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1.
Nat Commun ; 15(1): 7822, 2024 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-39242606

RESUMO

G protein-coupled receptors' conformational landscape can be affected by their local, microscopic interactions within the cell plasma membrane. We employ here a pleiotropic stimulus, namely osmotic swelling, to alter the cortical environment within intact cells and monitor the response in terms of receptor function and downstream signaling. We observe that in osmotically swollen cells the ß2-adrenergic receptor, a prototypical GPCR, favors an active conformation, resulting in cAMP transient responses to adrenergic stimulation that have increased amplitude. The results are validated in primary cell types such as adult cardiomyocytes, a model system where swelling occurs upon ischemia-reperfusion injury. Our results suggest that receptors' function is finely modulated by their biophysical context, and specifically that osmotic swelling acts as a potentiator of downstream signaling, not only for the ß2-adrenergic receptor, but also for other receptors, hinting at a more general regulatory mechanism.


Assuntos
AMP Cíclico , Miócitos Cardíacos , Receptores Adrenérgicos beta 2 , Transdução de Sinais , Receptores Adrenérgicos beta 2/metabolismo , Miócitos Cardíacos/metabolismo , Humanos , Animais , Ligantes , AMP Cíclico/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Camundongos
2.
Proc Natl Acad Sci U S A ; 121(39): e2411981121, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39284057

RESUMO

Bacterial biofilms have been implicated in several chronic infections. After initial attachment, a critical first step in biofilm formation is a cell inducing a surface-sensing response. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, two second messengers, cyclic diguanylate monophosphate (c-di-GMP) and cyclic adenosine monophosphate (cAMP), are produced by different surface-sensing mechanisms. However, given the disparate cellular behaviors regulated by these second messengers, how newly attached cells coordinate these pathways remains unclear. Some of the uncertainty relates to studies using different strains, experimental systems, and usually focusing on a single second messenger. In this study, we developed a tricolor reporter system to simultaneously gauge c-di-GMP and cAMP levels in single cells. Using PAO1, we show that c-di-GMP and cAMP are selectively activated in two commonly used experimental systems to study surface sensing. By further examining the conditions that differentiate a c-di-GMP or cAMP response, we demonstrate that an agarose-air interface activates cAMP signaling through type IV pili and the Pil-Chp system. However, a liquid-agarose interface favors the activation of c-di-GMP signaling. This response is dependent on flagellar motility and correlated with higher swimming speed. Collectively, this work indicates that c-di-GMP and cAMP signaling responses are dependent on the surface context.


Assuntos
Biofilmes , AMP Cíclico , GMP Cíclico , Pseudomonas aeruginosa , Pseudomonas aeruginosa/fisiologia , Pseudomonas aeruginosa/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Biofilmes/crescimento & desenvolvimento , Transdução de Sinais , Sistemas do Segundo Mensageiro/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética
3.
Gut Microbes ; 16(1): 2399215, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39284098

RESUMO

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in humans and animals, induced by enterotoxins produced by these pathogens. Despite the crucial role of neutrophils in combatting bacterial infections, our understanding of how enterotoxins impact neutrophil function is limited. To address this knowledge gap, we used heat-labile enterotoxin (LT) and heat-stable enterotoxin a (STa) to investigate their impact on the effector functions of neutrophils. Our study reveals that pSTa does not exert any discernible effect on the function of neutrophils. In contrast, LT altered the migration and phagocytosis of neutrophils and induced the production of inflammatory factors via activation of cAMP/PKA and ERK1/2 signaling. LT also attenuated the release of neutrophil extracellular traps by neutrophils via the PKA signaling pathway. Our findings provide novel insights into the impact of LT on neutrophil function, shedding light on the underlying mechanisms that govern its immunoregulatory effects. This might help ETEC in subverting the immune system and establishing infection.


Assuntos
Toxinas Bacterianas , Proteínas Quinases Dependentes de AMP Cíclico , AMP Cíclico , Escherichia coli Enterotoxigênica , Enterotoxinas , Infecções por Escherichia coli , Proteínas de Escherichia coli , Neutrófilos , Fagocitose , Enterotoxinas/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Humanos , AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/imunologia , Transdução de Sinais
4.
PLoS One ; 19(9): e0305312, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39259753

RESUMO

The primate-specific chemokine CCL18 is a potent chemoattractant for T cells and is expressed at elevated levels in several inflammatory diseases. However, the cognate receptor for CCL18 remains unconfirmed. Here, we describe attempts to validate a previous report that the chemokine receptor CCR8 is the human CCL18 receptor (Islam et al. J Exp Med. 2013, 210:1889-98). Two mouse pre-B cell lines (4DE4 and L1.2) exogenously expressing CCR8 exhibited robust migration in response to the known CCR8 ligand CCL1 but not to CCL18. Similarly, CCL1 but not CCL18 induced internalization of CCR8 on 4DE4 cells. CCR8 expressed on Chinese hamster ovarian (CHO) cells mediated robust G protein activation, inhibition of cAMP synthesis and ß-arrestin2 recruitment in response to CCL1 but not CCL18. Several N- and C-terminal variants of CCL18 also failed to stimulate CCR8 activation. On the other hand, and as previously reported, CCL18 inhibited CCL11-stimulated migration of 4DE4 cells expressing the receptor CCR3. These data suggest that CCR8, at least in the absence of unidentified cofactors, does not function as a high affinity receptor for CCL18.


Assuntos
Quimiocinas CC , Cricetulus , Receptores CCR8 , Animais , Receptores CCR8/metabolismo , Receptores CCR8/genética , Humanos , Células CHO , Camundongos , Quimiocinas CC/metabolismo , Quimiocinas CC/genética , Cricetinae , Quimiocina CCL1/metabolismo , Movimento Celular , Linhagem Celular , Quimiocina CCL11/metabolismo , AMP Cíclico/metabolismo
5.
Mol Plant Pathol ; 25(9): e70003, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39235122

RESUMO

Sugarcane smut fungus Sporisorium scitamineum produces polyamines putrescine (PUT), spermidine (SPD), and spermine (SPM) to regulate sexual mating/filamentous growth critical for pathogenicity. Besides de novo biosynthesis, intracellular levels of polyamines could also be modulated by oxidation. In this study, we identified two annotated polyamine oxidation enzymes (SsPAO and SsCuAO1) in S. scitamineum. Compared to the wild type (MAT-1), the ss1paoΔ and ss1cuao1Δ mutants were defective in sporidia growth, sexual mating/filamentation, and pathogenicity. The addition of a low concentration of cAMP (0.1 mM) could partially or fully restore filamentation of ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ. cAMP biosynthesis and hydrolysis genes were differentially expressed in the ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ cultures, further supporting that SsPAO- or SsCuAO1-based polyamine homeostasis regulates S. scitamineum filamentation by affecting the cAMP/PKA signalling pathway. During early infection, PUT promotes, while SPD inhibits, the accumulation of reactive oxygen species (ROS) in sugarcane, therefore modulating redox homeostasis at the smut fungus-sugarcane interface. Autophagy induction was found to be enhanced in the ss1paoΔ mutant and reduced in the ss1cuao1Δ mutant. Exogenous addition of cAMP, PUT, SPD, or SPM at low concentration promoted autophagy activity under a non-inductive condition (rich medium), suggesting a cross-talk between polyamines and cAMP signalling in regulating autophagy in S. scitamineum. Overall, our work proves that SsPAO- and SsCuAO1-mediated intracellular polyamines affect intracellular redox balance and thus play a role in growth, sexual mating/filamentation, and pathogenicity of S. scitamineum.


Assuntos
Oxirredução , Poliaminas , Poliaminas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , AMP Cíclico/metabolismo , Saccharum/microbiologia , Regulação Fúngica da Expressão Gênica , Ustilaginales/patogenicidade , Autofagia
6.
Nat Commun ; 15(1): 7684, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39227390

RESUMO

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cß (PLCß) isozymes to increase cytosolic Ca2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCß1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gßγ as driver of a PLCß2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCß3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCß3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.


Assuntos
Sinalização do Cálcio , Cálcio , Fosfolipase C beta , Receptores Acoplados a Proteínas G , Humanos , Fosfolipase C beta/metabolismo , Fosfolipase C beta/genética , Células HEK293 , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Cálcio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Sistemas CRISPR-Cas , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , AMP Cíclico/metabolismo , Animais , Edição de Genes , Citosol/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Adenilil Ciclases/metabolismo , Adenilil Ciclases/genética
7.
Int J Mol Sci ; 25(17)2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-39273390

RESUMO

Chronic kidney disease (CKD) is characterized by a steady decline in kidney function and affects roughly 10% of the world's population. This review focuses on the critical function of cyclic adenosine monophosphate (cAMP) signaling in CKD, specifically how it influences both protective and pathogenic processes in the kidney. cAMP, a critical secondary messenger, controls a variety of cellular functions, including transcription, metabolism, mitochondrial homeostasis, cell proliferation, and apoptosis. Its compartmentalization inside cellular microdomains ensures accurate signaling. In kidney physiology, cAMP is required for hormone-regulated activities, particularly in the collecting duct, where it promotes water reabsorption through vasopressin signaling. Several illnesses, including Fabry disease, renal cell carcinoma, nephrogenic diabetes insipidus, Bartter syndrome, Liddle syndrome, diabetic nephropathy, autosomal dominant polycystic kidney disease, and renal tubular acidosis, have been linked to dysfunction in the cAMP system. Both cAMP analogs and phosphodiesterase inhibitors have the potential to improve kidney function and reduce kidney damage. Future research should focus on developing targeted PDE inhibitors for the treatment of CKD.


Assuntos
AMP Cíclico , Insuficiência Renal Crônica , Transdução de Sinais , Humanos , AMP Cíclico/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia , Animais , Transdução de Sinais/efeitos dos fármacos , Terapia de Alvo Molecular , Rim/metabolismo , Rim/patologia , Inibidores de Fosfodiesterase/uso terapêutico , Inibidores de Fosfodiesterase/farmacologia
8.
Elife ; 132024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39255004

RESUMO

In birds and insects, the female uptakes sperm for a specific duration post-copulation known as the ejaculate holding period (EHP) before expelling unused sperm and the mating plug through sperm ejection. In this study, we found that Drosophila melanogaster females shortens the EHP when incubated with males or mated females shortly after the first mating. This phenomenon, which we termed male-induced EHP shortening (MIES), requires Or47b+ olfactory and ppk23+ gustatory neurons, activated by 2-methyltetracosane and 7-tricosene, respectively. These odorants raise cAMP levels in pC1 neurons, responsible for processing male courtship cues and regulating female mating receptivity. Elevated cAMP levels in pC1 neurons reduce EHP and reinstate their responsiveness to male courtship cues, promoting re-mating with faster sperm ejection. This study established MIES as a genetically tractable model of sexual plasticity with a conserved neural mechanism.


Assuntos
Drosophila melanogaster , Feromônios , Comportamento Sexual Animal , Animais , Feminino , Masculino , Drosophila melanogaster/fisiologia , Comportamento Sexual Animal/fisiologia , Feromônios/metabolismo , Neurônios/fisiologia , Neurônios/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , AMP Cíclico/metabolismo
9.
Biomolecules ; 14(8)2024 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-39199373

RESUMO

Sulfonylureas (SUs) are a class of antidiabetic drugs widely used in the management of diabetes mellitus type 2. They promote insulin secretion by inhibiting the ATP-sensitive potassium channel in pancreatic ß-cells. Recently, the exchange protein directly activated by cAMP (Epac) was identified as a new class of target proteins of SUs that might contribute to their antidiabetic effect, through the activation of the Ras-like guanosine triphosphatase Rap1, which has been controversially discussed. We used human embryonic kidney (HEK) 293 cells expressing genetic constructs of various Förster resonance energy transfer (FRET)-based biosensors containing different versions of Epac1 and Epac2 isoforms, alone or fused to different phosphodiesterases (PDEs), to monitor SU-induced conformational changes in Epac or direct PDE inhibition in real time. We show that SUs can both induce conformational changes in the Epac2 protein but not in Epac1, and directly inhibit the PDE3 and PDE4 families, thereby increasing cAMP levels in the direct vicinity of these PDEs. Furthermore, we demonstrate that the binding site of SUs in Epac2 is distinct from that of cAMP and is located between the amino acids E443 and E460. Using biochemical assays, we could also show that tolbutamide can inhibit PDE activity through an allosteric mechanism. Therefore, the cAMP-elevating capacity due to allosteric PDE inhibition in addition to direct Epac activation may contribute to the therapeutic effects of SU drugs.


Assuntos
AMP Cíclico , Fatores de Troca do Nucleotídeo Guanina , Compostos de Sulfonilureia , Humanos , Compostos de Sulfonilureia/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/química , Hipoglicemiantes/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Sítios de Ligação , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo
10.
J Ethnopharmacol ; 335: 118636, 2024 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-39089658

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke is an acute central nervous system disease that poses a threat to human health. It induces a series of severe pathological mechanisms, ultimately leading to neuronal cell death in the brain due to local ischemia and hypoxia. Buyang Huanwu decoction (BYHWD), as a representative formula for treating ischemic stroke, has shown good therapeutic effects in stroke patients. AIM OF THE STUDY: This study aimed to explore the mechanism of BYHWD in promoting neural remodeling after ischemic stroke from the perspective of neuronal synaptic plasticity, based on the cAMP/PKA/CREB signaling pathway. MATERIALS AND METHODS: A modified suture technique was employed to establish a rat model of MCAO. The rats were divided into sham, model, and BYHWD (20 g/kg) groups. After the corresponding intervention, rat brains from each group were collected. TMT quantitative proteomics technology was employed for the research. Following proteomics studies, we investigated the mechanism of BYHWD in the intervention of ischemic stroke through animal experiments and cell experiments. The experimental animals were divided into sham, model, and BYHWD (5 g/kg, 10 g/kg, and 20 g/kg) groups. Infarct volume and severity of brain injury were measured by TTC staining. HE staining was utilized to evaluate alterations in tissue morphology. The Golgi staining was used to observe changes in cell body, dendrites, and dendritic spines. Transmission electron microscopy was used to observe the ultrastructure of synapses in the cortex and hippocampus. TUNEL staining was conducted to identify apoptotic neurons. Meanwhile, a stable and reliable (OGD/R) SH-SY5Y cell model was established. The effect of BYHWD-containing serum on SH-SY5Y cell viability was measured by CCK-8 kit. The apoptosis situation of SH-SY5Y cells was determined by Annexin V-FITC/PI. Immunofluorescence was employed to measure the fluorescence intensity of synaptic-related factors Syt1, Psd95, and Syn1. Synaptic plasticity pathways were assessed by using RT-qPCR and Western blot to determine the expression levels of cAMP, Psd95, Prkacb, Creb1/p-Creb1, BDNF, Shank2, Syn1, Syt1, Bcl-2, Bcl-2/Bax mRNA and proteins. RESULTS: After treatment with BYHWD, notable alterations were detected in the signaling pathways linked to synaptic plasticity and the cAMP signaling pathway-related targets among the intervention targets. This trend of change was also reflected in other bioinformatics analyses, indicating the important role of synaptic plasticity changes before and after modeling and drug intervention. The results of vivo and vitro experiments showed that BYHWD improved local pathological changes, and reduced cerebral infarct volume, and neurological function scores in MCAO rats. It increased dendritic spine density, improved synaptic structural plasticity, and had a certain neuroprotective effect. BYHWD increased the postsynaptic membrane thickness, synaptic interface curvature, and synaptic quantity. 10% BYHWD-containing serum was determined as the optimal concentration for treatment. 10% BYHWD-containing serum significantly reduced the overall apoptotic rate of (OGD/R) SH-SY5Y cells. Immunofluorescence experiments demonstrated that 10% BYHWD-containing serum could improve synaptic plasticity and increase the relative expression levels of synaptic-related proteins Syt1, Psd95, and Syn1. BYHWD and decoction-containing serum upregulated the mRNA and protein expression levels in (OGD/R) SH-SY5Y cells and MCAO rats, suggesting its ability to improve damaged neuronal synaptic plasticity and enhance transmission efficiency, which might be achieved through the regulation of the cAMP/PKA/CREB pathway. CONCLUSIONS: This study may provide a basis for clinical medication by elucidating the underlying experimental evidence for the promotion of neural plasticity after ischemic stroke by BYHWD.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , AMP Cíclico , Medicamentos de Ervas Chinesas , AVC Isquêmico , Plasticidade Neuronal , Ratos Sprague-Dawley , Transdução de Sinais , Animais , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Plasticidade Neuronal/efeitos dos fármacos , AVC Isquêmico/tratamento farmacológico , Masculino , AMP Cíclico/metabolismo , Ratos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Fármacos Neuroprotetores/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico
11.
Int J Neuropsychopharmacol ; 27(8)2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39099166

RESUMO

BACKGROUND: Ethanol elicits a rapid stimulatory effect and a subsequent, prolonged sedative response, which are potential predictors of EtOH consumption by decreasing adenosine signaling; this phenomenon also reflects the obvious sex difference. cAMP (cyclic Adenosine Monophosphate)-PKA (Protein Kinase A) signaling pathway modulation can influence the stimulatory and sedative effects induced by EtOH in mice. This study's objective is to clarify the role of phosphodiesterase (PDE) in mediating the observed sex differences in EtOH responsiveness between male and female animals. METHODS: EtOH was administered i.p. for 7 days to identify the changes in PDE isoforms in response to EtOH treatment. Additionally, EtOH consumption and preference of male and female C57BL/6J mice were assessed using the drinking-in-the-dark and 2-bottle choice tests. Further, pharmacological inhibition of PDE7A heterozygote knockout mice was performed to investigate its effects on EtOH-induced stimulation and sedation in both male and female mice. Finally, Western blotting analysis was performed to evaluate the alterations in cAMP-PKA/Epac2 pathways. RESULTS: EtOH administration resulted in an immediate upregulation in PDE7A expression in female mice, indicating a strong association between PDE7A and EtOH stimulation. Through the pharmacological inhibition of PDE7A KD mice, we have demonstrated for the first time, to our knowledge, that PDE7A selectively attenuates EtOH responsiveness and consumption exclusively in female mice, whichmay be associated with the cAMP-PKA/Epac2 pathway and downstream phosphorylation of CREB and ERK1/2. CONCLUSIONS: Inhibition or knockdown of PDE7A attenuates EtOH responsivenessand consumption exclusively in female mice, which is associated with alterations in the cAMP-PKA/Epac2 signaling pathways, thereby highlighting its potential as a novel therapeutic target for alcohol use disorder.


Assuntos
Consumo de Bebidas Alcoólicas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7 , Etanol , Camundongos Endogâmicos C57BL , Camundongos Knockout , Animais , Masculino , Feminino , Etanol/farmacologia , Etanol/administração & dosagem , Consumo de Bebidas Alcoólicas/metabolismo , Camundongos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/metabolismo , Caracteres Sexuais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Depressores do Sistema Nervoso Central/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo
12.
Eur J Med Res ; 29(1): 433, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39192377

RESUMO

BACKGROUND: Reduction of inflammatory damage and inhibition of nucleus pulposus (NP) apoptosis are considered to be the main effective therapy idea to reverse the intervertebral disc degeneration (IDD) and alleviate the chronic low back pain. The adenosine A2A receptor (A2AR), as a member of G protein-coupled receptor families, plays an important role in the anti-inflammation and relieving pain. So far, the impact of A2AR on IDD therapy is unclear. The aim of this study was to explore the role of Adenosine A2A receptor (A2AR) in the intervertebral disc degeneration (IDD) and clarify potential mechanism. MATERIALS AND METHODS: IL-1ß and acupuncture was used to establish IDD model rats. A2AR agonist CGS-21680 and A2AR antagonist SCH442416 were used to investigate the therapeutical effects for IDD. Histological examination, western blotting analysis and RT-PCR were employed to evaluate the the association between A2AR and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway. RESULTS: A2AR activity of the intervertebral disc tissues was up-regulated in feedback way, and cAMP, PKA and CREB expression were also increased. But in general, IL-1ß-induced IDD promoted the significant up-regulation the expression of inflammatory factors. The nucleus pulposus (NP) inflammation was exacerbated in result of MMP3 and Col-II decline through activating NF-κB signaling pathway. A2AR agonist CGS-21680 exhibited a disc protective effect through significantly increasing A2AR activity, then further activated cAMP/PKA signaling pathway with attenuating the release of TNF-α and IL-6 via down-regulating NF-κB. In contrast, SCH442416 inhibited A2AR activation, consistent with lower expression levels of cAMP and PKA, further leading to the acceleration of IDD. CONCLUSIONS: The activation of A2AR can prevent inflammatory responses and mitigates degradation of IDD thus suggest a potential novel therapeutic strategy of IDD.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Inflamação , Degeneração do Disco Intervertebral , NF-kappa B , Receptor A2A de Adenosina , Transdução de Sinais , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/tratamento farmacológico , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor A2A de Adenosina/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ratos , Inflamação/metabolismo , Masculino , Ratos Sprague-Dawley , Fenetilaminas/farmacologia , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Núcleo Pulposo/efeitos dos fármacos , AMP Cíclico/metabolismo , Agonistas do Receptor A2 de Adenosina/farmacologia , Modelos Animais de Doenças , Adenosina/análogos & derivados
13.
Proc Natl Acad Sci U S A ; 121(34): e2405465121, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39145932

RESUMO

Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.


Assuntos
Analgésicos Opioides , AMP Cíclico , Gabapentina , Neuralgia , Transdução de Sinais , Traumatismos da Medula Espinal , Animais , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , AMP Cíclico/metabolismo , Ratos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Analgésicos Opioides/farmacologia , Gabapentina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ratos Sprague-Dawley , Masculino , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Pregabalina/farmacologia , Pregabalina/uso terapêutico , Sinergismo Farmacológico , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos
14.
BMC Neurosci ; 25(1): 38, 2024 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-39179957

RESUMO

Visual perception of X-radiation is a well-documented, but poorly understood phenomenon. Scotopic rod cells and rhodopsin have been implicated in visual responses to X-rays, however, some evidence suggests that X-rays excite the retina via a different mechanism than visible light. While rhodopsin's role in X-ray perception is unclear, the possibility that it could function as an X-ray receptor has led to speculation that it could act as a transgenically expressed X-ray receptor. If so, it could be used to transduce transcranial X-ray signals and control the activity of genetically targeted populations of neurons in a less invasive version of optogenetics, X-genetics. Here we investigate whether human rhodopsin (hRho) is capable of transducing X-ray signals when expressed outside of the retinal environment. We use a live-cell cAMP GloSensor luminescence assay to measure cAMP decreases in hRho-expressing HEK293 cells in response to visible light and X-ray stimulation. We show that cAMP GloSensor luminescence decreases are not observed in hRho-expressing HEK293 cells in response to X-ray stimulation, despite the presence of robust responses to visible light. Additionally, irradiation had no significant effect on cAMP GloSensor responses to subsequent visible light stimulation. These results suggest that ectopically expressed rhodopsin does not function as an X-ray receptor and is not capable of transducing transcranial X-ray signals into neural activity for X-ray mediated, genetically targeted neuromodulation.


Assuntos
AMP Cíclico , Rodopsina , Humanos , Células HEK293 , Rodopsina/metabolismo , Rodopsina/genética , Raios X , AMP Cíclico/metabolismo , Luz , Estimulação Luminosa/métodos
15.
Int J Mol Sci ; 25(15)2024 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-39125708

RESUMO

Single cilia, 100 nm in diameter and 10 µm in length, were isolated from mouse tracheae with Triton X-100 (0.02%) treatment, and the effects of pH on ciliary beating were examined by measuring the ciliary beat frequency (CBF) and the ciliary bend distance (CBD-an index of amplitude) using a high-speed video microscope (250 fps). ATP (2.5 mM) plus 8Br-cAMP (10 µM) reactivated the CBF and CBD in the isolated cilia, similar to the cilia of in vivo tracheae. In the reactivated isolated cilia, an elevation in pH from 7.0 to 8.0 increased the CBF from 3 to 15 Hz and the CBD from 0.6 to 1.5 µm. The pH elevation also increased the velocity of the effective stroke; however, it did not increase the recovery stroke, and, moreover, it decreased the intervals between beats. This indicates that H+ (pHi) directly acts on the axonemal machinery to regulate CBF and CBD. In isolated cilia priorly treated with 1 µM PKI-amide (a PKA inhibitor), 8Br-cAMP did not increase the CBF or CBD in the ATP-stimulated isolated cilia. pH modulates the PKA signal, which enhances the axonemal beating generated by the ATP-activated inner and outer dyneins.


Assuntos
Trifosfato de Adenosina , Cílios , AMP Cíclico , Traqueia , Animais , Cílios/efeitos dos fármacos , Cílios/metabolismo , Trifosfato de Adenosina/metabolismo , Concentração de Íons de Hidrogênio , Traqueia/metabolismo , Traqueia/efeitos dos fármacos , Camundongos , AMP Cíclico/metabolismo , Masculino
16.
Int J Mol Sci ; 25(15)2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-39126032

RESUMO

Cyclic nucleotide phosphodiesterases (PDEs) consist of a family of enzymes expressed in several types of cells, including inflammatory cells, that play a pivotal role in inflammation. Several studies have demonstrated that the inhibition of PDE4 results in a reduced inflammatory response via PKA and CREB signaling. Hence, PDE4 suppression improves the inflammatory feedback typical of several diseases, such as inflammatory bowel disease (IBD). In our previous studies, we have demonstrated that miR-369-3p regulates inflammatory responses, modulating different aspects of the inflammatory process. The aim of this study was to demonstrate an additional anti-inflammatory effect of miR-369-3p targeting PDE4B, one of the widely expressed isoforms in immune cells. We found that miR-369-3p was able to reduce the expression of PDE4B, elevating the intracellular levels of cAMP. This accumulation increased the expression of PKA and pCREB, mitigating the release of pro-inflammatory cytokines and promoting the release of anti-inflammatory cytokines. To prove that PDE4B is a good therapeutic target in IBD, we also demonstrate that the expression of PDE4B was increased in UC patients compared to healthy controls, affecting the immune infiltrate. PDE4B is considered an important player in inflammatory progression; hence, our results show the ability of miR-369-3p to ameliorate inflammation by targeting PDE4B, supporting its future application as a new therapeutic approach in IBD.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Doenças Inflamatórias Intestinais , MicroRNAs , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , AMP Cíclico/metabolismo , Inflamação/genética , Inflamação/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citocinas/metabolismo , Masculino , Transdução de Sinais , Feminino
17.
Bull Exp Biol Med ; 177(2): 212-216, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-39093471

RESUMO

The effect of a promising NO donor, a binuclear nitrosyl iron complex (NIC) with 3,4-dichlorothiophenolyls [Fe2(SC6H3Cl2)2(NO)4], on the adenylate cyclase and soluble guanylate cyclase enzymatic systems was studied. In in vitro experiments, this complex increased the concentration of important secondary messengers, such as cAMP and cGMP. An increase of their level by 2.4 and 4.5 times, respectively, was detected at NIC concentration of 0.1 mM. The ligand of the complex, 3,4-dichlorothiophenol, produced a less pronounced effect on adenylate cyclase. It was shown that the effect of this complex on the activity of soluble guanylate cyclase was comparable to the effect of anionic nitrosyl complex with thiosulfate ligands that exhibits vasodilating and cardioprotective properties.


Assuntos
AMP Cíclico , GMP Cíclico , GMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Animais , Ferro/metabolismo , Ferro/química , Adenilil Ciclases/metabolismo , Doadores de Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/química , Guanilil Ciclase Solúvel/metabolismo , Óxidos de Nitrogênio/farmacologia , Óxidos de Nitrogênio/metabolismo , Óxidos de Nitrogênio/química , Ratos
18.
Eur J Med Chem ; 277: 116769, 2024 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-39163778

RESUMO

Phosphodiesterases (PDEs) constitute a family of enzymes that play a pivotal role in the regulation of intracellular levels of cyclic nucleotides, including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Dysregulation of PDE activity has been implicated in diverse pathological conditions encompassing cardiovascular disorders, pulmonary diseases, and neurological disorders. Small-molecule inhibitors targeting PDEs have emerged as promising therapeutic agents for the treatment of these ailments, some of which have been approved for their clinical use. Despite their success, challenges such as resistance mechanisms and off-target effects persist, urging continuous research for the development of next-generation PDE inhibitors. The objective of this review is to provide an overview of the synthesis and clinical application of representative approved small-molecule PDE inhibitors, with the aim of offering guidance for further advancements in the development of novel PDE inhibitors.


Assuntos
Inibidores de Fosfodiesterase , Diester Fosfórico Hidrolases , Bibliotecas de Moléculas Pequenas , Animais , Humanos , Estrutura Molecular , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-Atividade , AMP Cíclico/química , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia
19.
Turk J Gastroenterol ; 35(6): 453-464, 2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-39114901

RESUMO

The pathogenesis mechanism of acute gastric mucosal lesions (AGML) is still unclear; further exploration is urgently needed to find a new therapeutic target. This study aimed to investigate whether morphine might regulate the expression and function of transient receptor potential ankyrin 1 (TRPA1) through a cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-dependent pathway, thereby alleviating gastric mucosal lesions caused by water-immersion restraint stress (WIRS). Rats were administered with intrathecal morphine, TRPA1 antagonist (HC-030031), µ-opioid receptor antagonist, or protein kinase A inhibitor (H-89), respectively, before WIRS. After 6 hours of WIRS, microscopic lesions, hematoxylin and eosin staining, and transmission electron microscopy were applied to assess the damage of the gastric mucosa. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were conducted to detect the levels of TRPA1 and substance P (SP) in the dorsal root ganglia (DRG) and gastric tissues. In addition, immunofluorescence was used to explore the possible co-expression of TRPA1 and µ-opioid receptors in the DRG. The results indicated that WIRS upregulated TRPA1 and SP in gastric mucosa, and HC-030031 or H-89 could alleviate gastric mucosal lesions caused by WIRS (P < .0001). Morphine was found to suppress both WIRS-induced gastric mucosal lesions (P < .0001) and the upregulation of TRPA1 (P = .0086) and SP (P = .0013). Both TRPA1 and SP play important roles in the pathogenesis of WIRS-induced AGML. Exogenous gastroprotective strategies reduce elevated levels of TRPA1 via the cAMP/PKA-dependent pathway. Inhibition of TRPA1 upregulation in the DRG is critical for intrathecal morphine preconditioning-induced gastric protection.


Assuntos
Gânglios Espinais , Mucosa Gástrica , Isoquinolinas , Morfina , Ratos Sprague-Dawley , Restrição Física , Canal de Cátion TRPA1 , Regulação para Cima , Animais , Morfina/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Regulação para Cima/efeitos dos fármacos , Canal de Cátion TRPA1/metabolismo , Masculino , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Restrição Física/efeitos adversos , Ratos , Isoquinolinas/farmacologia , Acetanilidas/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Purinas/farmacologia , Estresse Psicológico/complicações , Imersão , Receptores Opioides mu/metabolismo , AMP Cíclico/metabolismo , Sulfonamidas
20.
Int J Biol Macromol ; 278(Pt 2): 134783, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39153673

RESUMO

Glucagon-like peptide-1 (GLP-1) as a multifunctional hormone is secreted mainly from enteroendocrine L-cells, and enhancing its endogenous secretion has potential benefits of regulating glucose homeostasis and controlling body weight gain. In the present study, a novel polysaccharide (h-DHP) with high ability to enhance plasma GLP-1 level in mice was isolated from Dendrobium huoshanense protocorm-like bodies under the guidance of activity evaluation. Structural identification showed that h-DHP was an acidic polysaccharide with the molecular weight of 1.38 × 105 Da, and was composed of galactose, glucose, arabinose and glucuronic acid at a molar ratio of 15.7: 11.2: 4.5: 1.0 with a backbone consisting of →5)-α-L-Araf-(1→, →3)-α-D-Galp-(1→, →6)-α-D-Galp-(1→, →3,6)-α-D-Galp-(1→, →6)-ß-D-Glcp-(1→ and →4,6)-ß-D-Glcp-(1→ along with branches consisting of α-L-Araf-(1→, α-D-Galp-(1→, α-D-GlcAp-(1→, ß-D-Glcp-(1→ and →4)-ß-D-Glcp-(1→. Animal experiments with different administration routes demonstrated that h-DHP-enhanced plasma GLP-1 level was attributed to h-DHP-promoted GLP-1 secretion in the enteroendocrine L-cells, which was supported by h-DHP-enhanced extracellular GLP-1 level in STC-1 cells. Inhibition of adenylate cyclase and phospholipase C indicated that cAMP and cAMP-triggered intracellular Ca2+ increase participated in h-DHP-promoted GLP-1 secretion. These results suggested that h-DHP has the potential of enhancing endogenous GLP-1 level through h-DHP-promoted and cAMP-mediated GLP-1 secretion from enteroendocrine cells.


Assuntos
Dendrobium , Peptídeo 1 Semelhante ao Glucagon , Polissacarídeos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/sangue , Dendrobium/química , Animais , Polissacarídeos/farmacologia , Polissacarídeos/química , Camundongos , Masculino , Peso Molecular , Células Enteroendócrinas/metabolismo , Células Enteroendócrinas/efeitos dos fármacos , AMP Cíclico/metabolismo
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