Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.359
Filtrar
1.
J Biol Chem ; 294(31): 11677-11684, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31235524

RESUMO

The network of Wingless/Int-1 (WNT)-induced signaling pathways includes ß-catenin-dependent and -independent pathways. ß-Catenin regulates T cell factor/lymphoid enhancer-binding factor (TCF/LEF)-mediated gene transcription, and in response to WNTs, ß-catenin signaling is initiated through engagement of a Frizzled (FZD)/LDL receptor-related protein 5/6 (LRP5/6) receptor complex. FZDs are G protein-coupled receptors, but the question of whether heterotrimeric G proteins are involved in WNT/ß-catenin signaling remains unanswered. Here, we investigate whether acute activation of WNT/ß-catenin signaling by purified WNT-3A requires functional signaling through heterotrimeric G proteins. Using genome editing, we ablated expression of Gs/Golf/Gq/G11/G12/G13/Gz in HEK293 (ΔG7) cells, leaving the expression of pertussis toxin (PTX)-sensitive Gi/o proteins unchanged, to assess whether WNT-3A activates WNT/ß-catenin signaling in WT and ΔG7 cells devoid of functional G protein signaling. We monitored WNT-3A-induced activation by detection of phosphorylation of LDL receptor-related protein 6 (LRP6), electrophoretic mobility shift of the phosphoprotein Dishevelled (DVL), ß-catenin stabilization and dephosphorylation, and TCF-dependent transcription. We found that purified, recombinant WNT-3A efficiently induces WNT/ß-catenin signaling in ΔG7 cells in both the absence and presence of Gi/o-blocking PTX. Furthermore, cells completely devoid of G protein expression, so called Gα-depleted HEK293 cells, maintain responsiveness to WNT-3A with regard to the hallmarks of WNT/ß-catenin signaling. These findings corroborate the concept that heterotrimeric G proteins are not required for this FZD- and DVL-mediated signaling branch. Our observations agree with previous results arguing for FZD conformation-dependent functional selectivity between DVL and heterotrimeric G proteins. In conclusion, WNT/ß-catenin signaling through FZDs does not require the involvement of heterotrimeric G proteins.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/farmacologia , Proteínas Desgrenhadas , Edição de Genes , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Toxina Pertussis/farmacologia , Fosforilação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Fatores de Transcrição TCF/metabolismo , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo
2.
PLoS One ; 14(6): e0218110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31173603

RESUMO

PURPOSE: We previously reported that inhibitory G protein (Gi) exerts intrinsic receptor-independent inhibitory activity upon adenylyl cyclase (AC) that regulates contractile force in rat ventricle. The two major subtypes of AC in the heart are AC5 and AC6. The aim of this study was to determine if this intrinsic Gi inhibition regulating contractile force is AC subtype selective. METHODS: Wild-type (WT), AC5 knockout (AC5KO) and AC6 knockout (AC6KO) mice were injected with pertussis toxin (PTX) to inactivate Gi or saline (control).Three days after injection, we evaluated the effect of simultaneous inhibition of phosphodiesterases (PDE) 3 and 4 with cilostamide and rolipram respectively upon in vivo and ex vivo left ventricular (LV) contractile function. Also, changes in the level of cAMP were measured in left ventricular homogenates and at the membrane surface in cardiomyocytes obtained from the same mouse strains expressing the cAMP sensor pmEPAC1 using fluorescence resonance energy transfer (FRET). RESULTS: Simultaneous PDE3 and PDE4 inhibition increased in vivo and ex vivo rate of LV contractility only in PTX-treated WT and AC5KO mice but not in saline-treated controls. Likewise, Simultaneous PDE3 and PDE4 inhibition elevated total cAMP levels in PTX-treated WT and AC5KO mice compared to saline-treated controls. In contrast, simultaneous PDE3 and PDE4 inhibition did not increase in vivo or ex vivo rate of LV contractility or cAMP levels in PTX-treated AC6KO mice compared to saline-treated controls. Using FRET analysis, an increase of cAMP level was detected at the membrane of cardiomyocytes after simultaneous PDE3 and PDE4 inhibition in WT and AC5KO but not AC6KO. These FRET data are consistent with the functional data indicating that AC6 activity and PTX inhibition of Gi is necessary for simultaneous inhibition of PDE3 and PDE4 to elicit an increase in contractility. CONCLUSIONS: Together, these data suggest that AC6 is tightly regulated by intrinsic receptor-independent Gi activity, thus providing a mechanism for maintaining low basal cAMP levels in the functional compartment that regulates contractility.


Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Contração Miocárdica , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Feminino , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Toxina Pertussis/farmacologia
3.
Cell Physiol Biochem ; 52(3): 486-502, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30873823

RESUMO

BACKGROUND/AIMS: Cross-talk between different pancreatic islet cell types regulates islet function and somatostatin (SST) released from pancreatic delta cells inhibits insulin secretion from pancreatic beta cells. In other tissues SST exhibits both protective and pro-apoptotic properties in a tissue-specific manner, but little is known about the impact of the peptide on beta cell survival. Here we investigate the specific role of SST in the regulation of beta cell survival in response to physiologically relevant inducers of cellular stress including palmitate, cytokines and glucose. METHODS: Pancreatic MIN6 beta cells and primary mouse islet cells were pre-treated with SST with or without the Gi/o signalling inhibitor, pertussis toxin, and exposed to different cellular stress factors. Apoptosis and proliferation were assessed by measurement of caspase 3/7 activity, TUNEL and BrdU incorporation, respectively, and expression of target genes was measured by qPCR. RESULTS: SST partly alleviated upregulation of cellular stress markers (Hspa1a and Ddit3) and beta cell apoptosis in response to factors such as lipotoxicity (palmitate), pro-inflammatory cytokines (IL1ß and TNFα) and low glucose levels. This effect was mediated via a Gi/o protein-dependent pathway, but did not modify transcriptional upregulation of the specific NFκB-dependent genes, Nos2 and Ccl2, nor was it associated with transcriptional changes in SST receptor expression. CONCLUSION: Our results suggest an underlying protective effect of SST which modulates the beta cell response to ER stress and apoptosis induced by a range of cellular stressors associated with type 2 diabetes.


Assuntos
Proliferação de Células/efeitos dos fármacos , Glucose/antagonistas & inibidores , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Toxina Pertussis/antagonistas & inibidores , Somatostatina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Regulação da Expressão Gênica , Glucose/farmacologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/farmacologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ácido Palmítico/antagonistas & inibidores , Ácido Palmítico/farmacologia , Toxina Pertussis/farmacologia , Técnicas de Cultura de Tecidos , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia
4.
Auris Nasus Larynx ; 46(5): 790-796, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30739815

RESUMO

Objective The endocytosis of cationized feritin (CF) via a clathrin-mediated pathway is regulated by a signaling network. Marginal cells showed the active endocytosis of CF via a clathrin-mediated pathway. The internalization of receptors through this clathrin-mediated pathway is an important regulatory event in signal transduction. Numerous kinases are involved in endocytosis, and each endocytic route is subjected to high-order regulation by cellular signaling mechanisms. In this study, we investigated whether ROCK and MLCK signaling cascades and G-proteins regulate the endocytosis of CF in marginal cells of the stria vascularis. Methods CF was infused into the cochlear duct with pertussis toxin (PTX),Clostridium botulinum C3 toxin (BTX), guanosine(g-thio)-triphosphate (GTP-γS), ML-7, Y-27632. Endocytic activity was measured at 30 min after the start of infusion under an electron microscope. Results In marginal cells, CF was internalized via a clathrin-mediated pathway that depends on F-actin and microtubules (MT). Its processes were controlled by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK), but not affected by G-protein-coupled receptor (GPCR) or the RhoA signaling cascade. Conclusion Our previous study showed that the main endocytotic pathway of microperoxidase (MPO) did not depend on the Rho/ROCK molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade. The present study results indicate that these signaling cascades regulating CF internalization completely differ from the cascades for MPO internalization.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Ferritinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Estria Vascular/metabolismo , Quinases Associadas a rho/metabolismo , ADP Ribose Transferases/farmacologia , Amidas/farmacologia , Animais , Azepinas/farmacologia , Toxinas Botulínicas/farmacologia , Ducto Coclear , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Cobaias , Microscopia Eletrônica , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Fosfatase de Miosina-de-Cadeia-Leve/antagonistas & inibidores , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Naftalenos/farmacologia , Toxina Pertussis/farmacologia , Piridinas/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Estria Vascular/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismo
5.
Steroids ; 142: 6-13, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-28962850

RESUMO

The role played by progestogens in modulating Schwann cell pathophysiology is well established. Progestogens exert their effects in these cells through both classical genomic and non-genomic mechanisms, the latter mediated by the GABA-A receptor. However, there is evidence that other receptors may be involved. Membrane progesterone receptors (mPRs) are novel 7-transmembrane receptors coupled to G proteins that have been characterized in different tissues and cells, including the central nervous system (CNS). The mPRs were shown to mediate some of progestogens' neuroprotective effects in the CNS, and to be upregulated in glial cells after traumatic brain injury. Based on this evidence, this paper investigated the possible involvement of mPRs in mediating progestogen actions in S42 Schwann cells. All five mPR isoforms and progesterone receptor membrane component 1 (PGRMC1) were detected in Schwann cells, and were present on the cell membrane. Progesterone and the mPR-specific agonist, Org-OD-02-0 (02) bound to these membranes, indicating the presence of functional mPRs. The mPR agonist 02 rapidly increased cell migration in an in vitro assay, suggesting a putative role of mPRs in the nerve regeneration process. Treatment with pertussis toxin and 8-Br-cAMP blocked 02-induced cell migration, suggesting this progestogen action is mediated by activation of an inhibitory G protein, leading to a decrease in intracellular cAMP levels. In contrast, long-term mPR activation led to increased expression levels of myelin associated glycoprotein (MAG). Taken together, these findings show that mPRs are present and active in Schwann cells and have a role in modulating their physiological processes.


Assuntos
Membrana Celular/metabolismo , Movimento Celular , Glicoproteína Associada a Mielina/biossíntese , Neuroglia/citologia , Neuroglia/metabolismo , Receptores de Progesterona/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Neuroglia/efeitos dos fármacos , Toxina Pertussis/farmacologia , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Progesterona/agonistas , Receptores de Progesterona/análise , Tionucleotídeos/farmacologia , Células Tumorais Cultivadas
6.
Pharmacol Res Perspect ; 6(6): e00445, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30534379

RESUMO

Neuropeptide S (NPS) is the endogenous ligand of the neuropeptide S receptor (NPSR). NPS modulates several biological functions including anxiety, wakefulness, pain, and drug abuse. The aim of this study was the investigation of the pharmacological profile of NPSR using the dynamic mass redistribution (DMR) assay. DMR is a label-free assay that offers a holistic view of cellular responses after receptor activation. HEK293 cells stably transfected with the murine NPSR (HEK293mNPSR) have been used. To investigate the nature of the NPS-evoked DMR signaling, FR900359 (Gq inhibitor), pertussis toxin (Gi inhibitor), and rolipram (phosphodiesterase inhibitor) were used. To determine the pharmacology of NPSR, several selective ligands (agonists, partial agonists, antagonists) have been tested. NPS, through selective NPSR activation, evoked a robust DMR signal with potency in the nanomolar range. This signal was predominantly, but not completely, blocked by FR900359, suggesting the involvement of the Gq-dependent signaling cascade. NPSR ligands (agonists and antagonists) displayed potency values in DMR experiments similar, but not identical, to those reported in the literature. Furthermore, partial agonists produced a higher efficacy in DMR than in calcium experiments. DMR can be successfully used to study the pharmacology and signaling properties of novel NPSR ligands. This innovative approach will likely increase the translational value of in vitro pharmacological studies.


Assuntos
Bioensaio/métodos , Técnicas Biossensoriais/métodos , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Cálcio/metabolismo , Depsipeptídeos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Ligantes , Toxina Pertussis/farmacologia , Receptores de Neuropeptídeos/metabolismo , Rolipram/farmacologia
7.
Int Immunopharmacol ; 64: 298-307, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30243065

RESUMO

Activation of high affinity receptor for IgE (FcεRI) by IgE/antigen complexes in mast cells (MCs) leads to the release of preformed pro-inflammatory mediators stored in granules by a Ca2+-dependent process known as anaphylactic degranulation. Degranulation inhibition has been proposed as a strategy to control allergies and chronic inflammation conditions. Cannabinoids are important inhibitors of inflammatory reactions but their effects on IgE/Ag-mediated MCs responses are not well described. In this study, we analyzed the effect of the endocannabinoid anandamide (AEA), the selective CB2 receptor agonist HU308, and the GPR55 receptor agonist lysophosphatidylinositol (LPI) on FcεRI-induced activation in murine bone marrow-derived mast cells (BMMCs). Our results show that AEA, HU380 and LPI inhibited FcεRI-induced degranulation in a concentration-dependent manner. This effect was mediated by CB2 and GPR55 receptor activation through a mechanism insensitive to pertussis toxin. Degranulation inhibition was prevented by CB2 and GPR55 antagonism, but not by CB1 receptor blockage. AEA also inhibited calcium-dependent cytokine mRNA synthesis induced by FcεRI crosslinking, without affecting early phosphorylation events. In addition, AEA, HU308 and LPI inhibited intracellular Ca2+ rise in response to IgE/Ag. CB2 and GPR55 receptor antagonism could not prevent the inhibition produced by AEA and HU308, but partially blocked the one caused by LPI. These results indicate that AEA inhibits IgE/Ag-induced degranulation through a mechanism that includes the participation of CB2 and GPR55 receptors acting in close crosstalk, and show that CB2-GPR55 heteromers are important negative regulators of FcεRI-induced responses in MCs.


Assuntos
Ácidos Araquidônicos/farmacologia , Degranulação Celular/efeitos dos fármacos , Citocinas/biossíntese , Endocanabinoides/farmacologia , Mastócitos/efeitos dos fármacos , Alcamidas Poli-Insaturadas/farmacologia , Receptor CB2 de Canabinoide/fisiologia , Receptores de Canabinoides/fisiologia , Receptores de IgE/antagonistas & inibidores , Animais , Camundongos , Camundongos Endogâmicos C57BL , Toxina Pertussis/farmacologia , Receptor CB2 de Canabinoide/química , Receptores de Canabinoides/química , Receptores de IgE/fisiologia
8.
Sci Signal ; 11(548)2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30228224

RESUMO

G protein-coupled receptors (GPCRs) are major drug targets. Developing a method to measure the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface; thus, changes in the concentrations of downstream signaling molecules, which depend on the signaling pathway selectivity of the receptor, are often used as an index of receptor activity. We show that single-molecule imaging analysis provides an alternative method for assessing the effects of ligands on GPCRs. Using total internal reflection fluorescence microscopy (TIRFM), we monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions. Our single-molecule tracking analysis demonstrated that increases and decreases in the average diffusion coefficient of mGluR3 quantitatively reflected the ligand-dependent inactivation and activation of receptors, respectively. Through experiments with inhibitors and dual-color single-molecule imaging analysis, we found that the diffusion of receptor molecules was altered by common physiological events associated with GPCRs, including G protein binding, and receptor accumulation in clathrin-coated pits. We also confirmed that agonist also decreased the average diffusion coefficient for class A and B GPCRs, demonstrating that this parameter is a good index for estimating ligand effects on many GPCRs regardless of their phylogenetic groups, the chemical properties of the ligands, or G protein-coupling selectivity.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Microscopia de Fluorescência/métodos , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Aminoácidos/metabolismo , Células HEK293 , Humanos , Ligantes , Toxina Pertussis/metabolismo , Toxina Pertussis/farmacologia , Ligação Proteica/efeitos dos fármacos , Ensaio Radioligante/métodos , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/genética , Receptores de Glutamato Metabotrópico/análise , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Xantenos/metabolismo
9.
Cell Mol Neurobiol ; 38(8): 1479-1489, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30218403

RESUMO

Activation of inflammasome leads to the formation of an inflammatory microenvironment which plays an important role in the process of cancer development. Beta-hydroxybutyrate (BHB) is a ketone body that has recently been reported to exert anti-inflammatory effects via inhibition of NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome. Here, we investigated the potential influence of BHB on the in vitro migration of C6 glioma cells and the activation of NLRP3 inflammasome. Our results indicated that administration of BHB suppressed C6 cells migration and NLRP3 inflammasome activation, reducing the levels of activated cysteinyl aspartate-specific proteinase 1 (caspase-1) and mature Interleukin 1ß (IL-1ß). Fully activation of NLRP3 inflammasome was induced by lipopolysaccharide (LPS) prime plus adenosine triphosphate (ATP) stimulation in C6 cells, which promoted in vitro migration of C6 cell. BHB also counteracted the LPS/ATP-promoted cell migration by suppressing the activation of caspase-1 and the maturation of IL-1ß. The enhancement of phospho-signal transducer and activator of transcription 3 (p-STAT3), degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) as well as the overexpression of fibroblast growth factor 2 (FGF2) resulting from LPS/ATP treatment, and subsequent IL-1ß maturation could also be compensated by BHB. Our results suggested that BHB inhibits the activation of NLRP3 inflammasome in C6 glioma cells and consequently suppressed the C6 cell migration. These findings also implicated that by inhibiting NLRP3 inflammasome, BHB reduced the inflammatory microenvironment which provided ancillary therapeutic benefits for the intervention of glioma.


Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Movimento Celular/efeitos dos fármacos , Glioma/metabolismo , Glioma/patologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Toxina Pertussis/farmacologia , Ratos
10.
J Leukoc Biol ; 104(6): 1117-1132, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30134499

RESUMO

Acetate, an agonist for the free fatty acid receptor 2 (FFA2R/GPR43), triggers an increase in the cytosolic concentration of free Ca2+ in neutrophils without any assembly of the superoxide generating NADPH-oxidase. We show that the phenylacetamide compound 58 (Cmp 58; (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide), lacking a direct activating effect on neutrophils, acts as a positive FFA2R modulator that turns acetate into a potent activating agonist that triggers an assembly of the NADPH-oxidase. The NADPH-oxidase activity could be further increased in neutrophils treated with the pro-inflammatory cytokine TNF-α. Many neutrophil chemoattractant receptors are stored in secretory organelles but no FFA2R mobilization was induced in neutrophils treated with TNF-α. The receptor selectivity was demonstrated through the inhibition of the neutrophil response induced by the combined action of acetate and Cmp 58 by the FFA2R antagonist CATPB. Receptor modulators that positively co-operate with natural FFA2R agonists and prime neutrophils in their response to such agonists, may serve as good tools for further unraveling the physiological functions of FFA2R and its involvement in various diseases. In this study, we show that neutrophils primed with a presumed allosteric FFA2R modulator produce increased amounts of reactive oxygen species when activated by receptor specific agonists.


Assuntos
Acetanilidas/farmacologia , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Receptores de Superfície Celular/agonistas , Superóxidos/metabolismo , Tiazóis/farmacologia , Acetatos/farmacologia , Regulação Alostérica , Antígeno CD11b/biossíntese , Antígeno CD11b/genética , Sinalização do Cálcio , Células Cultivadas , Ciclopropanos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , Neutrófilos/enzimologia , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Toxina Pertussis/farmacologia , Receptores Acoplados a Proteínas-G/fisiologia , Fator de Necrose Tumoral alfa/farmacologia
11.
Sci Rep ; 8(1): 9466, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29930254

RESUMO

Activated platelets release micromolar concentrations of the chemokine CXCL4/Platelet Factor-4. Deposition of CXCL4 onto the vascular endothelium is involved in atherosclerosis, facilitating monocyte arrest and recruitment by an as yet, unidentified receptor. Here, we demonstrate that CXCL4 drives chemotaxis of the monocytic cell line THP-1. Migration and intracellular calcium responses induced by CXCL4 were pertussis toxin-sensitive, implicating a GPCR in signal transduction. Cell treatment with chondroitinase ABC ablated migration, suggesting that cis presentation of CXCL4 by cell surface glycosaminoglycans to a GPCR is required. Although CXCR3 has been previously described as a CXCL4 receptor, THP-1 cells were unresponsive to CXCR3 ligands and CXCL4-induced migration was insensitive to a CXCR3 antagonist, suggesting that an alternative receptor is involved. Interrogating CC-class chemokine receptor transfectants, we unexpectedly found that CXCL4 could induce the migration of CCR1-expressing cells and also induce CCR1 endocytosis. Extending our findings to primary human monocytes, we observed that CXCL4 induced CCR1 endocytosis and could induce monocyte chemotaxis in a CCR1 antagonist-sensitive manner. Collectively, our data identify CCR1 as a previously elusive monocyte CXCL4 receptor and suggest that CCR1 may play a role in inflammation where the release of CXCL4 is implicated.


Assuntos
Quimiotaxia , Monócitos/metabolismo , Fator Plaquetário 4/metabolismo , Receptores CCR1/metabolismo , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Condroitina ABC Liase/farmacologia , Endocitose , Humanos , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Toxina Pertussis/farmacologia , Fator Plaquetário 4/genética , Ligação Proteica
12.
Biochem Biophys Res Commun ; 503(1): 79-85, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-29852172

RESUMO

The noradrenergic neurons of the locus coeruleus (LC) are associated with various brain functions and psychiatric disorders, such as addiction and depression. It has been shown that neuropeptide galanin (GAL) inhibits neuronal excitability in LC, but the mechanisms remain unclear. In the present study, we investigated the ionic and signal transduction mechanisms underlying inhibitory effect of GAL on LC neurons using whole-cell patch clamp recording in rat brain slices. Bath application of GAL decreased the spontaneous firings and induced a dose-dependent hyperpolarization of LC neurons and this effect was attenuated by knockdown of Galr1, but not Galr2, confirming that mainly GALR1 mediates the inhibition effect of GAL. The inhibitory effect of GAL was also blocked by treatments of pertussis toxin (PTX), GTP-γ-s or GDP-ß-s, respectively, indicating that the functions of PTX sensitive Gi/o protein are required for GAL-induced hyperpolarization. Moreover, the blockers of GIRK (tertiapin-Q or SCH2 3390 hydrochloride) attenuated the GAL response while blocker of BK/SK/KATP channels or TASK-1/3 channels did not affect it significantly, suggesting that GIRK channels play an important role in GAL-induced hyperpolarization in LC neurons. Taken together, the inhibitory effect of GAL on LC neurons is mediated by GALR1 via PTX-sensitive Gi/o proteins, which activate GIRK channels.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Locus Cerúleo/metabolismo , Receptor Tipo 1 de Galanina/metabolismo , Neurônios Adrenérgicos/efeitos dos fármacos , Neurônios Adrenérgicos/metabolismo , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Galanina/metabolismo , Técnicas de Silenciamento de Genes , Locus Cerúleo/citologia , Locus Cerúleo/efeitos dos fármacos , Masculino , Técnicas de Patch-Clamp , Toxina Pertussis/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Precursores de Proteínas/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Galanina/antagonistas & inibidores , Receptor Tipo 1 de Galanina/genética , Receptor Tipo 2 de Galanina/antagonistas & inibidores , Receptor Tipo 2 de Galanina/genética , Receptor Tipo 2 de Galanina/metabolismo , Transdução de Sinais
13.
J Neurochem ; 146(5): 526-539, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29772059

RESUMO

The chemokine CCL5 prevents neuronal cell death mediated both by amyloid ß, as well as the human immunodeficiency virus viral proteins gp120 and Tat. Because CCL5 binds to CCR5, CCR3 and/or CCR1 receptors, it remains unclear which of these receptors plays a role in neuroprotection. Indeed, CCL5 also has neuroprotective activity in cells lacking these receptors. CCL5 may bind to a G-protein-coupled receptor 75 (GPR75), which encodes for a 540 amino-acid orphan receptor of the Gqα family. In this study, we have used SH-SY5Y human neuroblastoma cells to characterize whether CCL5 could activate a Gq signaling through GPR75. Both qPCR and flow cytometry show that these cells express GPR75 but do not express CCR5, CCR3 or CCR1 receptors. SY-SY5Y cells were then used to examine CCL5-mediated signaling. We report that CCL5 promotes a time- and concentration-dependent phosphorylation of protein kinase B (AKT), glycogen synthase kinase 3ß, and extracellular signal-regulated kinase (ERK) 1/2. Specific antagonists of CCR5, CCR3, and CCR1 did not prevent CCL5 from increasing phosphorylated AKT or ERK. Moreover, CCL5 promotes a time-dependent internalization of GPR75. Lastly, knocking down GPR75 expression by a CRISPR-Cas9 approach inhibited the ability of CCL5 to activate pERK in SH-SY5Y cells. Therefore, we propose that GPR75 is a novel receptor for CCL5 that could explain some of the pharmacological action of this chemokine. These findings may help in the development of small molecule GPR75 agonists that mimic CCL5. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.


Assuntos
Quimiocina CCL5/metabolismo , Regulação da Expressão Gênica/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/fisiologia , Animais , Antineoplásicos/farmacologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Quimiocina CCL5/genética , Quimiocina CCL5/farmacologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Humanos , Mutagênese/genética , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Toxina Pertussis/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Ratos , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/efeitos dos fármacos , Linfócitos T , Tretinoína/farmacologia
14.
Front Immunol ; 9: 687, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29696016

RESUMO

The follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen depends upon BCR, BAFF, and Notch2 signaling. Whether or how Gi signaling affects this fate decision is unknown. Here, we show that direct contact with Notch ligand expressing stromal cells (OP9-Delta-like 1) cannot promote normal MZ B cell development when progenitor B cells lack Gαi proteins, or if Gi signaling is disabled. Consistent with faulty ADAM10-dependent Notch2 processing, Gαi-deficient transitional B cells had low ADAM10 membrane expression levels and reduced Notch2 target gene expression. Immunoblotting Gαi-deficient B cell lysates revealed a reduction in mature, processed ADAM10. Suggesting that Gαi signaling promotes ADAM10 membrane expression, stimulating normal transitional B cells with CXCL12 raised it, while inhibiting Gαi nucleotide exchange blocked its upregulation. Surprisingly, inhibiting Gαi nucleotide exchange in transitional B cells also impaired the upregulation of ADAM10 that occurs following antigen receptor crosslinking. These results indicate that Gαi signaling supports ADAM10 maturation and activity in transitional B cells, and ultimately Notch2 signaling to promote MZ B cell development.


Assuntos
Proteína ADAM10/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Linfócitos B/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Proteínas de Membrana/fisiologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Toxina Pertussis/farmacologia , Receptor Notch2/fisiologia , Transdução de Sinais , Baço/citologia
15.
Cell Calcium ; 71: 53-64, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29604964

RESUMO

Intracellular Ca2+ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca2+ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca2+ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [3H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca2+ and cAMP signaling pathways. Histamine stimulated Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptors in hBASMCs. ß2-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca2+ signals. Responses to other Ca2+-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E2 (PGE2), through EP2 and EP4 receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca2+ signals. There was no consistent relationship between the inhibition of Ca2+ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that ß-adrenoceptors, EP2 and EP4 receptors, through cAMP and PKA, selectively inhibit Ca2+ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H1 receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.


Assuntos
Brônquios/citologia , Sinalização do Cálcio/efeitos dos fármacos , Compartimento Celular , AMP Cíclico/metabolismo , Histamina/farmacologia , Miócitos de Músculo Liso/metabolismo , Adulto , Criança , Pré-Escolar , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Isoproterenol/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Toxina Pertussis/farmacologia , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP4 , Fosfolipases Tipo C/metabolismo
16.
Epilepsia ; 59(2): 449-459, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29283181

RESUMO

OBJECTIVE: γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in adult central nervous system, and profound alterations of GABA receptor functions are linked to temporal lobe epilepsy (TLE). Here we describe the functional relationships between GABA receptors type B (GABAB R) and type A (GABAA R) in human temporal cortex and how TLE affects this aspect of GABAergic signaling. METHODS: Miniature inhibitory postsynaptic currents (mIPSCs) were recorded by patch-clamp techniques from human L5 pyramidal neurons in slices from temporal cortex tissue obtained from surgery. RESULTS: We describe a constitutive functional crosstalk between GABAB Rs and GABAA Rs in human temporal layer 5 pyramidal neurons, which is lost in epileptic tissues. The activation of GABAB Rs by baclofen, in addition to the expected reduction of mIPSC frequency, produced, in cortex of nonepileptic patients, the prolongation of mIPSC rise and decay times, thus increasing the inhibitory net charge associated with a single synaptic event. Block of K+ channels did not prevent the increase of decay time and charge. Protein kinase A (PKA) blocker KT5720 and pertussis toxin inhibited the action of baclofen, whereas 8Br-cAMP mimicked the GABAB R action. The same GABAB R-mediated modulation of GABAA Rs was observed in pyramidal neurons of rat temporal cortex, with both PKA and PKC involved in the process. In cortices from TLE patients and epileptic rats, baclofen lost its ability to modulate mIPSCs. SIGNIFICANCE: Our results highlight the association of TLE with functional changes of GABAergic signaling that may be related to seizure propagation, and suggest that the selective activation of a definite subset of nonpresynaptic GABAB Rs may be therapeutically useful in TLE.


Assuntos
Epilepsia do Lobo Temporal/metabolismo , Neocórtex/metabolismo , Células Piramidais/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Lobo Temporal/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adolescente , Adulto , Animais , Baclofeno/farmacologia , Carbazóis/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Modelos Animais de Doenças , Epilepsia Resistente a Medicamentos/metabolismo , Epilepsia Resistente a Medicamentos/fisiopatologia , Epilepsia Resistente a Medicamentos/cirurgia , Inibidores Enzimáticos/farmacologia , Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/cirurgia , Feminino , Agonistas dos Receptores de GABA-B/farmacologia , Humanos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Pessoa de Meia-Idade , Agonistas Muscarínicos/toxicidade , Neocórtex/efeitos dos fármacos , Neocórtex/fisiopatologia , Técnicas de Patch-Clamp , Toxina Pertussis/farmacologia , Pilocarpina/toxicidade , Proteína Quinase C/metabolismo , Células Piramidais/efeitos dos fármacos , Pirróis/farmacologia , Ratos , Lobo Temporal/efeitos dos fármacos , Lobo Temporal/fisiopatologia
18.
J Physiol ; 596(5): 921-940, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29280494

RESUMO

KEY POINTS: Neurotransmitter release is inhibited by metabotropic glutamate type 7 (mGlu7 ) receptors that reduce Ca2+ influx, yet synapses lacking this receptor also produce weaker release, suggesting that mGlu7 receptors may also prime synaptic vesicles for release. Prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs through a presynaptic effect. The inhibitory response is blocked by pertussis toxin, while the potentiating response is prevented by a phospholipase C inhibitor (U73122) and an inhibitor of diacylglycerol (DAG) binding (calphostin C), suggesting that this receptor also couples to pathways that generate DAG. Release potentiation is associated with an increase in the number of synaptic vesicles close to the plasma membrane, which was dependent on the Munc13-2 and RIM1α proteins. The Glu7 receptors activated by the glutamate released following high frequency stimulation provoke a bidirectional modulation of synaptic transmission. ABSTRACT: Neurotransmitter release is driven by Ca2+ influx at synaptic boutons that acts on synaptic vesicles ready to undergo exocytosis. Neurotransmitter release is inhibited when metabotropic glutamate type 7 (mGlu7 ) receptors provoke a reduction in Ca2+ influx, although the reduced release from synapses lacking this receptor suggests that they may also prime synaptic vesicles for release. These mGlu7 receptors activate phospholipase C (PLC) and generate inositol trisphosphate, which in turn releases Ca2+ from intracellular stores and produces diacylglycerol (DAG), an activator of proteins containing DAG-binding domains such as Munc13 and protein kinase C (PKC). However, the full effects of mGlu7 receptor signalling on synaptic transmission are unclear. We found that prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs, a presynaptic effect that changes the frequency but not the amplitude of the mEPSCs and the paired pulse ratio. Pertussis toxin blocks the inhibitory response, while the PLC inhibitor U73122, and the inhibitor of DAG binding calphostin C, prevent receptor mediated potentiation. Moreover, this DAG-dependent potentiation of the release machinery brings more synaptic vesicles closer to the active zone plasma membrane in a Munc13-2- and RIM1α-dependent manner. Electrically evoked release of glutamate that activates mGlu7 receptors also bidirectionally modulates synaptic transmission. In these conditions, potentiation now occurs rapidly and it overcomes any inhibition, such that potentiation prevails unless it is suppressed with the PLC inhibitor U73122.


Assuntos
Região CA1 Hipocampal/fisiologia , Diglicerídeos/metabolismo , Ácido Glutâmico/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/fisiologia , Transmissão Sináptica , Animais , Proteínas de Ligação ao GTP/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Naftalenos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Toxina Pertussis/farmacologia , Transdução de Sinais , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores
19.
Sci Rep ; 7(1): 16873, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29203889

RESUMO

Dimerization and oligomerization of G-protein coupled receptors (GPCRs) have emerged as important characters during their trans-membrane signal transduction. However, until now the relationship between GPCR dimerization and their trans-membrane signal transduction function is still uncovered. Here, using pertussis toxin (PTX) to decouple the receptor from G protein complex and with single-molecule imaging, we show that in the presence of agonist, cells treated with PTX showed a decrease in the number of dimers and oligomers on the cell surface compared with untreated ones, which suggests that oligomeric status of CXCR4 could be significantly influenced by the decoupling of G protein complex during its signal transduction process. Moreover, with chlorpromazine (CPZ) to inhibit internalization of CXCR4, it was found that after SDF-1α stimulation, cells treated with CPZ showed more dimers and oligomers on the cell surface than untreated ones, which suggest that dimers and oligomers of CXCR4 tend to internalize more easily than monomers. Taken together, our results demonstrate that dimerization and oligomerization of CXCR4 is closely related with its G protein mediated pathway and ß-arrestin mediated internalization process, and would play an important role in regulating its signal transduction functions.


Assuntos
Membrana Celular/metabolismo , Receptores CXCR4/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxia/efeitos dos fármacos , Clorpromazina/farmacologia , Dimerização , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Toxina Pertussis/farmacologia , Multimerização Proteica/efeitos dos fármacos , Receptores CXCR4/agonistas , Receptores CXCR4/genética
20.
J Neuroinflammation ; 14(1): 198, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28974234

RESUMO

BACKGROUND: Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood. METHODS: We used endogenous, natural, and surrogate agonists for GPR84 (capric acid, embelin, and 6-OAU, respectively) and examined their effect on mouse primary cultured microglia in vitro. RESULTS: 6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 gene. CONCLUSIONS: GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury.


Assuntos
Movimento Celular/efeitos dos fármacos , Microglia/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Animais Recém-Nascidos , Benzoquinonas/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , AMP Cíclico/metabolismo , Citocinas/genética , Citocinas/metabolismo , Ácidos Decanoicos/farmacologia , Deleção de Genes , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microglia/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Toxina Pertussis/farmacologia , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/efeitos dos fármacos , Uracila/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA