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Decidualization, a crucial process for successful pregnancy establishment and maintenance, involves endometrial stromal cell differentiation. This process is orchestrated by estradiol (E2), progesterone, and other stimuli that increase intracellular cyclic adenosine monophosphate (cAMP) levels. The intracellular progesterone receptor (PR), encoded by the PGR gene, has a key role in decidualization. This study aimed to understand the role of sex steroids and cAMP in regulating PGR expression during the in vitro decidualization of the human immortalized endometrial stromal cell line, T-HESC. We subjected the cells to individual and combined treatments of E2, medroxyprogesterone (MPA), and cAMP. Additionally, we treated cells with PR and estrogen receptor antagonists and a protein kinase A (PKA) inhibitor. We evaluated the expression of PGR isoforms and decidualization-associated genes by RT-qPCR. Our findings revealed that cAMP induced PGR-B and PGR-AB expression by activating the PKA signaling pathway, while MPA downregulated their expression through the PR. Furthermore, downstream genes involved in decidualization, such as those coding for prolactin (PRL), insulin-like growth factor-binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1), exhibited positive regulation via the cAMP-PKA pathway. Remarkably, MPA-activated PR signaling induced the expression of IGFBP1 and DKK1 but inhibited that of PRL. In conclusion, we have demonstrated that the PKA signaling pathway induces PGR gene expression during in vitro decidualization of the T-HESC human endometrial stromal cell line. This study has unraveled some of the intricate regulatory mechanisms governing PGR expression during this fundamental process for implantation and pregnancy maintenance.
Assuntos
Decídua , Receptores de Progesterona , Gravidez , Feminino , Humanos , Decídua/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Endométrio/metabolismo , Progesterona/farmacologia , Progesterona/metabolismo , AMP Cíclico/metabolismo , Células Estromais/metabolismo , Expressão Gênica , Células CultivadasRESUMO
The relationship between psoriasis severity and psychological stress has been described in several studies. However, the mechanism by which chronic stress exacerbates psoriasis is not completely understood. This study aimed at investigating whether chronic psychological stress can aggravate psoriasis-like skin inflammation. Mice were subjected to a restraint stress model and topically treated with imiquimod (IMQ). Differentiated human keratinocytes were treated with high epinephrine levels and IMQ in vitro. Stress aggravated macroscopic features and the increase in epidermal thickness induced by IMQ in mouse skin. The increase in NF-κB and IL-17A expression induced by IMQ was potentiated by chronic stress in mouse skin. The skin of stressed mice treated with IMQ showed higher levels of ß2-adrenergic receptors (ß2-AR). In human keratinocytes, high epinephrine levels exacerbated the increase in the levels of ß2-AR and IL-17A induced by IMQ. ß-AR antagonist reversed the effects of chronic stress in IMQ-induced inflammation both in vivo and in vitro. In conclusion, stress-stimulated overactivation of the ß2-AR and NF-κB pathways potentiates a Th1/Th17 profile leading to an exacerbation of psoriasis.
Assuntos
Imiquimode , Interleucina-17 , Queratinócitos , NF-kappa B , Psoríase , Receptores Adrenérgicos beta 2 , Transdução de Sinais , Estresse Psicológico , Animais , Humanos , Masculino , Camundongos , Modelos Animais de Doenças , Epinefrina , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-17/metabolismo , Queratinócitos/metabolismo , Queratinócitos/imunologia , NF-kappa B/metabolismo , Psoríase/imunologia , Psoríase/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Pele/patologia , Pele/imunologia , Pele/metabolismo , Estresse Psicológico/complicações , Estresse Psicológico/imunologia , Células Th1/imunologia , Células Th17/imunologiaRESUMO
A large amount of evidence has supported a clinical link between diabetes and inflammatory diseases, e.g., cancer, dementia, and hypertension. In addition, it is also suggested that dysregulations related to Ca2+ signaling could link these diseases, in addition to 3'-5'-cyclic adenosine monophosphate (cAMP) signaling pathways. Thus, revealing this interplay between diabetes and inflammatory diseases may provide novel insights into the pathogenesis of these diseases. Publications involving signaling pathways related to Ca2+ and cAMP, inflammation, diabetes, dementia, cancer, and hypertension (alone or combined) were collected by searching PubMed and EMBASE. Both signaling pathways, Ca2+ and cAMP signaling, control the release of neurotransmitters and hormones, in addition to neurodegeneration, and tumor growth. Furthermore, there is a clear relationship between Ca2+ signaling, e.g., increased Ca2+ signals, and inflammatory responses. cAMP also regulates pro- and anti-inflammatory responses. Due to the experience of our group in this field, this article discusses the role of Ca2+ and cAMP signaling in the correlation between diabetes and inflammatory diseases, including its pharmacological implications. As a novelty, this article also includes: (1) A timeline of the major events in Ca2+/cAMP signaling; and (2) As coronavirus disease 2019 (COVID-19) is an emerging and rapidly evolving situation, this article also discusses recent reports on the role of Ca2+ channel blockers for preventing Ca2+ signaling disruption due to COVID-19, including the correlation between COVID-19 and diabetes.
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Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Most of the different PDE variants play specific physiological functions; in fact, PDEs can associate with other proteins allowing them to be strategically anchored throughout the cell. In this regard, precise cellular expression and compartmentalization of these enzymes produce the specific control of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) gradients in cells and enable their integration with other signaling pathways.In trypanosomatids, some PDEs are essential for their survival and play fundamental roles in the adaptation of these parasites to different environmental stresses, as well as in the differentiation between their different life cycle forms. Given that these enzymes not only are similar to human PDEs but also have differential biochemical properties, and due to the great knowledge of drugs that target human PDEs, trypanosomatid PDEs could be postulated as important therapeutic targets through the repositioning of drugs.In this chapter, we describe a simple and sensitive radioisotope-based method to measure cyclic 3',5'-nucleotide phosphodiesterase using [3H]cAMP.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/isolamento & purificação , Ensaios Enzimáticos/métodos , Marcação por Isótopo/métodos , Proteínas de Protozoários/isolamento & purificação , Trypanosoma cruzi/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/química , AMP Cíclico/metabolismo , Estágios do Ciclo de Vida , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Trítio/químicaRESUMO
Interleukin (IL)-1ß is a potential target for treatment of several inflammatory diseases, including envenomation by the scorpion Tityus serrulatus. In this context, bioactive lipids such as prostaglandin (PG)E2 and leukotriene (LT)B4 modulate the production of IL-1ß by innate immune cells. Pattern recognition receptors (PRRs) that perceive T. serrulatus venom (TsV), and orchestrate LTB4, PGE2, and cyclic adenosine monophosphate (cAMP) production to regulate IL-1ß release are unknown. Furthermore, molecular mechanisms driving human cell responses to TsV remain uncharacterized. Here, we identified that both CD14 and CD36 control the synthesis of bioactive lipids, inflammatory cytokines, and mortality mediated by TsV. CD14 induces PGE2/cAMP/IL-1ß release and inflammation. By contrast, CD36 shunts eicosanoid metabolism toward production of LTB4, which represses the PGE2/cAMP/IL-1ß axis and mortality. Of importance, the molecular mechanisms observed in mice strongly correlate with those of human cell responses to TsV. Overall, this study provides major insights into molecular mechanisms connecting CD14 and CD36 with differential eicosanoid metabolism and inflammation mediated by IL-1ß.
Assuntos
Antígenos CD36/imunologia , Interleucina-1beta/imunologia , Receptores de Lipopolissacarídeos/imunologia , Picadas de Escorpião/imunologia , Venenos de Escorpião/imunologia , Adulto , Animais , Antígenos CD36/metabolismo , Modelos Animais de Doenças , Eicosanoides/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Interleucina-1beta/metabolismo , Leucócitos Mononucleares , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Cultura Primária de Células , Picadas de Escorpião/sangue , Picadas de Escorpião/mortalidade , Escorpiões/imunologia , Transdução de Sinais/imunologia , Adulto JovemRESUMO
Angiotensin-(3-4) (Ang-(3-4) or Val-Tyr) is the shorter angiotensin (Ang) II-derived peptide, formed through successive hydrolysis that culminates with the release of Val-Tyr as a dipeptide. It is formed both in plasma and in kidney from Ang II and Ang III, and can be considered a component of the systemic and organ-based renin-angiotensin system. It is potently antihypertensive in humans and rats, and its concerted actions on proximal tubule cells culminate in the inhibition of fluid reabsorption, hyperosmotic urinary excretion of Na+. At the renal cell signaling level, Ang-(3-4) counteracts Ang II-type 1 receptor-mediated responses by acting as an allosteric enhancer in Ang II-type 2 receptor populations that target adenosine triphosphate-dependent Ca2+ and Na+ transporters through a cyclic adenosine monophosphate-activated protein kinase pathway.
Assuntos
Angiotensina II/metabolismo , Sistema Renina-Angiotensina , Animais , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Peptídeos/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismoRESUMO
OBJECTIVES: Empiric treatment with milrinone, a phosphodiesterase (PDE) 3 inhibitor, has become increasingly common in patients with single ventricle heart disease of right ventricular (RV) morphology (SRV); our objective was to characterize the myocardial response to PDE3 inhibition (PDE3i) in the pediatric population with SRV. STUDY DESIGN: Cyclic adenosine monophosphate levels, PDE activity, and phosphorylated phospholamban (PLN) were determined in explanted human ventricular myocardium from nonfailing pediatric donors (n = 10) and pediatric patients transplanted secondary to SRV. Subjects with SRV were further classified by PDE3i treatment (n = 13 with PDE3i and n = 12 without PDE3i). RESULTS: In comparison with nonfailing RV myocardium (n = 8), cyclic adenosine monophosphate levels are lower in patients with SRV treated with PDE3i (n = 12, P = .021). Chronic PDE3i does not alter total PDE or PDE3 activity in SRV myocardium. Compared with nonfailing RV myocardium, SRV myocardium (both with and without PDE3i) demonstrates equivalent phosphorylated PLN at the protein kinase A phosphorylation site. CONCLUSIONS: As evidenced by preserved phosphorylated PLN, the molecular adaptation associated with SRV differs significantly from that demonstrated in pediatric heart failure because of dilated cardiomyopathy. These alterations support a pathophysiologically distinct mechanism of heart failure in pediatric patients with SRV, which has direct implications regarding the presumed response to PDE3i treatment in this population.
Assuntos
Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/metabolismo , Ventrículos do Coração/anormalidades , Milrinona/uso terapêutico , Miocárdio/metabolismo , Inibidores da Fosfodiesterase 3/uso terapêutico , Monofosfato de Adenosina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatia Dilatada/tratamento farmacológico , Criança , Pré-Escolar , Feminino , Transplante de Coração , Humanos , Lactente , Masculino , Diester Fosfórico Hidrolases/metabolismoRESUMO
cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/fisiologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Serina-Treonina Quinases TOR/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Isoquinolinas/farmacologia , Sistema de Sinalização das MAP Quinases , Ácido Okadáico/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Sirolimo/farmacologia , Sulfonamidas/farmacologiaRESUMO
The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of soluble mediators from resident cells (e.g., mast cells, macrophages) and/or recruited blood cells. The interaction of the mediators with receptors expressed on the surface of endothelial cells diminishes barrier function either by altering the expression of adhesive proteins in the inter-endothelial junctions, by altering the organization of the cytoskeleton, or both. Reactive oxygen species (ROS), proteolytic enzymes (e.g., matrix metalloproteinase, elastase), oncostatin M, and VEGF are part of a long list of mediators that have been implicated in endothelial barrier failure. In this review, we address the role of blood borne cells, including, neutrophils, lymphocytes, monocytes, and platelets, in the regulation of endothelial barrier function in health and disease. Attention is also devoted to new targets for therapeutic intervention in disease states with morbidity and mortality related to endothelial barrier dysfunction.
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The pedunculopontine nucleus (PPN) is a major component of the reticular activating system (RAS) that regulates waking and REM sleep, states of high-frequency EEG activity. Recently, we described the presence of high threshold, voltage-dependent N- and P/Q-type calcium channels in RAS nuclei that subserve gamma band oscillations in the mesopontine PPN, intralaminar parafascicular nucleus (Pf), and pontine subcoeruleus nucleus dorsalis (SubCD). Cortical gamma band activity participates in sensory perception, problem solving, and memory. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. That is, the RAS may play an early permissive role in volition. Our latest results suggest that (1) the manifestation of gamma band activity during waking may employ a separate intracellular pathway compared to that during REM sleep, (2) neuronal calcium sensor (NCS-1) protein, which is over expressed in schizophrenia and bipolar disorder, modulates gamma band oscillations in the PPN in a concentration-dependent manner, (3) leptin, which undergoes resistance in obesity resulting in sleep dysregulation, decreases sodium currents in PPN neurons, accounting for its normal attenuation of waking, and (4) following our discovery of electrical coupling in the RAS, we hypothesize that there are cell clusters within the PPN that may act in concert. These results provide novel information on the mechanisms controlling high-frequency activity related to waking and REM sleep by elements of the RAS.
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Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that modulate immune responses in various diseases. Due their complexity, standardized methods to identify their physicochemical properties and determine that production batches are biologically active must be established. We aimed to develop and validate a size exclusion ultra performance chromatographic (SE-UPLC) method to characterize Transferon™, a DLE that is produced under good manufacturing practices (GMPs). We analyzed an internal human DLE standard and 10 representative batches of Transferon™, all of which had a chromatographic profile characterized by 8 main peaks and a molecular weight range between 17.0 and 0.2kDa. There was high homogeneity between batches with regard to retention times and area percentages, varying by less than 0.2% and 30%, respectively, and the control chart was within 3 standard deviations. To analyze the biological activity of the batches, we studied the ability of Transferon™ to stimulate IFN-γ production in vitro. Transferon™ consistently induced IFN-γ production in Jurkat cells, demonstrating that this method can be included as a quality control step in releasing Transferon™ batches. Because all analyzed batches complied with the quality attributes that were evaluated, we conclude that the DLE Transferon™ is produced with high homogeneity.
Assuntos
Interferon gama/química , Adjuvantes Imunológicos/isolamento & purificação , Movimento Celular/efeitos dos fármacos , Quimiotaxia , Cromatografia Líquida de Alta Pressão , Citocinas/metabolismo , Humanos , Inflamação , Células Jurkat , Leucócitos/citologia , Peso Molecular , Peptídeos/química , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
UNLABELLED: The effect of long-term administration of imipramine, a tricyclic antidepressant, on the phosphorylation status of cyclic adenosine monophosphate-responsive element-binding protein (CREB), mitogen-activated protein kinase family members, and phospholipase γ-1 (PLCγ-1) was investigated in the dorsal horn of the spinal cord following peripheral nerve lesion. Nerve injury induced an ipsilateral long-lasting increased phosphorylation of CREB and PLCγ-1 but not extracellular signal-regulated kinase (ERK1,2), p38, and c-Jun N-terminal kinase. Daily administration of imipramine (5, 10, or 30 mg/kg) for 21 days progressively reduced both tactile-induced neuropathic pain hypersensitivity and thermal hyperalgesia. After withdrawal of treatment, the antinociceptive effect of imipramine was gradually abolished but still remained for at least 3 weeks. Conversely, no analgesic effect was observed with short-term imipramine treatment. Moreover, imipramine therapy reversed nerve injury-induced CREB and PLCγ-1 phosphorylation but had no effect on ERK1,2, p38, and c-Jun N-terminal kinase activity. These results indicate that long-term administration of imipramine may prevent some of the harmful changes in the spinal cord dorsal horn following nerve injury. However, imipramine analgesic effect takes time to develop and mature, which might explain in part why the clinical analgesic effect of tricyclic antidepressants develops with a delay after the beginning of treatment. Our data also provide evidence that prolonged imipramine treatment may induce antinociception in neuropathic pain conditions because of its action on the PLCγ-1/CREB-signaling pathway. PERSPECTIVE: This article demonstrates that long-term treatment with imipramine reverses some of the marked effects induced by peripheral nerve injury in the spinal dorsal horn that contribute to long-term maintenance of sensory disorder, providing a new view to the mechanisms of action of these drugs.
Assuntos
Antidepressivos/uso terapêutico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Fosfolipase C gama/metabolismo , Células do Corno Posterior/metabolismo , Animais , Antidepressivos Tricíclicos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Western Blotting , Imipramina/uso terapêutico , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Medição da Dor/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ciática/tratamento farmacológico , Ciática/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Learning and memory are basic functions of the brain that allowed human evolution. It is well accepted that during learning and memory formation the dynamic establishment of new active synaptic connections is crucial. Persistent synaptic activation leads to molecular events that include increased release of neurotransmitters, increased expression of receptors on the postsynaptic neuron, thus creating a positive feedback that results in the activation of distinct signaling pathways that temporally and permanently alter specific patterns of gene expression. However, the epigenetic changes that allow the establishment of long term genetic programs that control learning and memory are not completely understood. Even less is known regarding the signaling events triggered by synaptic activity that regulate these epigenetic marks. Here we review the current understanding of the molecular mechanisms controlling activity-dependent gene transcription leading synaptic plasticity and memory formation. We describe how Ca(2+) entry through N-methyl-d-aspartate-type glutamate neurotransmitter receptors result in the activation of specific signaling pathways leading to changes in gene expression, giving special emphasis to the recent data pointing out different epigenetic mechanisms (histone acetylation, methylation and phosphorylation as well as DNA methylation and hydroxymethylation) underlying learning and memory.
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Epigênese Genética/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Transcrição Gênica , Animais , Humanos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The cyclic adenosine 3, 5-monophosphate (cAMP) production was evaluated in liver thin sections of broiler chicks fed on a experimental diet containing bicalcium phosphate or Araxá rock phosphate (ARP) as source of P, with a high content of fluorine, at different ages: from the first to the 42nd and from the 21st to the 42nd day of age. The intake of the ARP formulated diet starting from birth elicited an increase of cAMP production in broiler liver. However, when this diet was offered after the 21st day of age, the hepatic cAMP production in broilers was not significantly (P>0.05) affected, suggesting that the effect of high fluorine present in Araxá rock phosphate, on hepatic cAMP of broiler chicks depends on the age in which the experimental diet is started.
A produção de adenosina 3, 5- monofosfato cíclico (AMPc) foi avaliada em fatias de fígado de frangos de corte que receberam dieta experimental contendo fosfato bicálcico ou fosfato de rocha de Araxá, com alto teor de flúor, como fonte de fósforo, em dois períodos distintos: do 1º ao 42º e do 21º ao 42º dias de idade. A ingestão da dieta formulada com fosfato de rocha de Araxá desde o 1º dia de vida, provocou elevação da produção de AMPc no fígado dos frangos de corte. Entretanto, a utilização da mesma dieta a partir do 21º de idade não alterou significativamente (P>0,05) a produção hepática de AMPc nas aves, sugerindo que a influência da alta ingestão do flúor presente no fosfato de rocha de Araxá sobre o AMPc hepático dos frangos de corte é dependente da idade em que a administração da dieta é iniciada.