observed in the mRNA of SERCA2a. CONCLUSION: Collectively, these results demonstrate that E2 modulates the expression of genes related to the cAMP-LTCC pathway and contributes to sex differences in cardiac contraction and responses to stress. We also show that estrogen confers cardioprotection against cardiac stress by non-genomic acute signaling via GPR30.


Assuntos
Canais de Cálcio Tipo L/metabolismo , AMP Cíclico/metabolismo , Estradiol/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Caracteres Sexuais , Função Ventricular Esquerda/fisiologia , Animais , Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio , Cardiotônicos/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Receptores Adrenérgicos beta 1/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos
18.
Environ Toxicol ; 34(7): 844-852, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30951242

RESUMO

Our goals were to investigate whether environmentally relevant doses of T-2 toxin can affect human ovarian granulosa cells' function and to reveal the potential mechanism of T-2 toxin's action. Results showed that T-2 toxin strongly attenuated luteinizing hormone/choriogonadotropin receptor (LHCGR) mRNA expression in follicle-stimulating hormone (FSH)-stimulated human cumulus granulosa cells. Addition of human chorionic gonadotropin was not able to elicit maximal response of ovulatory genes amphiregulin, epiregulin, and progesterone receptor. T-2 toxin reduced mRNA levels of CYP19A1 and steroidogenic acute regulatory protein (STAR) and lowered FSH-stimulated estradiol and progesterone production. Mechanistic experiments demonstrated that T-2 toxin decreased FSH-stimulated cyclic adenosine monophosphate (cAMP) production. Addition of total PDE inhibitor 3-isobutyl-1-methylxanthine prevented T-2 toxin's action on LHCGR, STAR, and CYP19A1 mRNA expression in FSH-stimulated human cumulus granulosa cells. Furthermore, T-2 toxin partially decreased 8-bromoadenosine 3'5'-cyclic monophosphate (8-Br-cAMP)-stimulated LHCGR and STAR, but did not affect 8-Br-cAMP-stimulated CYP19A1 mRNA expression in human cumulus granulosa cells. Overall, our data indicate that environmentally relevant dose of T-2 toxin decreases steroidogenesis and ovulatory potency in human cumulus granulosa cells probably through activation of PDE, thus posing a significant risk for female fertility.


Assuntos
Aromatase/genética , Células do Cúmulo/efeitos dos fármacos , AMP Cíclico/metabolismo , Hormônios Esteroides Gonadais/biossíntese , Fosfoproteínas/genética , Receptores do LH/genética , Toxina T-2/farmacologia , Adulto , Aromatase/metabolismo , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Células do Cúmulo/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Fosfoproteínas/metabolismo , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Receptores do LH/metabolismo , Adulto Jovem
19.
Methods Mol Biol ; 1947: 217-237, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969419

RESUMO

A variety of FRET-based biosensors are currently in use for real-time monitoring of dynamic changes of intracellular cAMP. Due to differences in sensor properties, unique features of the cell type under examination and diverse specifications of the imaging setups in different laboratories, data generated using these sensors may not be immediately comparable within the same study or across studies. To facilitate comparison, often FRET data are normalized and expressed as fractional change of the maximal FRET response at sensor saturation. However, this approach may lead to misinterpretation of the underlying cAMP change. In this chapter, we provide examples of the problems that may arise when using normalized FRET data and present a method based on the conversion of FRET ratio changes into actual cAMP concentrations that mitigates these issues.


Assuntos
Técnicas Biossensoriais/métodos , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Humanos , Transdução de Sinais
20.
Int J Mol Sci ; 20(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018568

RESUMO

Despite numerous studies on major depressive disorder (MDD) susceptibility, the precise underlying molecular mechanism has not been elucidated which restricts the development of etiology-based disease-modifying drug. Major depressive disorder treatment is still symptomatic and is the leading cause of (~30%) failure of the current antidepressant therapy. Here we comprehended the probable genes and pathways commonly associated with antidepressant response and MDD. A systematic review was conducted, and candidate genes/pathways associated with antidepressant response and MDD were identified using an integrative genetics approach. Initially, single nucleotide polymorphisms (SNPs)/genes found to be significantly associated with antidepressant response were systematically reviewed and retrieved from the candidate studies and genome-wide association studies (GWAS). Also, significant variations concerning MDD susceptibility were extracted from GWAS only. We found 245 (Set A) and 800 (Set B) significantly associated genes with antidepressant response and MDD, respectively. Further, gene set enrichment analysis revealed the top five co-occurring molecular pathways (p ≤ 0.05) among the two sets of genes: Cushing syndrome, Axon guidance, cAMP signaling pathway, Insulin secretion, and Glutamatergic synapse, wherein all show a very close relation to synaptic plasticity. Integrative analyses of candidate gene and genome-wide association studies would enable us to investigate the putative targets for the development of disease etiology-based antidepressant that might be more promising than current ones.


Assuntos
Antidepressivos/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/genética , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Inibidores de Captação de Serotonina/uso terapêutico , Antidepressivos/farmacologia , AMP Cíclico/metabolismo , Transtorno Depressivo Maior/metabolismo , Estudo de Associação Genômica Ampla , Genômica/métodos , Humanos , Inibidores de Captação de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fluxo de Trabalho
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