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1.
Nat Commun ; 11(1): 4981, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020469

RESUMO

Antagonism or agonism of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) prevents weight gain and leads to dramatic weight loss in combination with glucagon-like peptide-1 receptor agonists in preclinical models. Based on the genetic evidence supporting GIPR antagonism, we previously developed a mouse anti-murine GIPR antibody (muGIPR-Ab) that protected diet-induced obese (DIO) mice against body weight gain and improved multiple metabolic parameters. This work reconciles the similar preclinical body weight effects of GIPR antagonists and agonists in vivo, and here we show that chronic GIPR agonism desensitizes GIPR activity in primary adipocytes, both differentiated in vitro and adipose tissue in vivo, and functions like a GIPR antagonist. Additionally, GIPR activity in adipocytes is partially responsible for muGIPR-Ab to prevent weight gain in DIO mice, demonstrating a role of adipocyte GIPR in the regulation of adiposity in vivo.


Assuntos
Adipócitos/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Adipócitos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/uso terapêutico , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Peso Corporal/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Obesidade/patologia , Receptores dos Hormônios Gastrointestinais/deficiência , Receptores dos Hormônios Gastrointestinais/metabolismo
2.
Gene ; 763: 145059, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32858177

RESUMO

Osteopontin (OPN) is not only a marker of osteoblasts but it is also related to cancer progression and inflammation. The expression of OPN increases in response to inflammatory cytokines, hormones, and mechanical stress. Among them, cyclic-AMP (cAMP) elevating agents stimulate OPN expression in the presence of 1, 25-OH vitamin D3 (VD3). We aimed to clarify the mechanism by which cAMP enhances OPN expression in osteoblastic cells. The OPN promoter (-2335 to +76, OPNp2335) exerted a cell type specific response to forskolin (FK) and VD3. Sequential deletion analysis of OPNp revealed that the OPNp (-833 to +76) contained essential responsive regions to respond to cAMP signaling. In particular, both Vitamin D response element (VDRE, -758 to -743) and osteoblast-specific cis- acting element 2 (OSE2, -695 to -690) were essential for cAMP-mediated OPNp activity. The expression of vitamin D receptor (VDR), but not runt-related transcription factor 2 (Runx2), a nuclear receptor for OSE2, was induced by the treatment of the cells with FK. Although, VD3-induced OPNp activity was slightly enhanced in VDR-overexpressing osteoblasts, it reached the same level as that of osteoblasts induced by both VD3 and FK in the presence of histone deacetylase (HDAC) inhibitor. Moreover, we identified histone acetylation on the OPN promoter region by FK treatment. These results strongly suggest that OPNp activity is controlled by the cAMP signaling via genetic and epigenetic regulations.


Assuntos
AMP Cíclico/metabolismo , Epigênese Genética , Osteoblastos/metabolismo , Osteopontina/genética , Acetilação , Animais , Células HEK293 , Código das Histonas , Humanos , Camundongos , Osteopontina/química , Osteopontina/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo
3.
PLoS One ; 15(8): e0236727, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750068

RESUMO

Low-power laser irradiation (LPLI) is clinically used to modulate inflammation, proliferation and apoptosis. However, its molecular mechanisms are still not fully understood. This study aimed to describe the effects of LPLI upon inflammatory, apoptotic and proliferation markers in submandibular salivary glands (SMGs) in an experimental model of chronic disorder, 24h after one time irradiation. Diabetes was induced in rats by the injection of streptozotocin. After 29 days, these animals were treated with LPLI in the SMG area, and euthanized 24h after this irradiation. Treatment with LPLI significantly decreased diabetes-induced high mobility group box 1 (HMGB1) and tumor necrosis factor alpha (TNF-α) expression, while enhancing the activation of the transcriptional factor cAMP response element binding (CREB) protein. LPLI also reduced the expression of bax, a mitochondrial apoptotic marker, favoring the cell survival. These findings suggest that LPLI can hamper the state of chronic inflammation and favor homeostasis in diabetic rats SMGs.


Assuntos
Diabetes Mellitus Experimental/radioterapia , Terapia com Luz de Baixa Intensidade , Transdução de Sinais/efeitos da radiação , Glândula Submandibular/efeitos da radiação , Animais , Apoptose , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Feminino , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismo
4.
Cells ; 9(9)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32854430

RESUMO

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.


Assuntos
Inibidores de Adenilil Ciclases/uso terapêutico , Adenilil Ciclases/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , AMP Cíclico/antagonistas & inibidores , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
5.
Int J Nanomedicine ; 15: 4139-4149, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606669

RESUMO

Introduction: A correlation is established between the efficacy of Chinese herbal medicine and its charcoal drugs. Lonicerae japonicae Flos (LJF) is commonly used to treat fever, carbuncle, and tumors, among others. LJF Carbonisatas (LJFC) is preferred for detoxifying and relieving dysentery and its related symptoms. However, the mechanisms underlying the effects of LJFC remain unknown. Aim: The aim of this study was to explore the effects of LJFC-derived carbon dots (LJFC-CDs) on lipopolysaccharide (LPS)-induced fever and hypothermia rat models. Methods: LJFC-CDs were characterized using transmission electron microscopy, high-resolution transmission electron microscopy, Fourier-transform infrared, ultraviolet, fluorescence, X-ray photoelectron spectroscopy, X-ray diffraction and high-performance liquid chromatography. The anti-inflammatory effects of LJFC-CDs were evaluated and confirmed using rat models of LPS-induced fever or hypothermia. Results: The LJFC-CDs ranged from 1.0 to 10.0 nm in diameter, with a yield of 0.5%. LJFC-CDs alleviated LPS-induced inflammation, as demonstrated by the expression of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 and the recovery of normal body temperature. Conclusion: LJFC-CDs may have an anti-inflammatory effect and a potential to alleviate fever and hypothermia caused by inflammation.


Assuntos
Carbono/química , Febre/tratamento farmacológico , Hipotermia/tratamento farmacológico , Lonicera/química , Extratos Vegetais/uso terapêutico , Animais , Temperatura Corporal/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , Citocinas/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Mediadores da Inflamação/sangue , Lipopolissacarídeos , Masculino , Camundongos , Extratos Vegetais/toxicidade , Células RAW 264.7 , Ratos Sprague-Dawley , Espectrometria de Fluorescência
6.
PLoS One ; 15(7): e0232963, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730272

RESUMO

Mammalian cardiomyocytes exit the cell cycle shortly after birth. As a result, an occurrence of coronary occlusion-induced myocardial infarction often results in heart failure, postinfarction LV dilatation, or death, and represents one of the most significant public health morbidities worldwide. Interestingly however, the hearts of neonatal pigs have been shown to regenerate following an acute myocardial infarction (MI) occuring on postnatal day 1 (P1); a recovery period which is accompanied by an increased expression of markers for cell-cycle activity, and suggests that early postnatal myocardial regeneration may be driven in part by the MI-induced proliferation of pre-existing cardiomyocytes. In this study, we identified signaling pathways known to regulate the cell cycle, and determined of these, the pathways persistently upregulated in response to MI injury. We identified five pathways (mitogen associated protein kinase [MAPK], Hippo, cyclic [cAMP], Janus kinase/signal transducers and activators of transcription [JAK-STAT], and Ras) which were comprehensively upregulated in cardiac tissues collected on day 7 (P7) and/or P28 of the P1 injury hearts. Several of the initiating master regulators (e.g., CSF1/CSF1R, TGFB, and NPPA) and terminal effector molecules (e.g., ATF4, FOS, RELA/B, ITGB2, CCND1/2/3, PIM1, RAF1, MTOR, NKF1B) in these pathways were persistently upregulated at day 7 through day 28, suggesting there exists at least some degree of regenerative activity up to 4 weeks following MI at P1. Our observations provide a list of key regulators to be examined in future studies targeting cell-cycle activity as an avenue for myocardial regeneration.


Assuntos
Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Animais , Animais Recém-Nascidos , AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases , Infarto do Miocárdio/metabolismo , Suínos , Fatores de Tempo
7.
Proc Natl Acad Sci U S A ; 117(30): 18079-18090, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32647060

RESUMO

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways.


Assuntos
Retículo Endoplasmático/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Família Multigênica , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Nó Sinoatrial/fisiologia , Nó Sinoatrial/fisiopatologia
8.
Life Sci ; 257: 118073, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32663574

RESUMO

AIMS: The preservation of pancreatic beta-cell function is crucial for the treatment of type 2 diabetes. Inhibition of class I histone deacetylase (HDAC) has been proved to protect beta-cells from palmitate- or cytokine-induced apoptosis and increase insulin secretion. However, the underlying molecular mechanism is unclear. MAIN METHODS: Rat islets were isolated for insulin secretion, real-time PCR, RNA- sequencing, ChIP-PCR, and oxygen consumption rate analysis after treated with the HDAC1 and HDAC3 inhibitor MS-275. KEY FINDINGS: MS-275 pretreatment significantly potentiated insulin secretion from rat islets. RNA-sequencing revealed that multiple signaling pathways were involved in MS-275-regulated islet function. Cacna1g and Adcy1 in calcium and cAMP signaling pathways were up-regulated in MS-275-treated islets, which was validated by real-time PCR. The expressions of the two genes displayed a similar increase in islets isolated from mice treated with MS-275. Knockdown of HDAC1 elevated Cacna1g and Adcy1 expressions in islets. ChIP-sequencing analysis showed that the pan-HDAC inhibitor sodium butyrate increased H3K27 acetylation level in the upstream region of Adcy1 and the promoter region of Cacna1g. ChIP-PCR revealed a similar result in MS-275-treated rat islets. However, MS-275 had minor effect on glucose-induced oxygen consumption rate in rat islets. Unlike glucose, MS-275 did not alter the expressions of glucose-sensitive genes such as Glut2 and Gck, but elevated intracellular Ca2+ concentration in beta-cells. SIGNIFICANCE: Our findings support the notion that MS-275-potentiated insulin secretion is involved in calcium and cAMP signaling-mediated gene expressions independent of glucose oxidation. Therefore, HDAC inhibition may serve as a therapeutic strategy for type 2 diabetes.


Assuntos
Benzamidas/farmacologia , Glucose/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Secreção de Insulina/efeitos dos fármacos , Piridinas/farmacologia , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Técnicas de Silenciamento de Genes , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilases/efeitos dos fármacos , Células Secretoras de Insulina , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
9.
Proc Natl Acad Sci U S A ; 117(25): 14220-14230, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513741

RESUMO

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Animais , Animais Geneticamente Modificados , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Humanos , Fosforilação , Tauopatias , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Peixe-Zebra , Proteínas tau/metabolismo
10.
Metabolism ; 109: 154282, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32497535

RESUMO

The most severe presentation of COVID-19 is characterized by a hyperinflammatory state attributed to the massive pro-inflammatory cytokine release, called "cytokine storm". Several specific anti-inflammatory/immunosuppressive agents are being evaluated by ongoing clinical trials; however, there is currently insufficient evidence for their efficacy and safety in COVID-19 treatment. Given the role of phosphodiesterase 4 (PDE) 4 and cyclic adenosine monophosphate in the inflammatory response, we hypothesize that selective PDE4 inhibition may attenuate the cytokine storm in COVID-19, through the upstream inhibition of pro-inflammatory molecules, particularly TNF-α, and the regulation of the pro-inflammatory/anti-inflammatory balance. Conversely, other anti-cytokine agents lead to the downstream inhibition of specific targets, such as IL-1, IL-6 or TNF-α, and may not be efficient in blocking the cytokine storm, once it has been triggered. Due to their mechanism of action targeting an early stage of the inflammatory response and ameliorating lung inflammation, we believe that selective PDE4 inhibitors may represent a promising treatment option for the early phase of COVID-19 pneumonia before the cytokine storm and severe multiorgan dysfunction take place. Furthermore, PDE4 inhibitors present several advantages including an excellent safety profile; the oral route of administration; the convenient dosing; and beneficial metabolic properties. Interestingly, obesity and diabetes mellitus type 2 have been reported to be risk factors for the severity of COVID-19. Therefore, randomized clinical trials of PDE4 inhibitors are necessary to explore their potential therapeutic effect as an adjunct to supportive measures and other therapeutic regiments.


Assuntos
Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Síndrome da Liberação de Citocina/tratamento farmacológico , Inibidores da Fosfodiesterase 4/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Infecções por Coronavirus/etiologia , Infecções por Coronavirus/imunologia , AMP Cíclico/metabolismo , Humanos , Interleucina-17/antagonistas & inibidores , Obesidade/complicações , Pandemias , Pneumonia Viral/etiologia , Pneumonia Viral/imunologia
11.
Arterioscler Thromb Vasc Biol ; 40(6): e166-e179, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32349534

RESUMO

OBJECTIVE: Recent studies suggest that the P2Y12 (P2Y purinoceptor 12) receptor of vascular smooth muscle cells in atherosclerotic plaques aggravates atherosclerosis, and P2Y12 receptor inhibitors such as CDL (clopidogrel) may effectively treat atherosclerosis. It is imperative to identify an effective biomarker for reflecting the P2Y12 receptor expression on vascular smooth muscle cells in plaques. Approach and Results: We found that there was a positive correlation between the level of circulating sLRP1 (soluble low-density lipoprotein receptor-related protein 1) and the number of LRP1+ α-SMA+ (α-smooth muscle actin), P2Y12+, or P2Y12+ LRP1+ cells in plaques from apoE-/- mice fed a high-fat diet. Furthermore, activation of the P2Y12 receptor increased the expression and shedding of LRP1 in vascular smooth muscle cells by inhibiting cAMP (3'-5'-cyclic adenosine monophosphate)/PKA (protein kinase A)/SREBP-2 (sterol regulatory element binding transcription factor 2). Conversely, genetic knockdown or pharmacological inhibition of the P2Y12 receptor had the opposite effects. Additionally, CDL decreased the number of lesional LRP1+ α-SMA+ cells and the levels of circulating sLRP1 by activating cAMP/PKA/SREBP-2 in apoE-/- mice fed a high-fat diet. CONCLUSIONS: Our study suggests that sLRP1 may be a biomarker that reflects the P2Y12 receptor level in plaques and has the potential to be an indicator for administering P2Y12 receptor inhibitors for patients with atherosclerosis.


Assuntos
Biomarcadores/análise , Expressão Gênica , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/análise , Placa Aterosclerótica/metabolismo , Receptores Purinérgicos P2Y12/genética , Actinas/análise , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/fisiologia , Clopidogrel/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dieta Hiperlipídica , Técnicas de Silenciamento de Genes , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/química , Músculo Liso Vascular/metabolismo , Placa Aterosclerótica/química , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Receptores Purinérgicos P2Y12/fisiologia , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(20): 10839-10847, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32358188

RESUMO

Cyclic nucleotide-gated (CNG) ion channels are essential components of mammalian visual and olfactory signal transduction. CNG channels open upon direct binding of cyclic nucleotides (cAMP and/or cGMP), but the allosteric mechanism by which this occurs is incompletely understood. Here, we employed double electron-electron resonance (DEER) spectroscopy to measure intersubunit distance distributions in SthK, a bacterial CNG channel from Spirochaeta thermophila Spin labels were introduced into the SthK C-linker, a domain that is essential for coupling cyclic nucleotide binding to channel opening. DEER revealed an agonist-dependent conformational change in which residues of the B'-helix displayed outward movement with respect to the symmetry axis of the channel in the presence of the full agonist cAMP, but not with the partial agonist cGMP. This conformational rearrangement was observed both in detergent-solubilized SthK and in channels reconstituted into lipid nanodiscs. In addition to outward movement of the B'-helix, DEER-constrained Rosetta structural models suggest that channel activation involves upward translation of the cytoplasmic domain and formation of state-dependent interactions between the C-linker and the transmembrane domain. Our results demonstrate a previously unrecognized structural transition in a CNG channel and suggest key interactions that may be responsible for allosteric gating in these channels.


Assuntos
Sítio Alostérico/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Spirochaeta/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Escherichia coli/metabolismo , Ativação do Canal Iônico/fisiologia , Modelos Moleculares , Nucleotídeos Cíclicos , Conformação Proteica
13.
Nat Commun ; 11(1): 2188, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366818

RESUMO

Olfactory receptor neurons (ORNs) use odour-induced intracellular cAMP surge to gate cyclic nucleotide-gated nonselective cation (CNG) channels in cilia. Prolonged exposure to cAMP causes calmodulin-dependent feedback-adaptation of CNG channels and attenuates neural responses. On the other hand, the odour-source searching behaviour requires ORNs to be sensitive to odours when approaching targets. How ORNs accommodate these conflicting aspects of cAMP responses remains unknown. Here, we discover that olfactory marker protein (OMP) is a major cAMP buffer that maintains the sensitivity of ORNs. Upon the application of sensory stimuli, OMP directly captured and swiftly reduced freely available cAMP, which transiently uncoupled downstream CNG channel activity and prevented persistent depolarization. Under repetitive stimulation, OMP-/- ORNs were immediately silenced after burst firing due to sustained depolarization and inactivated firing machinery. Consequently, OMP-/- mice showed serious impairment in odour-source searching tasks. Therefore, cAMP buffering by OMP maintains the resilient firing of ORNs.


Assuntos
AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteína de Marcador Olfatório/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Animais , Butorfanol/farmacologia , Cílios/metabolismo , Células HEK293 , Humanos , Masculino , Medetomidina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Midazolam/farmacologia , Odorantes , Proteína de Marcador Olfatório/genética , Mucosa Olfatória/citologia , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/fisiologia , Técnicas de Patch-Clamp
14.
Life Sci ; 254: 117780, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32407844

RESUMO

AIMS: In vivo studies suggest a positive influence of fresh frozen plasma (FFP) on endothelial properties and vascular barrier function, leading to improved outcomes in animal sepsis models as well as in major abdominal surgery. However, those effects are incompletely described. It was our aim to evaluate in vitro effects of FFP on endothelial key functions and to identify underlying mechanisms. MATERIALS AND METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were prestimulated with LPS, followed by incubation with FFP. Permeability for FITC-dextran was assessed, and intercellular gap formation was visualized. NF-κB nuclear translocation and expression of pro-inflammatory, pro-adhesion, and leakage-related genes were evaluated, and monocyte adhesion to ECs was assessed. Intracellular cAMP levels as well as phosphorylation of functional proteins were analyzed. In patients undergoing major abdominal surgery, Syndecan-1 serum levels were assessed prior to and following FFP transfusion. KEY FINDINGS: Post-incubation of HPMVECs with FFP increased intracellular cAMP levels that had been decreased by preceding LPS stimulation. On one hand, this reduced endotoxin-mediated upregulation of IL-8, ICAM-1, VCAM-1, VEGF, and ANG-2. Impaired phosphorylation of functional proteins was restored, and intercellular cohesion and barrier function were rescued. On the other hand, NF-κB nuclear translocation as well as monocyte adhesion was markedly increased by the combination of LPS and FFP. Syndecan-1 serum levels were lower in surgery patients that were transfused with FFP compared to those that were not. SIGNIFICANCE: Our data provide evidence for a differential modulation of crucial endothelial properties by FFP, potentially mediated by elevation of intracellular cAMP levels.


Assuntos
Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Plasma/fisiologia , Idoso , Adesão Celular/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Junções Comunicantes/fisiologia , Humanos , Lipopolissacarídeos , Pessoa de Meia-Idade , Monócitos/fisiologia , NF-kappa B/metabolismo , Fosforilação , Sindecana-1/sangue
15.
Adv Pharmacol ; 88: 173-191, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32416867

RESUMO

The glucagon-like peptide-1 receptor (GLP-1R) is a significant therapeutic target for small molecule drug discovery given the therapeutic impact of peptide agonists in the diabetes sphere. We review the discovery and subsequent characterization of the small molecule GLP-1R allosteric modulator 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP). BETP is a covalent modulator of the GLP-1R, and we discuss the pharmacological implications and possible structural basis of this novel mode of action. We highlight the insights into class B G-protein coupled receptor pharmacology and biology provided by studies conducted with BETP. These include the descriptions of exquisite allosteric modulator probe dependence and biased signaling in vitro and in vivo. We conclude with an analysis of the utility of BETP as a chemical probe for the GLP-1R.


Assuntos
Descoberta de Drogas , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Pirimidinas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Animais , AMP Cíclico/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Humanos , Pirimidinas/química , Bibliotecas de Moléculas Pequenas/farmacologia
16.
Vascul Pharmacol ; 131: 106690, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32407896

RESUMO

Cutaneous cold-induced vasoconstriction is a normal physiological reaction mediated by alpha 2C-adrenergic receptors (α2C-ARs) expressed in vascular smooth muscle cells (VSMCs). When this reaction is exaggerated, Raynaud's phenomenon (RP) ensues. RP is more prevalent in females compared to age-matched men. We previously established that 17-ß estradiol (estrogen) upregulates α2C-ARs in human VSMCs via a cAMP/Epac/Rap pathway. We also showed that cAMP acts through JNK to increase α2C-AR expression. However, whether estrogen employs JNK to regulate α2C-AR is not investigated. Knowing that the α2C-AR promoter harbors an activator protein-1 (AP-1) binding site that can be potentially activated by JNK, we hypothesized that estrogen regulates α2C-AR expression through an Epac/JNK/AP-1 pathway. Our results show that estrogen (10-10 M) activated JNK in human VSMCs extracted from cutaneous arterioles. Pretreatment with ESI09 (10 µM; an Epac inhibitor), abolished estrogen-induced JNK activation. In addition, pre-treatment with SP600125 (3 µM; a JNK specific inhibitor) abolished estrogen-induced expression of α2C-AR. Importantly, estrogen-induced activation of α2C-AR promoter was attenuated with SP600125. Moreover, transient transfection of VSMCs with an Epac dominant negative mutant (Epac-DN) abolished estrogen-induced activation of α2C-AR promoter. However, co-transfection of constitutively active JNK mutant overrode the inhibitory effect of Epac-DN on α2C-AR promoter. Moreover, estrogen caused a concentration-dependent increase in the activity of AP-1-driven reporter construct. Mutation of AP-1 site in the α2C-AR promoter abolished its activation by estrogen. This in vitro estrogen-increased α2C-AR expression was mirrored by an increase in the ex vivo functional responsiveness of arterioles. Indeed, estrogen potentiated α2C-AR-mediated cold-induced vasoconstriction, which was abolished by SP600125. Collectively, these results indicate that estrogen upregulates α2C-AR expression via an EPAC-mediated JNK/AP-1- dependent mechanism. These results provide an insight into the mechanism by which exaggerated cold-induced vasoconstriction occurs in estrogen-replete females and identify Epac and JNK as potential targets for the treatment of RP.


Assuntos
Temperatura Baixa , AMP Cíclico/metabolismo , Estradiol/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Cauda/irrigação sanguínea , Fator de Transcrição AP-1/metabolismo , Vasoconstrição/efeitos dos fármacos , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/enzimologia , Células Cultivadas , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Doença de Raynaud/tratamento farmacológico , Doença de Raynaud/enzimologia , Doença de Raynaud/fisiopatologia , Receptores Adrenérgicos alfa 2/genética , Transdução de Sinais , Fator de Transcrição AP-1/genética , Regulação para Cima
17.
Zhongguo Zhen Jiu ; 40(4): 397-404, 2020 Apr 12.
Artigo em Chinês | MEDLINE | ID: mdl-32275369

RESUMO

OBJECTIVE: To observe the direct intervention effects of electroacupuncture (EA) and non-steroid anti-inflammatory drugs (NSAIDs) on pain memory, and to explore their effects on cAMP/PKA/cAMP pathway in anterior cingulate gyrus (ACC). METHODS: Fifty clean healthy male SD rats were randomly divided into a control group, a model group, an indomethacin group, an EA group and a sham EA group, 10 rats in each group. Except the control group, the pain memory model was established in the remaining four groups by twice injection of carrageenan at foot; 0.1 mL of 2%λ-carrageenan was subcutaneously injected at the left foot of rats; 14 days later, when the pain threshold of rats of each group returned to the basic level, the second injection was performed with the same procedure. The rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36) for 30 min; the rats in the indomethacin group was treated with indomethacin intragastric administration with the dose of 3 mg/kg; the rats in the sham EA group was treated with EA without electricity at the point 0.3 mm forward "Zusanli" (ST 36) with the depth of 2 mm for 30 min; the rats in the control group was not given any invention. All the above interventions were performed 5 h, 1 d, 2 d and 3 d after the second injection of 2% λ-carrageenan. The left-side paw withdrawal thresholds (PWT) were observed before the first injection, 4 h, 3 d, 5 d after the first injection, before the second injection and 4 h, 1 d, 2 d, 3 d after the second injection. Three days after the second injection, the number of positive cells of cAMP, p-PKA, p-CREB and the number of positive cells of protein co-expression in the right ACC brain area were detected by immunofluorescence, and the relative protein expression of p-PKA and p-CREB were detected by Western blot. RESULTS: Compared with the control group, the PWTs in the model group decreased significantly 4 h, 3 d and 5 d after the first injection and 1 d, 2 d and 3 d after the second injection (P<0.05); compared with the control group, the positive expression of cAMP, p-PKA and p-CREB in the right ACC brain area in the model group increased significantly (P<0.05), and the number of positive cells of the co-expression of cAMP/p-PKA and p-PKA/p-CREB also increased significantly (P<0.05). Compared with the model group, indomethacin group and sham EA group, the PWTs in the EA group were increased significantly 1 d, 2 d and 3 d after the second injection (P<0.05); compared with the model group, indomethacin group and sham EA group, the positive expression of p-PKA and p-CREB in the right ACC brain area in the EA group decreased significantly (P<0.05), and the number of positive cells of co-expression of cAMP/p-PKA and p-PKA/p-CREB was decreased significantly (P<0.05). Compared with the model group and sham EA group, the positive expression of cAMP in the right ACC brain area was decreased in the EA group (P<0.05). CONCLUSION: EA have a direct intervention effect on pain memory, which have significant advantage over NSAIDs in the treatment of chronic pain. The advantage effect of EA on pain memory may be related to the inhibition of cAMP/PKA/CREB pathway in ACC area.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Eletroacupuntura , Giro do Cíngulo/metabolismo , Limiar da Dor , Transdução de Sinais , Animais , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
18.
J Med Chem ; 63(10): 5159-5184, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32340447

RESUMO

Exchange proteins directly activated by cAMP (EPAC) play a central role in various biological functions, and activation of the EPAC1 protein has shown potential benefits for the treatment of various human diseases. Herein, we report the synthesis and biochemical evaluation of a series of noncyclic nucleotide EPAC1 activators. Several potent EPAC1 binders were identified including 25g, 25q, 25n, 25u, 25e, and 25f, which promote EPAC1 guanine nucleotide exchange factor activity in vitro. These agonists can also activate EPAC1 protein in cells, where they exhibit excellent selectivity toward EPAC over protein kinase A and G protein-coupled receptors. Moreover, 25e, 25f, 25n, and 25u exhibited improved selectivity toward activation of EPAC1 over EPAC2 in cells. Of these, 25u was found to robustly inhibit IL-6-activated signal transducer and activator of transcription 3 (STAT3) and subsequent induction of the pro-inflammatory vascular cell adhesion molecule 1 (VCAM1) cell-adhesion protein. These novel EPAC1 activators may therefore act as useful pharmacological tools for elucidation of EPAC function and promising drug leads for the treatment of relevant human diseases.


Assuntos
AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , AMP Cíclico/agonistas , Avaliação Pré-Clínica de Medicamentos/métodos , Fatores de Troca do Nucleotídeo Guanina/agonistas , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Nucleotídeos/síntese química , Nucleotídeos/química , Nucleotídeos/farmacologia , Ligação Proteica/fisiologia
19.
PLoS Biol ; 18(3): e3000614, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32126082

RESUMO

The reproductive hormones that trigger oocyte meiotic maturation and release from the ovary vary greatly between animal species. Identification of receptors for these maturation-inducing hormones (MIHs) and understanding how they initiate the largely conserved maturation process remain important challenges. In hydrozoan cnidarians including the jellyfish Clytia hemisphaerica, MIH comprises neuropeptides released from somatic cells of the gonad. We identified the receptor (MIHR) for these MIH neuropeptides in Clytia using cell culture-based "deorphanization" of candidate oocyte-expressed G protein-coupled receptors (GPCRs). MIHR mutant jellyfish generated using CRISPR-Cas9 editing had severe defects in gamete development or in spawning both in males and females. Female gonads, or oocytes isolated from MIHR mutants, failed to respond to synthetic MIH. Treatment with the cAMP analogue Br-cAMP to mimic cAMP rise at maturation onset rescued meiotic maturation and spawning. Injection of inhibitory antibodies to the alpha subunit of the Gs heterodimeric protein (GαS) into wild-type oocytes phenocopied the MIHR mutants. These results provide the molecular links between MIH stimulation and meiotic maturation initiation in hydrozoan oocytes. Molecular phylogeny grouped Clytia MIHR with a subset of bilaterian neuropeptide receptors, including neuropeptide Y, gonadotropin inhibitory hormone (GnIH), pyroglutamylated RFamide, and luqin, all upstream regulators of sexual reproduction. This identification and functional characterization of a cnidarian peptide GPCR advances our understanding of oocyte maturation initiation and sheds light on the evolution of neuropeptide-hormone systems.


Assuntos
Hidrozoários/fisiologia , Neuropeptídeos/metabolismo , Oócitos/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , AMP Cíclico/metabolismo , Feminino , Expressão Gênica , Hidrozoários/genética , Masculino , Mutação , Filogenia , Receptores Acoplados a Proteínas-G/genética , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo
20.
Nat Commun ; 11(1): 1145, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123179

RESUMO

Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.


Assuntos
Receptores Opioides kappa/química , Receptores Opioides kappa/metabolismo , Anticorpos de Domínio Único/química , Sítio Alostérico , Sítios de Ligação , Técnicas Biossensoriais , Cristalografia por Raios X , AMP Cíclico/metabolismo , Dinorfinas/química , Dinorfinas/farmacologia , Células HEK293 , Humanos , Medições Luminescentes/métodos , Piperazinas/química , Piperazinas/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Conformação Proteica , Pirrolidinas/química , Pirrolidinas/farmacologia , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/metabolismo , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/farmacologia
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