RESUMO
This study aimed to re-examine the receptor subtype that mediates the fever-producing effects of prostaglandin E2 (PGE2) in the rostral ventromedial preoptic area (rvmPOA) of the hypothalamus. Among the four subtypes of PGE2 receptors (EP1, EP2, EP3, and EP4), EP3 receptor is crucially involved in the febrile effects of PGE2. However, it is possible for other subtypes of PGE2 receptor to contribute in the central mechanism of fever generation. Accordingly, effects of microinjection of PGE2 receptor subtype-specific agonists or antagonists were examined at the locus where a microinjection of a small amount (420 fmol) of PGE2 elicited prompt increases in the O2 consumption rate (VO2), heart rate, and colonic temperature (Tc) in the rvmPOA of urethane-chloralose-anesthetized rats. The EP3 agonist sulprostone mimicked, whereas its antagonist L-798,106 reduced, the febrile effects of PGE2 microinjected into the same site. Similarly, the EP4 agonist rivenprost mimicked, whereas its antagonist ONO-AE3-208 reduced, the effects of PGE2 microinjected into the same site. In contrast, microinjection of the EP1 agonist iloprost induced a very small increase in VO2 but did not have significant influences on the heart rate and Tc, whereas its antagonist, AH6809, did not affect the PGE2-induced responses. Microinjection of the EP2 agonist butaprost had no effects on the VO2, heart rate, and Tc. The results suggest that the EP3 and EP4 receptor subtypes are both involved in the fever generated by PGE2 in the rvmPOA.
Assuntos
Área Pré-Óptica , Receptores de Prostaglandina E , Animais , Hipotálamo/metabolismo , Área Pré-Óptica/metabolismo , Ratos , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP4RESUMO
Hypothalamic feeding circuits have been identified as having innate synaptic plasticity, mediating adaption to the changing metabolic milieu by controlling responses to feeding and obesity. However, less is known about the regulatory principles underlying the dynamic changes in agouti-related protein (AgRP) perikarya, a region crucial for gating of neural excitation and, hence, feeding. Here we show that AgRP neurons activated by food deprivation, ghrelin administration, or chemogenetics decreased their own inhibitory tone while triggering mitochondrial adaptations in neighboring astrocytes. We found that it was the inhibitory neurotransmitter GABA released by AgRP neurons that evoked this astrocytic response; this in turn resulted in increased glial ensheetment of AgRP perikarya by glial processes and increased excitability of AgRP neurons. We also identified astrocyte-derived prostaglandin E2, which directly activated - via EP2 receptors - AgRP neurons. Taken together, these observations unmasked a feed-forward, self-exciting loop in AgRP neuronal control mediated by astrocytes, a mechanism directly relevant for hunger, feeding, and overfeeding.
Assuntos
Proteína Relacionada com Agouti , Astrócitos/metabolismo , Fome , Hipotálamo/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , Masculino , Camundongos , Camundongos Transgênicos , Receptores de Prostaglandina E Subtipo EP2/metabolismoRESUMO
To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.
Assuntos
Adipogenia/efeitos dos fármacos , Alprostadil/análogos & derivados , Glicina/análogos & derivados , Isoquinolinas/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Sulfonamidas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Células 3T3-L1 , Alprostadil/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Glicina/farmacologia , Camundongos , Organoides , Receptores de Prostaglandina E Subtipo EP2/agonistasRESUMO
Exposure to manganese (Mn) can cause male reproductive damage and lead to abnormal secretion of sex hormones. Gonadotropin-releasing hormone (GnRH) plays an important role in the neuromodulation of vertebrate reproduction. Astrocytes can indirectly regulate the secretion of GnRH by binding paracrine prostaglandin E2 (PGE2) specifically to the EP1 and EP2 receptors on GnRH neurons. Prior studies assessed the abnormal secretion of GnRH caused by Mn exposure, but the specific mechanism has not been reported in detail. This study investigated the effects of Mn exposure on the reproductive system of male mice to clarify the role of PGE2 in the abnormal secretion of GnRH in the hypothalamus caused by exposure to Mn. Our data demonstrate that antagonizing the EP1 and EP2 receptors of PGE2 can restore abnormal levels of GnRH caused by Mn exposure. Mn exposure causes reduced sperm count and sperm shape deformities. These findings suggest that EP1 and EP2, the receptors of PGE2, may be the key to abnormal GnRH secretion caused by Mn exposure. Antagonizing the PGE2 receptors may reduce reproductive damage caused by Mn exposure.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/efeitos dos fármacos , Manganês/toxicidade , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Reprodução/efeitos dos fármacos , Animais , Hipotálamo/metabolismo , Masculino , Manganês/metabolismo , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de Prostaglandina E Subtipo EP1/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidoresRESUMO
The inflammatory prostaglandin E2 (PGE2) EP2 receptor is a master suppressor of beneficial microglial function, and myeloid EP2 signaling ablation reduces pathology in models of inflammatory neurodegeneration. Here, we investigated the role of PGE2 EP2 signaling in a model of stroke in which the initial cerebral ischemic event is followed by an extended poststroke inflammatory response. Myeloid lineage cell-specific EP2 knockdown in Cd11bCre;EP2lox/lox mice attenuated brain infiltration of Cd11b+CD45hi macrophages and CD45+Ly6Ghi neutrophils, indicating that inflammatory EP2 signaling participates in the poststroke immune response. Inducible global deletion of the EP2 receptor in adult ROSA26-CreERT2 (ROSACreER);EP2lox/lox mice also reduced brain myeloid cell trafficking but additionally reduced stroke severity, suggesting that nonimmune EP2 receptor-expressing cell types contribute to cerebral injury. EP2 receptor expression was highly induced in neurons in the ischemic hemisphere, and postnatal deletion of the neuronal EP2 receptor in Thy1Cre;EP2lox/lox mice reduced cerebral ischemic injury. These findings diverge from previous studies of congenitally null EP2 receptor mice where a global deletion increases cerebral ischemic injury. Moreover, ROSACreER;EP2lox/lox mice, unlike EP2-/- mice, exhibited normal learning and memory, suggesting a confounding effect from congenital EP2 receptor deletion. Taken together with a precedent that inhibition of EP2 signaling is protective in inflammatory neurodegeneration, these data lend support to translational approaches targeting the EP2 receptor to reduce inflammation and neuronal injury that occur after stroke.
Assuntos
Isquemia Encefálica/metabolismo , Dinoprostona/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Animais , Isquemia Encefálica/imunologia , Isquemia Encefálica/prevenção & controle , Cognição , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Imunidade Inata , Masculino , Camundongos Endogâmicos C57BL , Células Mieloides/fisiologia , Neurônios/metabolismo , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidoresRESUMO
BACKGROUND: Osthole (7-methoxy-8-isopentenylcoumarin) is natural coumarin isolated from the fruit of Cnidium monnieri (L.) Cusson, which is commonly used in medical practice of traditional Chinese medicine (TCM) in various diseases including allergies and asthma disorders. PURPOSE: Osthole was tested for the anti-histamine, anti-allergic, and inhibitory effects of COX-2 (cyclooxygenase-2) in children with diagnosed allergies. Additionally, we hypothesize that stated alterations in children with diagnosed allergies including increased expression of interleukin 1-ß receptor type 1 (IL-1 type I) and E-prostanoid (EP) 2 receptors, as well as raised expression, production, and activity of COX-2 and IL-1ß in incubated medium are approximately connected. Furthermore, we establish the mechanisms included in the changed regulation of the COX-2 pathway and determine whether osthole may be COX-2 inhibitor in peripheral blood mononuclear cells (PBMCs). METHOD: PBMCs were obtained from peripheral blood of healthy children (control, n = 28) and patients with diagnosed allergies (allergy, n = 30). Expression of the autocrine loop components regulating PGE2 production and signaling namely IL-1 type I receptor (IL-1RI), cyclooksygenaze-2 (COX-2), E-prostanoid (EP) 2, and also histamine receptor-1 (HRH-1) was assessed at baseline and after stimulation with histamine, osthole, and a mixture of histamine/osthole 1:2 (v/v). This comprised the expression of histamine receptor 1 (HRH-1), IL-1RI, COX-2, EP2 receptor, and the secretion of IL-1ß and COX-2 in cultured media and sera. RESULTS: Compared with control group, basal mRNA expression levels of HRH-1, IL-1RI, COX-2, and EP2 were higher in the allergy group. Histamine-induced EP2 and COX-2 expression mRNA levels were also increased. CONCLUSIONS: Osthole successively inhibits PGE2 and COX-2 mRNA expression. Furthermore, osthole reduces the secretion of COX-2 protein in signaling cellular mechanisms. Changed EP2 expression in children with allergies provides higher IL-1RI induction, increasing IL-1ß capacity to increase COX-2 expression. This effects in higher PGE2 production, which in turn increases its capability to induce IL-1RI.
Assuntos
Cumarínicos/farmacologia , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Interleucina-1beta/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Cumarínicos/química , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Histamina/química , Histamina/farmacologia , Humanos , Hipersensibilidade/genética , Hipersensibilidade/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Receptores Tipo I de Interleucina-1/genética , Receptores de Prostaglandina E Subtipo EP2/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Pathological features of pulmonary fibrosis include accumulation of myofibroblasts and increased extracellular matrix (ECM) deposition in lung tissue. Contractile α-smooth muscle actin (α-SMA)-expressing myofibroblasts that produce and secrete ECM are key effector cells of the disease and therefore represent a viable target for potential novel anti-fibrotic treatments. We used primary normal human lung fibroblasts (NHLF) in two novel high-throughput screening assays to discover molecules that inhibit or revert fibroblast-to-myofibroblast differentiation. A phenotypic high-content assay (HCA) quantified the degree of myofibroblast differentiation, whereas an impedance-based assay, multiplexed with MS / MS quantification of α-SMA and collagen 1 alpha 1 (COL1) protein, provided a measure of contractility and ECM formation. The synthetic prostaglandin E1 (PGE1) alprostadil, which very effectively and potently attenuated and even reversed TGF-ß1-induced myofibroblast differentiation, was identified by screening a library of approved drugs. In TGF-ß1-induced myofibroblasts the effect of alprostadil was attributed to activation of prostanoid receptor 2 and 4 (EP2 and EP4, respectively). However, selective activation of the EP2 or the EP4 receptor was already sufficient to prevent or reverse TGF-ß1-induced NHLF myofibroblast transition. Our high-throughput assays identified chemical structures with potent anti-fibrotic properties acting through potentially novel mechanisms.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP4/agonistas , Desdiferenciação Celular/efeitos dos fármacos , Feminino , Humanos , Pessoa de Meia-Idade , Miofibroblastos/patologia , Fenótipo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Aprendizado de Máquina SupervisionadoRESUMO
The prostaglandin E2 receptor, EP2, plays an important role in physiology and in a variety of pathological conditions. Studies indicate that EP2 is pro-inflammatory in chronic peripheral and central nervous system disease and cancer models. Thus, targeting the EP2 receptor with small molecules could be a therapeutic strategy for treating inflammatory diseases and cancer. We recently reported a novel class of competitive antagonists of the EP2 receptor. However, earlier leads displayed low selectivity against the DP1 prostanoid receptor, moderate plasma half-life, and low aqueous solubility, which renders them suboptimal for testing in animal models of disease. We now report a novel compound TG8-69, which has suitable drug-like properties. We present synthesis, lead-optimization studies, pharmacological characterization, and anti-inflammatory properties of this compound that support its use in chronic peripheral inflammatory diseases, including rheumatoid arthritis, endometriosis, and cancer, in which EP2 appears to play a pathogenic role.
Assuntos
Anti-Inflamatórios/farmacologia , Mediadores da Inflamação/metabolismo , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Regulação para Cima/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Linhagem Celular , Dinoprostona/imunologia , Dinoprostona/metabolismo , Avaliação Pré-Clínica de Medicamentos , Endometriose/tratamento farmacológico , Endometriose/imunologia , Feminino , Meia-Vida , Mediadores da Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Ratos , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina E Subtipo EP2/imunologia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Solubilidade , Regulação para Cima/imunologia , Água/químicaRESUMO
Considering possible effects of poly-unsaturated fatty acids (PUFA) on embryo implantation more likely through PGs, we investigated effects of dietary omega-3 and -6 PUFA on prostaglandin E2 (PGE2) signaling in mice uterus during pre-implantation period. The mRNA expressions of microsomal- and cytosolic- PGE synthase (mPGES and cPGES) and protein expressions of PGE receptor 2 and 4 (EP2 and EP4) were evaluated in uterus tissues of control as well as omega 3 and omega 6 received mice at days 1-5 of pregnancy. Expression of cPGES gene was not significantly different between groups but the mPGES expression on days 4 and 5 of pregnancy in supplemented groups was higher than controls. Omega-3 significantly decreased EP2 levels on days 3 and 4, while omega-6 caused an increase on days 3-5 of pregnancy. The levels of EP4 were significantly higher in the omega-6 group than other groups on days 4 and 5 of pregnancy. Also the implantation rate was higher in omega -6 compared to omega-3 group (pâ¯=â¯0.006). Moreover, there were significant correlations between implantation rate with expression levels of mPGES and EP2. Our results showed negative and positive effects of respectively dietary omega-3 and -6 PUFA on PGE2 signaling and implantation rate.
Assuntos
Implantação do Embrião/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Prostaglandina-E Sintases/biossíntese , Receptores de Prostaglandina E Subtipo EP2/biossíntese , Útero/efeitos dos fármacos , Útero/metabolismo , Animais , Suplementos Nutricionais , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-6/administração & dosagem , Feminino , Masculino , CamundongosRESUMO
Polymicrobial intra-abdominal infections (IAI) involving Candida albicans and Staphylococcus aureus are associated with severe morbidity and mortality (â¼80%). Our laboratory discovered that the immunomodulatory eicosanoid prostaglandin E2 (PGE2) plays a key role in the lethal inflammatory response during polymicrobial IAI using a mouse model of infection. In studies designed to uncover key PGE2 biosynthesis/signaling components involved in the response, selective eicosanoid enzyme inhibitors and receptor antagonists were selected and prescreened for antimicrobial activity against C. albicans or S. aureus Unexpectedly, we found that the EP4 receptor antagonist L-161,982 had direct growth-inhibitory effects on S. aureusin vitro at the physiological concentration required to block the PGE2 interaction with EP4 This antimicrobial activity was observed with methicillin-sensitive S. aureus and methicillin-resistant S. aureus (MRSA) strains, with the MIC and minimum bactericidal concentration values for planktonic cells being 50 µg/ml and 100 µg/ml, respectively. In addition, L-161,982 inhibited S. aureus biofilm formation and had activity against preformed mature biofilms. More importantly, treatment of mice with L-161,982 following intraperitoneal inoculation with a lethal dose of MRSA significantly reduced the bioburden and enhanced survival. Furthermore, L-161,982 protected mice against the synergistic lethality induced by coinfection with C. albicans and S. aureus The antimicrobial activity of L-161,982 is independent of EP4 receptor inhibitory activity; an alternative EP4 receptor antagonist exerted no antimicrobial or protective effects. Taken together, these findings demonstrate that L-161,982 has potent antimicrobial activity against MRSA and may represent a significant therapeutic alternative in improving the prognosis of mono- or polymicrobial infections involving MRSA.
Assuntos
Antibacterianos/uso terapêutico , Anti-Infecciosos/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Animais , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Feminino , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Testes de Sensibilidade Microbiana , Peritonite/tratamento farmacológico , Peritonite/microbiologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidadeRESUMO
Epoxyeicosatrienoic acids (EETs) are potent vasodilators that play important roles in cardiovascular physiology and disease, yet the molecular mechanisms underlying the biological actions of EETs are not fully understood. Multiple lines of evidence suggest that the actions of EETs are in part mediated via G protein-coupled receptor (GPCR) signaling, but the identity of such a receptor has remained elusive. We sought to identify 14,15-EET-responsive GPCRs. A set of 105 clones were expressed in Xenopus oocyte and screened for their ability to activate cAMP-dependent chloride current. Several receptors responded to micromolar concentrations of 14,15-EET, with the top five being prostaglandin receptor subtypes (PTGER2, PTGER4, PTGFR, PTGDR, PTGER3IV). Overall, our results indicate that multiple low-affinity 14,15-EET GPCRs are capable of increasing cAMP levels following 14,15-EET stimulation, highlighting the potential for cross-talk between prostanoid and other ecosanoid GPCRs. Our data also indicate that none of the 105 GPCRs screened met our criteria for a high-affinity receptor for 14,15-EET.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Receptores Acoplados a Proteínas G/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Ácido 8,11,14-Eicosatrienoico/farmacologia , Animais , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Avaliação Pré-Clínica de Medicamentos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Camundongos , Oócitos/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Vasoconstrição/efeitos dos fármacos , Xenopus , beta-Arrestinas/metabolismoRESUMO
Heme (Fe(2+) protoporphyrin IX) and hemin (Fe(3+)), the prosthetic group of hemoprotein, are cytotoxic due to their ability to contribute to the production of reactive oxygen species, increased intracellular calcium levels, and stimulate glutamate-mediated excitotoxicity. Previous work by our group showed that blockade of the prostaglandin E2 (PGE2)-EP1 receptor reduced hemin-induced cytotoxicity in primary cortical neuronal cultures. However, the role of the prostaglandin E2 (PGE2)-EP2 receptor in hemin neurotoxicity remains unclear. Activation of the EP2 receptor in neurons results in increased cyclic AMP (cAMP) and protein kinase A signaling; therefore, we hypothesized that the activation of the EP2 receptor decreases hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wildtype-control (WT) and EP2(-/-) mice, we investigated the role of the EP2 receptor in hemin neurotoxicity by monitoring cell survival with the Calcein-AM live-cell and lactate dehydrogenase assays. MitoTracker staining was also performed to determine how mitochondria were affected by hemin. Hemin neurotoxicity in EP2(-/-) neurons was 37.2 ± 17.0% greater compared to WT neurons. Of interest, cotreatment with the EP2 receptor agonist, butaprost (1 and 10 µM), significantly attenuated hemin neurotoxicity by 55.7 ± 21.1% and 60.1 ± 14.8%, respectively. To further investigate signaling mechanisms related to EP2 receptor mediating cytoprotection, neurons were cotreated with hemin and activators/inhibitors of both the cAMP-protein kinase A/exchange protein directly activated by cAMP (Epac) pathways. Forskolin, a cAMP activator, and 8-pCPT-cAMP, an Epac activator, both attenuated hemin neurotoxicity by 78.8 ± 22.2% and 58.4 ± 9.8%, respectively, as measured using the lactate dehydrogenase assay. Together, the results reveal that activation of the EP2 receptor is protective against hemin neurotoxicity in vitro and these findings suggest that neuroprotection occurs through the cAMP-Epac pathway in neuronal cultures. Therefore, activation of the EP2 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.
Assuntos
Hemina/toxicidade , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Adjuvantes Imunológicos/farmacologia , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , L-Lactato Desidrogenase/metabolismo , Camundongos , Camundongos Knockout , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/genética , Tionucleotídeos/farmacologiaRESUMO
Incidence of kidney cancer is on the rise, and a better understanding of molecular mechanisms involved in the cancer invasion and metastasis is required for the development of curative therapeutics. In this study, we report that the proinflammatory cytokine prostaglandin E2 (PGE2) induces the malignant SN12C, but not benign HK2 kidney cell invasion. The PGE2 increases SN12C cell invasion through a signal pathway that encompasses EP2 and EP4, Akt, small GTPase RalA and Ral·GTP inactivator RGC2. The results support the idea that targeted interference of EP2/EP4 signal to RalA·GTP may provide benefit to patients diagnosed with advanced kidney cancer.
Assuntos
Carcinoma de Células Renais/patologia , Dinoprostona/farmacologia , Neoplasias Renais/patologia , Proteínas ral de Ligação ao GTP/fisiologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , RNA Interferente Pequeno/farmacologia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP2/fisiologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Receptores de Prostaglandina E Subtipo EP4/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas ral de Ligação ao GTP/antagonistas & inibidores , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismoAssuntos
Degranulação Celular/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Imunoglobulina E/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transdução de Sinais , Animais , Feminino , HumanosRESUMO
To identify topically effective EP4 agonists and EP2/EP4 dual agonists with excellent subtype selectivity, further optimization of the 16-phenyl ω-chain moiety of the γ-lactam 5-thia prostaglandin E analog and the 2-mercaptothiazole-4-carboxylic acid analog were undertaken. Rat in vivo evaluation of these newly identified compounds as their poly (lactide-co-glycolide) microsphere formulation, from which sustained release of the test compound is possible, led us to discover compounds that showed efficacy in a rat bone fracture healing model after its topical administration without serious influence on blood pressure and heart rate. A structure-activity relationship study is also presented.
Assuntos
Lactamas/síntese química , Lactamas/farmacologia , Prostaglandinas E Sintéticas/síntese química , Prostaglandinas E Sintéticas/farmacologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP4/agonistas , Administração Tópica , Animais , Pressão Sanguínea/efeitos dos fármacos , Células CHO , Cricetinae , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Dinoprostona/química , Avaliação Pré-Clínica de Medicamentos/métodos , Consolidação da Fratura/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Lactamas/administração & dosagem , Masculino , Camundongos , Microesferas , Estrutura Molecular , Poliglactina 910/administração & dosagem , Poliglactina 910/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tiazolidinas/químicaRESUMO
To identify potent EP2/EP4 dual agonists with excellent subtype selectivity, a series of γ-lactam prostaglandin E analogs bearing a 16-phenyl ω-chain were synthesized and evaluated. Structural hybridization of 1 and 2, followed by more detailed chemical modification of the benzoic acid moiety, led us to the discovery of a 2-mercaptothiazole-4-carboxylic acid analog 3 as the optimal compound in the series. An isomer of this compound, the 2-mercaptothiazole-5-carboxylic acid analog 13, showed 34-fold and 13-fold less potent EP2 and EP4 receptor affinities, respectively. Structure activity relationship data from an in vitro mouse receptor binding assay are presented. Continued evaluation in an in vivo rat model of another 2-mercaptothiazole-4-carboxylic acid analog 17, optimized for sustained compound release from PLGA microspheres, demonstrated its effectiveness in a rat bone fracture-healing model following topical administration.
Assuntos
Prostaglandinas Sintéticas/química , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP4/agonistas , Tiazolidinas/química , Administração Tópica , Animais , Células CHO , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Fraturas Ósseas/tratamento farmacológico , Isomerismo , Camundongos , Prostaglandinas Sintéticas/síntese química , Prostaglandinas Sintéticas/uso terapêutico , Ratos , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Relação Estrutura-Atividade , Tiazolidinas/síntese química , Tiazolidinas/uso terapêuticoRESUMO
Melanoma is the most serious type of skin disease and a leading cause of death from skin disease due to its highly metastatic ability. To develop more effective chemopreventive agents for the prevention of melanoma, we have determined the effect of green tea catechins on the invasive potential of human melanoma cells and the molecular mechanisms underlying these effects using A375 (BRAF-mutated) and Hs294t (Non-BRAF-mutated) melanoma cell lines as an in vitro model. Employing cell invasion assays, we found that the inhibitory effects of green tea catechins on the cell migration were in the order of (-)-epigallocatechin-3-gallate (EGCG)>(-)-epigallocatechin>(-)-epicatechin-3-gallate>(-)-gallocatechin>(-)-epicatechin. Treatment of A375 and Hs294t cells with EGCG resulted in a dose-dependent inhibition of cell migration or invasion of these cells, which was associated with a reduction in the levels of cyclooxygenase (COX)-2, prostaglandin (PG) E(2) and PGE(2) receptors (EP2 and EP4). Treatment of cells with celecoxib, a COX-2 inhibitor, also inhibited melanoma cell migration. EGCG inhibits 12-O-tetradecanoylphorbol-13-acetate-, an inducer of COX-2, and PGE(2)-induced cell migration of cells. EGCG decreased EP2 agonist (butaprost)- and EP4 agonist (Cay10580)-induced cell migration ability. Moreover, EGCG inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A375 melanoma cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited cell migration. Inhibition of melanoma cell migration by EGCG was associated with transition of mesenchymal stage to epithelial stage, which resulted in an increase in the levels of epithelial biomarkers (E-cadherin, cytokeratin and desmoglein 2) and a reduction in the levels of mesenchymal biomarkers (vimentin, fibronectin and N-cadherin) in A375 melanoma cells. Together, these results indicate that EGCG, a major green tea catechin, has the ability to inhibit melanoma cell invasion/migration, an essential step of metastasis, by targeting the endogenous expression of COX-2, PGE(2) receptors and epithelial-to-mesenchymal transition.
Assuntos
Catequina/farmacologia , Ciclo-Oxigenase 2/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Melanoma/enzimologia , Melanoma/patologia , Receptores de Prostaglandina E/metabolismo , Chá/química , Catequina/análogos & derivados , Catequina/química , Celecoxib , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/biossíntese , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , NF-kappa B/metabolismo , Invasividade Neoplásica , Pirazóis/farmacologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Sulfonamidas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição RelA/metabolismoRESUMO
Better control of intraocular pressure (IOP) is the most effective way to preserve visual field function in glaucomatous patients. While prostaglandin FP analogs are leading the therapeutic intervention for glaucoma, new target classes also are being identified with new lead compounds being developed for IOP reduction. One target class currently being investigated includes the prostaglandin EP receptor agonists. Recently PF-04217329 (Taprenepag isopropyl), a prodrug of CP-544326 (active acid metabolite), a potent and selective EP(2) receptor agonist, was successfully evaluated for its ocular hypotensive activity in a clinical study involving patients with primary open angle glaucoma. In the current manuscript, the preclinical attributes of CP-544326 and PF-0421329 have been described. CP-544326 was found to be a potent and selective EP(2) agonist (IC(50) = 10 nM; EC(50) = 2.8 nM) whose corneal permeability and ocular bioavailability were significantly increased when the compound was dosed as the isopropyl ester prodrug, PF-04217329. Topical ocular dosing of PF-04217329 was well tolerated in preclinical species and caused an elevation of cAMP in aqueous humor/iris-ciliary body indicative of in vivo EP(2) target receptor activation. Topical ocular dosing of PF-04217329 resulted in ocular exposure of CP-544326 at levels greater than the EC(50) for the EP(2) receptor. PF-04217329 when dosed once daily caused between 30 and 50% IOP reduction in single day studies in normotensive Dutch-belted rabbits, normotensive dogs, and laser-induced ocular hypertensive cynomolgus monkeys and 20-40% IOP reduction in multiple day studies compared to vehicle-dosed eyes. IOP reduction was sustained from 6 h through 24 h following a single topical dose. In conclusion, preclinical data generated thus far appear to support the clinical development of PF-04217329 as a novel compound for the treatment of glaucoma.
Assuntos
Acetatos/farmacologia , Anti-Hipertensivos/farmacologia , Modelos Animais de Doenças , Glaucoma de Ângulo Aberto/tratamento farmacológico , Glaucoma/tratamento farmacológico , Pressão Intraocular/efeitos dos fármacos , Pró-Fármacos/farmacologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Sulfonamidas/farmacologia , Acetatos/farmacocinética , Administração Tópica , Animais , Anti-Hipertensivos/farmacocinética , Humor Aquoso/metabolismo , Disponibilidade Biológica , Cálcio/metabolismo , Corpo Ciliar/metabolismo , Córnea/metabolismo , AMP Cíclico/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Glaucoma/metabolismo , Humanos , Iris/metabolismo , Macaca fascicularis , Masculino , Soluções Oftálmicas/farmacocinética , Soluções Oftálmicas/farmacologia , Pró-Fármacos/farmacocinética , Coelhos , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Sulfonamidas/farmacocinética , Tonometria OcularRESUMO
A novel series of EP(4) ligands, based on a benzyl indoline scaffold, has been discovered. It was found that agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl group. The pharmacokinetic, metabolic and pharmacological profiles of these compounds was explored. It was found that these compounds show good pharmacokinetics in rat and are efficacious in pre-clinical models of pain and inflammation.
Assuntos
Indóis/química , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Indóis/farmacocinética , Indóis/uso terapêutico , Ligantes , Ratos , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Relação Estrutura-AtividadeRESUMO
Activation of the Galphas-coupled EP2 receptor for prostaglandin E2 (PGE(2)) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 microM (i) potentiated the cAMP response to a low concentration of PGE(2) by > 50%; (ii) had no effect on EP4 or beta2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE(2) on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE(2) on EP2 receptors 4- to 5-fold at 10 to 20 microM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE(2) receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE(2) released in a cell-injury setting is neuroprotective.