RESUMEN
RBM12 is a high-penetrance risk factor for familial schizophrenia and psychosis, yet its precise cellular functions and the pathways to which it belongs are not known. We utilize two complementary models, HEK293 cells and human iPSC-derived neurons, and delineate RBM12 as a novel repressor of the G protein-coupled receptor/cAMP/PKA (GPCR/cAMP/PKA) signaling axis. We establish that loss of RBM12 leads to hyperactive cAMP production and increased PKA activity as well as altered neuronal transcriptional responses to GPCR stimulation. Notably, the cAMP and transcriptional signaling steps are subject to discrete RBM12-dependent regulation. We further demonstrate that the two RBM12 truncating variants linked to familial psychosis impact this interplay, as the mutants fail to rescue GPCR/cAMP signaling hyperactivity in cells depleted of RBM12. Lastly, we present a mechanism underlying the impaired signaling phenotypes. In agreement with its activity as an RNA-binding protein, loss of RBM12 leads to altered gene expression, including that of multiple effectors of established significance within the receptor pathway. Specifically, the abundance of adenylyl cyclases, phosphodiesterase isoforms, and PKA regulatory and catalytic subunits is impacted by RBM12 depletion. We note that these expression changes are fully consistent with the entire gamut of hyperactive signaling outputs. In summary, the current study identifies a previously unappreciated role for RBM12 in the context of the GPCR-cAMP pathway that could be explored further as a tentative molecular mechanism underlying the functions of this factor in neuronal physiology and pathophysiology.
Asunto(s)
AMP Cíclico , Neuronas , Trastornos Psicóticos , Proteínas de Unión al ARN , Transducción de Señal , Humanos , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Trastornos Psicóticos/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genética , Neuronas/fisiología , Regulación Enzimológica de la Expresión Génica/genéticaRESUMEN
The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape.
Asunto(s)
Inhibidores de Adenilato Ciclasa/farmacología , Adenilil Ciclasas/genética , Antineoplásicos/farmacología , AMP Cíclico/antagonistas & inhibidores , Melanoma Experimental/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Inhibidores de Adenilato Ciclasa/síntesis química , Adenilil Ciclasas/inmunología , Animales , Antineoplásicos/síntesis química , Compuestos de Bencilo/química , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Ésteres , Femenino , Expresión Génica , Humanos , Inmunidad Innata/efectos de los fármacos , Inyecciones Intralesiones , Proteína Antagonista del Receptor de Interleucina 1/genética , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Micelas , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Células Mieloides/patología , Péptidos/química , Ácido Poliglutámico/química , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Sarcosina/análogos & derivados , Sarcosina/química , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Carga Tumoral/efectos de los fármacos , Escape del Tumor/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Abnormal melanogenesis and melanosome transport can cause skin pigmentation disorders that are often treated using ginseng-based formulation. We previously found that phenolic acid compounds in ginseng root could inhibit melanin production and as a skin-whitening agents. However, mechanisms of action underlying effects of ginseng phenolic acid monomers on melanogenesis remain unclear. This study was conducted to investigate effects of salicylic acid, a main ginseng root phenolic acid component, on melanogenesis and melanosome functions in melanocytes of zebrafish and other species. Salicylic acid exhibited no cytotoxicity and reduced melanin levels and tyrosinase activity in B16F10 murine melanoma cells and normal human epidermal melanocytes regardless of prior cell stimulation with α-melanocyte stimulating hormone. Additionally, salicylic acid treatment reduced expression of melanogenic enzymes tyrosinase, tyrosinase-related protein 1 and tyrosinase-related protein 2, while reducing expression of their master transcriptional regulator, microphthalmia-associated transcription factor. Moreover, reduced phosphorylation of cAMP response-element binding protein was observed due to reduced cAMP levels resulting from salicylic acid inhibition of upstream signal regulators (adenylyl cyclase and protein kinase A). Furthermore, salicylic acid treatment suppressed expression of transport complex-associated proteins melanophilin and myosin Va in two UVB-treated melanocytic cell lines, suppressed phagocytosis of fluorescent microspheres by UVB-stimulated human keratinocytes (HaCaT), inhibited protease-activated receptor 2 activation by reducing both Ca2+ release and activation of phosphoinositide 3 kinase/AKT and mitogen-activated protein kinases and induced anti-melanogenic effects in zebrafish. Collectively, these results indicate that salicylic acid within ginseng root can inhibit melanocyte melanogenesis and melanin transport, while also suppressing keratinocyte phagocytic function.
Asunto(s)
Hiperpigmentación/tratamiento farmacológico , Melaninas/metabolismo , Melanosomas/metabolismo , Panax/química , Ácido Salicílico/farmacología , Animales , Calcio/metabolismo , Línea Celular , AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Queratinocitos/efectos de los fármacos , Melaninas/antagonistas & inhibidores , Melanocitos/efectos de los fármacos , Melanosomas/efectos de los fármacos , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Oxidorreductasas/metabolismo , Fagocitosis/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptor PAR-2/metabolismo , Transducción de Señal/efectos de los fármacos , Rayos Ultravioleta , Pez Cebra , alfa-MSH/farmacologíaRESUMEN
The present study investigated hyperalgesia during sickness syndrome in female rats. Hyperalgesia was induced by an intraperitoneal injection of lipopolysaccharide (LPS) or an intracerebroventricular injection of prostaglandin E2 (PGE2). No differences were found in basal mechanical and thermal thresholds or in LPS-induced hyperalgesia in sham-operated animals in the diestrus or proestrus phase or in ovariectomized (OVX) animals. However, higher levels of PGE2 where found in the cerebrospinal fluid of OVX animals compared to sham-operated females. Intracerebroventricular injection of PGE2 produced rapid mechanical hyperalgesia in sham-operated rats while these responses were observed at later times in OVX animals. The protein kinase A (PKA) inhibitor H-89 reduced mechanical PGE2-induced hyperalgesia in OVX female rats, whereas no effect was observed in sham-operated animals. In contrast, the exchange protein activated by cyclic adenosine monophosphate (cAMP; Epac) inhibitor ESI-09 reduced mechanical PGE2-induced hyperalgesia, whereas no effect was observed in OVX animals. PGE2 also induced thermal hyperalgesia in sham-operated and OVX female rats and a similar effect of ESI-09 was observed. These results suggest that PGE2-induced hyperalgesia that is observed during sickness syndrome has different signaling mechanisms in cycling and OVX female rats involving the activation of the cAMP-Epac or cAMP-PKA pathways, respectively.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Dinoprostona/farmacología , Ciclo Estral/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Conducta de Enfermedad/efectos de los fármacos , Animales , AMP Cíclico/antagonistas & inhibidores , Dinoprostona/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Hidrazonas/farmacología , Isoquinolinas/farmacología , Isoxazoles/farmacología , Lipopolisacáridos/farmacología , Ovariectomía , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacologíaRESUMEN
Immunosuppression is one of the main mechanisms facilitating tumor expansion. It may be driven by immune checkpoint protein expression, anti-inflammatory cytokine secretion or enhanced metabolic enzyme production, leading to the subsequent build-up of metabolites such as adenosine. Under physiological conditions, adenosine prevents the development of tissue damage resulting from a prolonged immune response; the same mechanism might be employed by tumor tissue to promote immunosuppression. Immune cells expressing A2A and A2B adenosine receptors present in an adenosine-rich environment have suppressed effector functions, such as cytotoxicity, proinflammatory cytokine release, antigen presentation and others, making them inert to cancer cells. This study was designed to investigate the dual antagonist potential of SEL330-639 to abolish adenosine-driven immunosuppression. SEL330-639 has slow dissociation kinetics. It inhibits cAMP production in human CD4+ cells, CD8+ cells and moDCs, which leads to diminished CREB phosphorylation and restoration of antitumor cytokine production (IL-2, TNFα, IL-12) in multiple primary human immune cells. The aforementioned results were additionally validated by gene expression analysis and functional assays in which NK cell line cytotoxicity was recovered by SEL330-639. Adenosine-driven immunosuppression is believed to preclude the effectiveness of immune checkpoint inhibitor therapies. Hence, there is an urgent need to develop new immuno-oncological strategies. Here, we comprehensively characterize SEL330-639, a novel dual A2A/A2B receptor antagonist effective in both lymphoid and myeloid cell populations with nanomolar potency. Due to its tight binding to the A2A and A2B receptors, this binding is sustained even at high adenosine concentrations mimicking the upper limit of the range of adenosine levels observed in the tumor microenvironment.
Asunto(s)
Antagonistas del Receptor de Adenosina A2/farmacología , Adenosina/inmunología , Terapia de Inmunosupresión/métodos , Animales , Línea Celular , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Células Asesinas Naturales/efectos de los fármacos , Cinética , Fosforilación/efectos de los fármacos , Ratas , Receptor de Adenosina A2A/efectos de los fármacos , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/inmunología , Receptor de Adenosina A2B/efectos de los fármacos , Receptor de Adenosina A2B/genética , Receptor de Adenosina A2B/inmunología , Linfocitos T/metabolismoRESUMEN
The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling acts a pivotal part in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels-mediated neuropathic and inflammatory pain. However, there has been no evidence of cAMP-PKA signaling is involved in regulation of spinal HCN channels function in the occurrence of diabetic neuropathic pain (DNP). The study aimed to elucidate the impact of HCN channels on neuropathic pain in a rat model of diabetes induced by streptozotocin, and whether cAMP-PKA signaling is involved in regulation of HCN channels function. In this report, we evaluated the effect of intrathecal administration of HCN channel blockers ZD7288, cAMP inhibitor SQ22536 and PKA inhibitor H-89 on nociceptive behavior in DNP rats. The mechanical withdrawal threshold (MWT) was measured to evaluate pain behavior in rats. Protein expression levels of HCN2, HCN4 channels and PKA in the spinal dorsal horn of rats were assessed. Furthermore, the levels of cAMP in rat spinal dorsal horn was analyzed. We discovered that DNP rats showed significant mechanical allodynia and are related to the increased HCN2 and HCN4 channels expression, enhanced cAMP production and elevated the expression of PKA protein in the spinal dorsal horn, which were attenuated by intrathecal ZD7288. Furthermore, intrathecal injection of SQ22536 and H-89 significantly reduced the HCN2 and HCN4 channels expression in the spinal dorsal horn of DNP rats. Our findings indicate that HCN channels of the spinal dorsal horn participate in the pathogenesis of allodynia in rats with DNP, which could be regulated by cAMP-PKA signaling. Therefore, HCN channels and cAMP-PKA signaling are potential targets for hyperalgesia treatment in DNP patients.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Neuropatías Diabéticas/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Nocicepción , Médula Espinal/metabolismo , Animales , AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Neuropatías Diabéticas/fisiopatología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/antagonistas & inhibidores , Isoquinolinas/farmacología , Masculino , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Médula Espinal/fisiología , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND: Survivors of sepsis must often endure significant cognitive and behavioral impairments after discharge, but research on the relevant mechanisms and interventions remains lacking. TGR5, a member of the class A GPCR family, plays an important role in many physiological processes, and recent studies have shown that agonists of TGR5 show neuroprotective effects in a variety of neurological disorders. To date, no studies have assessed the effects of TGR5 on neuroinflammatory, cognitive, or behavioral changes in sepsis models. METHODS: A total of 267 eight-week-old male Sprague-Dawley rats were used in this study. Sepsis was induced via cecal ligation and puncture (CLP). All animals received volume resuscitation. The rats were given TGR5 CRISPR oligonucleotide intracerebroventricularly 48 h before CLP surgery. INT-777 was administered intranasally 1 h after CLP, and the cAMP inhibitor, SQ22536, was administered intracerebroventricularly 1 h after CLP. Survival rate, bodyweight change, and clinical scores were assessed, and neurobehavioral tests, western blot, and immunofluorescence staining were performed. The cognitive function of rats was measured using the Morris water maze during 15-20 days after CLP. RESULTS: The expression of TGR5 in the rat hippocampus was upregulated, and peaked at 3 days after CLP. The survival rate of rats after CLP was less than 50%, and the growth rate, in terms of weight, was significantly decreased. While INT-777 treatment did not improve these changes, the treatment did reduce the clinical scores of rats at 24 h after CLP. On day 15 and later, the surviving mice completed a series of behavioral tests. CLP rats showed spatial and memory deficits and anxiety-like behaviors, but INT-777 treatment significantly improved these effects. Mechanistically, immunofluorescence analysis showed that INT-777 treatment reduced the number of microglia in the hippocampus, neutrophilic infiltration, and the expression of inflammatory factors after CLP in rats. Moreover, INT-777 treatment significantly increased the expression of TGR5, cAMP, p-PKA, and p-CREB, but downregulated the expression of IL-1ß, IL-6, and TNF-α. CRISPR-mediated TGR5 knockdown and SQ22536 treatment abolished the neuroprotective effects of TGR5 activation after CLP. CONCLUSION: This study demonstrates that INT-777 treatment reduced neuroinflammation and microglial cell activation, but improved cognitive impairment in the experimental sepsis rats. TGR5 has translational potential as a therapeutic target to improve neurological outcomes in sepsis survivors.
Asunto(s)
Ácidos Cólicos/uso terapéutico , Encefalitis/tratamiento farmacológico , Nootrópicos/uso terapéutico , Receptores Acoplados a Proteínas G/agonistas , Sepsis/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Adenina/análogos & derivados , Adenina/farmacología , Animales , Ansiedad/psicología , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/etiología , Disfunción Cognitiva/psicología , AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico , Citocinas/biosíntesis , Encefalitis/patología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Sepsis/complicaciones , Sepsis/psicología , Análisis de SupervivenciaRESUMEN
Adiponectin (APN) is an adipokine secreted from adipose tissue and exhibits biological functions such as microcirculation-regulating, hearing-protective, and antiapoptotic. However, the effect of APN on the apoptosis of spiral arterial smooth muscle cells (SMCs) under hypoxic conditions in vitro is not clear. We used cobalt chloride (CoCl2) to simulate chemical hypoxia in vitro, and the SMCs were pretreated with APN and then stimulated with CoCl2. The viability of cells and apoptosis were assessed by CCK-8 and flow cytometry, respectively. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) levels, cAMP level, and the activity of PKA were detected by ELISA. Protein expression and localization were studied by Western blot and immunofluorescence analysis. In the present study, we found that APN exhibits antiapoptosis effects. CoCl2 exhibited decreased cell viability, increased apoptosis and MDA levels, and decreased SOD activity in a concentration-dependent manner, compared with the control group. Moreover, CoCl2 upregulated the expression levels of Bax and cleaved caspase-3 and then downregulated Bcl-2 levels in a time-dependent manner. Compared with the CoCl2 group, the group pretreated with APN had increased cell viability, SOD activity, PKA activity, cAMP level, and PKA expression, but decreased MDA levels and apoptosis. Lastly, the protective effect of APN was blocked by cAMP inhibitor SQ22536 and PKA inhibitor H 89. These results showed that APN protected SMCs against CoCl2-induced hypoxic injury via the cAMP/PKA signaling pathway.
Asunto(s)
Adiponectina/metabolismo , Apoptosis/efectos de los fármacos , Cobalto/toxicidad , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Adiponectina/farmacología , Animales , Apoptosis/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cóclea/irrigación sanguínea , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cobayas , Miocitos del Músculo Liso/patología , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
While searching for a counterpart to cyclic AMP, a new compound was found to inhibit adenylate cyclase. It was identified as prostaglandyl-(15-4')-myo-inositol (1':2'-cyclic)-phosphate (cyclic PIP). The substrates for its biosynthesis are prostaglandin E (PGE) and the novel inositol phosphate, guanosine diphospho-4-myo-inositol 1:2-cyclic phosphate (n-IP). The basic regulatory properties of cyclic PIP are to inhibit dose-dependently protein kinase A (PKA) and to seven-fold activate protein ser/thr phosphatase holoenzyme. These regulations occur as rapidly as the activation of PKA by cyclic AMP. Such regulatory properties are essential for the meticulous regulation of the equilibrium between the phospho- and de-phospho-form of interconvertible enzymes. The synthesis of cyclic PIP is stimulated by insulin and noradrenaline (α-receptor action). The insulin-stimulated cyclic PIP synthase is active in a tyrosine-phosphorylated state. A comparable characterization of the adrenaline-stimulated cyclic PIP synthase is still incomplete. In streptozotocin-diabetic rats, the hormonal stimulation of cyclic PIP synthesis decreases with time. Cyclic PIP synthesis is activated by biguanides as metformin two to four-fold and by antihypertensive drugs two-fold. Inhibition of cyclic PIP synthesis leads to a metabolic state as observed in early-stage type-2 diabetes. In summary, all living cells synthesize cyclic PIP, which switches on anabolism, whereas cyclic AMP triggers catabolism.
Asunto(s)
AMP Cíclico/antagonistas & inhibidores , Diabetes Mellitus/patología , Fosfatos de Inositol/farmacología , Prostaglandinas E/farmacología , Animales , Diabetes Mellitus/metabolismo , HumanosRESUMEN
An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.
Asunto(s)
Inhibidores de Adenilato Ciclasa/uso terapéutico , Adenilil Ciclasas/metabolismo , Betacoronavirus/fisiología , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/prevención & control , AMP Cíclico/antagonistas & inhibidores , Pandemias/prevención & control , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/prevención & control , Antivirales/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , COVID-19 , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Neumonía Viral/metabolismo , Neumonía Viral/virología , SARS-CoV-2 , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The discovery of a novel 3H-pyrido[2,3-d]pyrimidin-4-one series as potent and biased sst2 agonists is described. This class of molecules exhibits excellent sst2 potency and selectivity against sst1, sst3, and sst5 receptors, and they are significantly more potent at inhibiting cAMP production than inducing internalization. The orally bioavailable 6-(3-chloro-5-methylphenyl)-3-(3-fluoro-5-hydroxyphenyl)-5-({methyl[(2S)-pyrrolidin-2-ylmethyl]amino}methyl)-3H,4H-pyrido[2,3-d]pyrimidin-4-one (36) also suppresses GH secretion in GHRH-challenged rats in a dose-dependent manner.
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Descubrimiento de Drogas , Pirimidinonas/farmacología , Receptores de Interleucina-1/agonistas , Administración Oral , Animales , Disponibilidad Biológica , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/biosíntesis , Relación Dosis-Respuesta a Droga , Masculino , Estructura Molecular , Pirimidinonas/administración & dosificación , Pirimidinonas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.
Asunto(s)
Caspasa 1/genética , AMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Pseudomonas aeruginosa/metabolismo , Transducción de Señal , 1-Metil-3-Isobutilxantina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Alprostadil/análogos & derivados , Alprostadil/farmacología , Animales , Caspasa 1/metabolismo , Proliferación Celular/efectos de los fármacos , Colforsina/farmacología , AMP Cíclico/agonistas , AMP Cíclico/antagonistas & inhibidores , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Dinoprostona/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/microbiología , Células Endoteliales/patología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Inflamasomas/efectos de los fármacos , Inflamasomas/genética , Inflamasomas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Cultivo Primario de Células , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Ratas , Rolipram/farmacología , Análisis de la Célula IndividualRESUMEN
Epilepsy is one of the most common chronic neurological conditions. Today, close to 30 different medications to prevent epileptic seizures are in use; yet, far from all patients become seizure free upon medical treatment. Thus, there is a need for new pharmacological approaches including novel drug targets for the management of epilepsy. Despite the fact that a role for cAMP signaling in epileptogenesis and seizures was first suggested some four decades ago, none of the current medications target the cAMP signaling system. The reasons for this are probably many including limited knowledge of the underlying biology and pathology as well as difficulties in designing selective drugs for the different components of the cAMP signaling system. This review explores selected aspects of cAMP signaling in the context of epileptogenesis and seizures including cAMP response element binding (CREB)-mediated transcriptional regulation. We discuss the therapeutic potential of targeting cAMP signaling in epilepsy and point to an increased knowledge of the A-kinase anchoring protein-based signaling hubs as being of seminal importance for future drug discovery within the field. Further, in terms of targeting CREB, we argue that targeting upstream cAMP signals might be more fruitful than targeting CREB itself. Finally, we point to astrocytes as cellular targets in epilepsy since cAMP signals may regulate astrocytic K+ clearance affecting neuronal excitability.
Asunto(s)
Anticonvulsivantes/metabolismo , AMP Cíclico/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Epilepsia/metabolismo , Convulsiones/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Anticonvulsivantes/administración & dosificación , AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Sistemas de Liberación de Medicamentos/tendencias , Epilepsia/tratamiento farmacológico , Humanos , Convulsiones/tratamiento farmacológico , Transducción de Señal/fisiologíaRESUMEN
Autophagy is critical for maintaining cellular function via clearance of excess nutrients and damaged organelles. In pancreatic ß-cells, it helps counter the endoplasmic reticulum (ER) stress that impairs insulin secretory capacity during Type 2 diabetes. Chronic exposure of ß-cells to saturated fatty acids (FAs) such as palmitate stimulates ER stress and modulates autophagy, but the effects of unsaturated FAs such as oleate, which are also elevated during obesity, are less well understood. We therefore treated MIN6 cells and mouse islets for 8-48 h with either palmitate or oleate, and then monitored autophagic flux, signaling pathways, lysosomal biology, and phospholipid profiles. Compared with palmitate, oleate more effectively stimulated both autophagic flux and clearance of autophagosomes. The flux stimulation occurred independently of ER stress, nutrient-sensing (mTOR) and signaling pathways (protein kinases A, C, and D). Instead the mechanism involved the exchange factor directly activated by cAMP 2 (EPAC2). Oleate reduced cellular cAMP, and its effects on autophagic flux were reproduced or inhibited, respectively, by Epac2 knockdown or activation. Oleate also increased lysosomal acidity and increased phospholipid saturation, consistent with improved autophagosomal fusion with lysosomes. We conclude that a potent stimulation of autophagy might help explain the known benefits of unsaturated FAs in countering the toxicity of saturated FAs in ß-cells during obesity and lipid loading.
Asunto(s)
Apoptosis/efectos de los fármacos , AMP Cíclico/antagonistas & inhibidores , Células Secretoras de Insulina/efectos de los fármacos , Ácido Oléico/farmacología , Transducción de Señal/efectos de los fármacos , Animales , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Tumorales CultivadasRESUMEN
BACKGROUND: It has been suggested that protein directly activated by cAMP (Epac), one of the downstream signaling molecules of ß-adrenergic receptor (ß-AR), may be an effective target for the treatment of arrhythmia. However, there have been no reports on the anti-arrhythmic effects or cardiac side-effects of Epac1 inhibitors in vivo. MethodsâandâResults: In this study, the roles of Epac1 in the development of atrial and ventricular arrhythmias are examined. In addition, we examined the usefulness of CE3F4, an Epac1-selective inhibitor, in the treatment of the arrhythmias in mice. In Epac1 knockout (Epac1-KO) mice, the duration of atrial fibrillation (AF) was shorter than in wild-type mice. In calsequestrin2 knockout mice, Epac1 deficiency resulted in a reduction of ventricular arrhythmia. In both atrial and ventricular myocytes, sarcoplasmic reticulum (SR) Ca2+ leak, a major trigger of arrhythmias, and spontaneous SR Ca2+ release (SCR) were attenuated in Epac1-KO mice. Consistently, CE3F4 treatment significantly prevented AF and ventricular arrhythmia in mice. In addition, the SR Ca2+ leak and SCR were significantly inhibited by CE3F4 treatment in both atrial and ventricular myocytes. Importantly, cardiac function was not significantly affected by a dosage of CE3F4 sufficient to exert anti-arrhythmic effects. CONCLUSIONS: These findings indicated that Epac1 is involved in the development of atrial and ventricular arrhythmias. CE3F4, an Epac1-selective inhibitor, prevented atrial and ventricular arrhythmias in mice.
Asunto(s)
Fibrilación Atrial/prevención & control , AMP Cíclico/antagonistas & inhibidores , Fibrilación Ventricular/prevención & control , Animales , Fibrilación Atrial/etiología , Calcio/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/fisiología , Ratones , Ratones Noqueados , Quinolinas/uso terapéutico , Retículo Sarcoplasmático/metabolismo , Fibrilación Ventricular/etiologíaRESUMEN
The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element-binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP-PKA-CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)-CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC-specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p-PKA, and p-CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC-cAMP-PKA-CREB signaling pathway of osteoblasts directly or indirectly.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Magnetoterapia , Osteogénesis/efectos de la radiación , Osteoporosis/terapia , Inhibidores de Adenilato Ciclasa/farmacología , Adenilil Ciclasas/genética , Adenilil Ciclasas/farmacología , Animales , Densidad Ósea/efectos de la radiación , Diferenciación Celular/efectos de la radiación , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Modelos Animales de Enfermedad , Fémur/crecimiento & desarrollo , Fémur/patología , Fémur/efectos de la radiación , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Osteoblastos/efectos de la radiación , Osteoporosis/genética , Osteoporosis/patología , Ratas , Transducción de Señal/efectos de la radiaciónRESUMEN
Decrease of chloride concentration contributes to cardiovascular diseases, however, whether decrease of chloride concentration is involved in platelet activation remains elusive. In the present study, we found that ACI patients had lower serum chloride which would be rescued after Aspirin administration. ADP induced chloride concentration reduction in platelets. Blockade of chloride channel prevented ADP-induced platelet adhesion, activation and aggregation, however, decreasing the extracellular chloride concentration promoted ADP-induced platelet adhesion and activation. Decrease of the extracellular chloride concentration facilitated the inactivation of Src family kinase Lyn, which was not involved in PI3K/Akt phosphorylation. Nevertheless, low chloride concentration promoted the production of platelet cytosol Gαi2 subunit. This subunit prevents AC from converting ATP into cAMP, which therefore, inhibited the phosphorylation of PKA to promote platelet activation. In conclusion, decreased intracellular chloride promotes ADP induced platelet activation through the Gαi2/cAMP/PKA pathway instead of the Lyn/PI3K/Akt signal pathway.
Asunto(s)
Adenosina Difosfato/metabolismo , Cloruros/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/antagonistas & inhibidores , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Cloruros/sangre , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Agregación Plaquetaria/sangre , Proteínas Proto-Oncogénicas c-akt/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
ABC transporters are key players in drug excretion with alterations in their expression and activity by therapeutic agents potentially leading to drug-drug interactions. The interaction potential of nomegestrol acetate (NMGA), a synthetic progestogen increasingly used as oral contraceptive, had never been explored. In this work we evaluated (1) the effect of NMGA on ABC transporters in the human hepatic cell line HepG2 and (2) the underlying molecular mechanism. NMGA (5, 50 and 500â¯nM) increased P-glycoprotein (P-gp) expression at both protein and mRNA levels and reduced intracellular calcein accumulation, indicating an increase also in transporter activity. This up-regulation of P-gp was corroborated in Huh7 cells and was independent of the classical progesterone receptor. Instead, using a siRNA-mediated silencing approach, we demonstrated the involvement of membrane progesterone receptor α. Moreover, we found that the activation of this receptor by NMGA led to a falling-rising profile in intracellular cAMP levels and protein kinase A activity over time, ultimately leading to transcriptional P-gp up-regulation. Finally, we identified inhibitory G protein and phosphodiesterases as mediators of this novel biphasic modulation. These results demonstrate the ability of NMGA to selectively up-regulate hepatic P-gp expression and activity and constitute the first report of ABC transporter modulation by membrane progesterone receptor α. If a similar regulation took place in vivo, decreased bioavailability and therapeutic efficacy of NMGA-coadministered P-gp substrates could be expected. This holds special importance considering long-term administration of NMGA and broad substrate specificity of P-gp.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Anticonceptivos/farmacología , AMP Cíclico/metabolismo , Hepatocitos/metabolismo , Megestrol/farmacología , Norpregnadienos/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/agonistas , AMP Cíclico/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Expresión Génica , Células Hep G2 , Hepatocitos/efectos de los fármacos , HumanosRESUMEN
Anthrax is a highly lethal disease caused by the Gram-(+) bacteria Bacillus anthracis. Edema toxin (ET) is a major contributor to the pathogenesis of disease in humans exposed to B. anthracis. ET is a bipartite toxin composed of two proteins secreted by the vegetative bacteria, edema factor (EF) and protective antigen (PA). Our work towards identifying a small molecule inhibitor of anthrax edema factor is the subject of this letter. First we demonstrate that the small molecule probe 5'-Fluorosulfonylbenzoyl 5'-adenosine (FSBA) reacts irreversibly with EF and blocks enzymatic activity. We then show that the adenosine portion of FSBA can be replaced to provide more drug-like molecules which are up to 1000-fold more potent against EF relative to FSBA, display low cross reactivity when tested against a panel of kinases, and are nanomolar inhibitors of EF in a cell-based assay of cAMP production.
Asunto(s)
Carbunco/tratamiento farmacológico , Bacillus anthracis/efectos de los fármacos , Toxinas Bacterianas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antígenos Bacterianos/farmacología , Toxinas Bacterianas/farmacología , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Estructura Molecular , Proteínas Quinasas/metabolismo , Células RAW 264.7 , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule-induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs.