RESUMEN
Actin nucleotide-dependent actin remodeling is essential to orchestrate signal transduction and cell adaptation. Rapid energy starvation requires accurate and timely reorganization of the actin network. Despite distinct treadmilling mechanisms of ADP- and ATP-actin filaments, their filament structures are nearly identical. How other actin-binding proteins regulate ADP-actin filament assembly is unclear. Here, we show that Spa2 which is the polarisome scaffold protein specifically remodels ADP-actin upon energy starvation in budding yeast. Spa2 triggers ADP-actin monomer nucleation rapidly through a dimeric core of Spa2 (aa 281-535). Concurrently, the intrinsically disordered region (IDR, aa 1-281) guides Spa2 undergoing phase separation and wetting on the surface of ADP-G-actin-derived F-actin and bundles the filaments. Both ADP-actin-specific nucleation and bundling activities of Spa2 are actin D-loop dependent. The IDR and nucleation core of Spa2 are evolutionarily conserved by coexistence in the fungus kingdom, suggesting a universal adaptation mechanism in the fungal kingdom in response to glucose starvation, regulating ADP-G-actin and ADP-F-actin with high nucleotide homogeneity.
Asunto(s)
Actinas , Adenosina Difosfato , Glucosa , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Difosfato/análogos & derivados , Glucosa/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/química , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Combination of tumor immunotherapy with photothermal therapy (PTT) is a feasible tactic to overcome the drawback of immunotherapy such as poor immune response. Via triggering the immunogenic cells death (ICD), PTT can stimulate the activity of immune cells, but meanwhile, the level of adenosine is elevated via the CD73-induced decomposition of ATP which is overexpressed accompanying with the PTT process, resulting in negative feedback to impair the immune stimulation. Herein, we developed a novel biomimetic photothermal nanodrug to specifically block CD73 for inhibition of adenosine production and more efficient priming of the suppressive immune microenvironments. The nanodrug, named as AptEM@CBA, is constructed by encapsulation of photothermal agent black phosphorus quantum dots (BPQDs) and selective CD73 inhibitor α, ß-Methyleneadenosine 5'-diphosphate (AMPCP) in chitosan nanogels, which are further covered with aptamer AS1411 modified erythrocyte membrane (EM) for biomimetic camouflage. With AS1411 induced active targeting and EM induced long blood circulation time, the enrichment of the nanodrug tumor sites is promoted. The photothermal treatment promotes the maturation of dendritic cells. Meanwhile, the release of AMPCP suppress the adenosine generation via CD73 blockade, alleviating the impairment of adenosine to dendritic cells and suppressing regulatory T cells, synergically stimulate the activity of T cells. The combination of CD73 blockade with PTT, not only suppresses the growth of primary implanted tumors, but also boosts strong antitumor immunity to inhibit the growth of distal tumors, providing good potential for tumor photoimmunotherapy.
Asunto(s)
5'-Nucleotidasa , Adenosina Difosfato , Adenosina , Inmunoterapia , Terapia Fototérmica , Animales , Humanos , Ratones , 5'-Nucleotidasa/antagonistas & inhibidores , Adenosina/química , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina Difosfato/análogos & derivados , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Biomimética/métodos , Línea Celular Tumoral , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Inmunoterapia/métodos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanopartículas/química , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Terapia Fototérmica/métodos , Puntos Cuánticos/química , Microambiente Tumoral/efectos de los fármacos , MasculinoRESUMEN
Platelet activation by adenosine diphosphate (ADP) is mediated through two G-protein-coupled receptors, P2Y1 and P2Y12, which signal through Gq and Gi, respectively. P2Y1 stimulation leads to phospholipase C activation and an increase in cytosolic calcium necessary for CalDAG-GEF1 activation. Engagement of P2Y12 inhibits adenylate cyclase, which reduces cAMP, and activation of PI3-kinase, which inhibits RASA3 resulting in sustained activated Rap1b. In this study we activated human platelets with 2-MeSADP in the presence of LY294002, a PI3-kinase inhibitor, AR-C69931MX, a P2Y12 antagonist or MRS2179, a P2Y1 antagonist. We measured the phosphorylation of Akt on Ser473 as an indicator of PI3-kinase activity. As previously shown, LY294002 and ARC69931MX abolished 2MeSADP-induced Akt phosphorylation. MRS2179 reduced ADP-induced Akt phosphorylation but did not abolish it. Rap1b activation, however, was only reduced, but not ablated, using LY294002 and was completely inhibited by ARC69931MX or MRS2179. Furthermore, 2MeSADP-induced Rap1b activation was abolished in either P2Y1 or P2Y12 null platelets. These data suggest that ADP-induced Rap1b activation requires both P2Y1 and P2Y12. In addition, although stimulation of P2Y12 results in PI3-kinase activation leading to Akt phosphorylation and Rap1b activation, Rap1b activation can occur independently of PI3-kinase downstream of P2Y12. Thus, we propose that the P2Y12 receptor can regulate Rap1b, possibly through RASA3, in a pathway independent of PI3-kinase.
Asunto(s)
Fosfatidilinositol 3-Quinasas , Receptores Purinérgicos P2 , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Adenilil Ciclasas/metabolismo , Plaquetas/metabolismo , Calcio/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Antagonistas del Receptor Purinérgico P2Y , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Tionucleótidos , Fosfolipasas de Tipo C/metabolismo , Proteínas de Unión al GTP rap/metabolismoRESUMEN
ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5'-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local "purinergic junctions" with CD39low/CD73- neuronal cell bodies and CD39high/CD73- retinal blood vessels. The relevance of the CD73-adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.
Asunto(s)
5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Luz , Retina/efectos de la radiación , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/genética , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Apirasa/genética , Apirasa/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microglía/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Retina/fisiología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismoRESUMEN
The aim of the current study was to determine the effects of cerebral contusion injury with purinergic adenosine triphosphate Y1 (P2Y1) receptor blockers on postinjury inflammatory responses. Adenosine triphosphate (ATP) is released into the extracellular space in several in vivo models, including traumatic brain injury. Released ATP triggers neuroinflammation via activation of microglial cells. P2Y1 receptor blockers were reported to suppress extracellular ATP elevation in several disease models through inhibition of cellular ATP release. In addition to the beneficial effects of inflammation, excess inflammatory reactions cause secondary damage and aggravate outcomes. Here, we assessed the effect of the selective P2Y1 receptor blocker MRS2179 on its potential to prevent posttraumatic inflammation in a rat cerebral contusion model. Cerebral contusion injury was induced in the rat cerebral cortex. Either MRS2179 or artificial cerebral spinal fluid as a control was administered in situ into the center of contused tissue via a subcutaneously implanted osmotic pump. Galectin 3, a marker of microglia and proinflammatory cytokines, was measured 1, 3 and 7 days following injury. Another group of rats was assessed for behavioral performance up to 28 days after injury, including the beam walk test, neurological response test and plus maze test. The Galectin 3 levels in the cortex around the contusion cavity and in the cortex far from the contusion cavity were significantly suppressed by MRS2179 administration on postinjury Days 1 and 3 (pâ¯<â¯0.05). However, administration of MRS2179 failed to improve behavioral outcome. Administration of MRS2179 successfully suppressed microglial activation in a traumatic brain injury model, which will be a potent treatment option in the future. Further study is required to conclude its therapeutic effects.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Antiinflamatorios/farmacología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/lesiones , Galectina 3/efectos de los fármacos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Antagonistas del Receptor Purinérgico P2Y/farmacología , Adenosina Difosfato/farmacología , Animales , Conducta Animal/efectos de los fármacos , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/inmunología , Corteza Cerebral/inmunología , Modelos Animales de Enfermedad , Microglía/efectos de los fármacos , Enfermedades Neuroinflamatorias/etiología , Enfermedades Neuroinflamatorias/inmunología , RatasRESUMEN
A previous study showed that 2'-3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) was a weak allosteric activator of Rhizobium etli pyruvate carboxylase (RePC) in the absence of acetyl-CoA. On the other hand, TNP-ATP inhibited the allosteric activation of RePC by acetyl-CoA. Here, we aimed to study the role of triphosphate group of TNP-ATP on its allosteric activation of the enzyme and inhibition of acetyl-CoA-dependent activation of RePC using TNP-ATP and its derivatives, including TNP-ADP, TNP-AMP and TNP-adenosine. The pyruvate carboxylation activity was assayed to determine the effect of reducing the number of phosphate groups in TNP-ATP derivatives on allosteric activation and inhibition of acetyl-CoA activation of RePC and chicken liver pyruvate carboxylase (CLPC). Reducing the number of phosphate groups in TNP-ATP derivatives decreased the activation efficacy for both RePC and CLPC compared to TNP-ATP. The apparent binding affinity and inhibition of activation of the enzymes by acetyl-CoA were also diminished when the number of phosphate groups in the TNP-ATP derivatives was reduced. Whilst TNP-AMP activated RePC, it did not activate CLPC, but it did inhibit acetyl-CoA activation of both RePC and CLPC. Similarly, TNP-adenosine did not activate RePC; however, it did inhibit acetyl-CoA activation using a different mechanism compared to phosphorylated TNP-derivatives. These findings indicate that mechanisms of PC activation and inhibition of acetyl-CoA activation by TNP-ATP and its derivatives are different. This study provides the basis for possible drug development for treatment of metabolic diseases and cancers with aberrant expression of PC.
Asunto(s)
Acetilcoenzima A/química , Adenosina Trifosfato/análogos & derivados , Regulación Alostérica/efectos de los fármacos , Activadores de Enzimas/química , Piruvato Carboxilasa/química , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/química , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Adenosina Trifosfato/química , Animales , Pollos , Pruebas de Enzimas , Cinética , Hígado/enzimología , Estructura MolecularRESUMEN
The aim of this study was to examine the effects of glycosaminoglycan (GAG) from Urechis unicinctus on the P2Y1 receptor pathway and expression of related factors in rat platelets. The concentration of calcium ion (Ca2+) in rat platelets was determined by double wavelength Fura-2 fluorescence spectrophotometry, and the concentrations of inositol trisphosphate (IP3) and glycoprotein IIb/IIIa (GPIIb/IIIa) in rat platelets were measured using the enzymatic immunoassay method. The phosphorylation levels of phospholipase C (PLC), phospholipase A2 (PLA2), protein kinase C (PKC), and p38 mitogen-activated protein kinase (p38MAPK) were also detected by Western blot. It was found that the GAG from U. unicinctus significantly reduced the Ca2+ and IP3 levels in rat platelets (p<0.05, p<0.01). Moreover, medium and high concentrations of GAG significantly reduced the concentration of the platelet membrane GPIIb/IIIa in rats (p<0.05, p<0.01). The phosphorylation levels of PLC, PLA)2), PKC and p38MAPK in rat platelets were also inhibited by GAG and P)2)Y)1) receptor blocker MRS2179 (p<0.05, p<0.01). However, the degree of inhibition of GAG was lower than that of MRS2179. The results laid a foundation for further utilization of the glycosaminoglycan.
Asunto(s)
Productos Biológicos/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Glicosaminoglicanos/farmacología , Receptores Purinérgicos P2Y1/biosíntesis , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Productos Biológicos/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Glicosaminoglicanos/aislamiento & purificación , Nematodos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/biosíntesis , Antagonistas del Receptor Purinérgico P2Y/farmacología , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesisRESUMEN
The functional role of ATP released from sympathetic nerve terminals was examined in isolated guinea pig ventricular papillary muscles. The contractile force of papillary muscles was increased by field electrical stimulation of sympathetic nerve endings. This increase was attenuated by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or suramin, blockers of the P2X receptor, and was abolished by propranolol and prazosin. PPADS, suramin, and ATP affected neither the basal contractile force nor the positive inotropic effect of noradrenaline. These results provide functional evidence that ATP released from sympathetic nerve terminals enhances noradrenaline release and contributes to sympathetic nerve-induced inotropy.
Asunto(s)
Adenosina Trifosfato/fisiología , Retroalimentación Fisiológica , Músculos Papilares/fisiología , Sistema Nervioso Simpático , Función Ventricular , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Cobayas , Ventrículos Cardíacos , Masculino , Contracción Muscular , Norepinefrina/fisiología , Prazosina/farmacología , Propranolol/farmacología , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Suramina/farmacologíaRESUMEN
Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y1 and P2Y2 receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young's modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y2 receptor stimulation with UTP. Such exposure to UTP doubled the Young's modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y1 and P2Y13 receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y13 receptor, although they were negatively modulated by P2Y1 receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Astrocitos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2/metabolismo , Tionucleótidos/metabolismo , Uridina Trifosfato/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Astrocitos/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Microscopía de Fuerza Atómica , Transducción de Señal , Tionucleótidos/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
Alcoholic liver fibrosis (ALF) is commonly associated with long-term alcohol consumption and the activation of hepatic stellate cells (HSCs). Inhibiting the activation and proliferation of HSCs is a critical step to alleviate liver fibrosis. Increasing evidence indicates that ecto-5'-nucleotidase (CD73) plays a vital role in liver disease as a critical component of extracellular adenosine pathway. However, the regulatory role of CD73 in ALF has not been elucidated. In this study, both ethanol plus CCl4-induced liver fibrosis mice model and acetaldehyde-activated HSC-T6 cell model were employed and the expression of CD73 was consistently elevated in vivo and in vitro. C57BL/6 J mice were intraperitoneally injected with CD73 inhibitor Adenosine 5'-(α, ß-methylene) diphosphate sodium salt (APCP) from 5th week to the 8th week in the development of ALF. The results showed APCP could inhibit the activation of HSCs, reduce fibrogenesis marker expression and thus alleviate ALF. Silencing of CD73 inhibited the activation of HSC-T6 cells and promoted apoptosis of activated HSC-T6 cells. What's more, the proliferation of HSC-T6 cells was inhibited, which was characterized by decreased cell viability and cycle arrest. Mechanistically, Wnt/ß-catenin pathway was activated in acetaldehyde-activated HSC-T6 cells and CD73 silencing or overexpression could regulate Wnt/ß-catenin signaling pathway. Collectively, our study unveils the role of CD73 in HSCs activation, and Wnt/ß-catenin signaling pathway might be involved in this progression.
Asunto(s)
5'-Nucleotidasa/biosíntesis , Proliferación Celular/fisiología , Células Estrelladas Hepáticas/metabolismo , Vía de Señalización Wnt/fisiología , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/deficiencia , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Estrelladas Hepáticas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
BACKGROUND: Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disease. Recent studies have reported the close association between cognitive function in AD and purinergic receptors in the central nervous system. In the current study, we investigated the effect of CD73 inhibitor α, ß-methylene ADP (APCP) on cognitive impairment of AD in mice, and to explore the potential underlying mechanisms. RESULTS: We found that acute administration of Aß142 (i.c.v.) resulted in a significant increase in adenosine release by using microdialysis study. Chronic administration of APCP (10, 30 mg/kg) for 20 d obviously mitigated the spatial working memory impairment of Aß142-treated mice in both Morris water maze (MWM) test and Y-maze test. In addition, the extracellular adenosine production in the hippocampus was inhibited by APCP in Aß-treated mice. Further analyses indicated expression of acetyltransferase (ChAT) in hippocampus of mice of was significantly reduced, while acetylcholinesterase (AChE) expression increased, which compared to model group. We observed that APCP did not significantly alter the NLRP3 inflammasome activity in hippocampus, indicating that anti-central inflammation seems not to be involved in APCP effect. CONCLUSIONS: In conclusion, we report for the first time that inhibition of CD73 by APCP was able to protect against memory loss induced by Aß142 in mice, which may be due to the decrease of CD73-driven adenosine production in hippocampus. Enhancement of central cholinergic function of the central nervous system may also be involved in the effects of APCP.
Asunto(s)
Animales , Masculino , Ratones , Adenosina Difosfato/análogos & derivados , Enfermedades Neurodegenerativas/prevención & control , Hipocampo , Nucleotidasas/antagonistas & inhibidores , Acetilcolinesterasa , Adenosina Difosfato/administración & dosificación , Enfermedad de Alzheimer/prevención & control , Prueba del Laberinto Acuático de Morris , Ratones Endogámicos C57BLRESUMEN
The behavioural impacts of prenatal exposure to ethanol include a lower IQ, learning problems, anxiety and conduct disorders. Several components of the neurochemical network could contribute to the long-lasting effects of ethanol embryonic exposure. Adenosine is an important neuromodulator, that has been indicated to be affected by acute and chronic exposure to ethanol. Here, embryos of zebrafish exposed to 1% ethanol during the developmental stages of gastrula/segmentation or pharyngula exhibited anxiolytic effect, increased aggressiveness, and decreased social interaction. The exposure during pharyngula stage was able to affect all behavioural parameters analysed at 3 months-post fertilization (mpf), while the treatment during gastrula stage affected the anxiety and social interaction parameters. The aggressiveness was the only behavioural effect of early ethanol exposure that lasted to 12 mpf. The use of a specific inhibitor of adenosine production, the inhibitor of ecto-5'-nucleotidase (AMPCP/150 mg/kg), and the specific inhibitor of adenosine degradation, the inhibitor of adenosine deaminase, EHNA (100 mg/kg) did not affect the effects over anxiety. However, AMPCP at 3 mpf, but not EHNA, reversed aggressive parameters. AMPCP also recovered the social interaction parameter at 3 mpf in animals treated in both stages, while EHNA recovered this parameter just in those animals treated with ethanol during the gastrula stage. These results suggest that long-lasting behavioural effects of ethanol can be modulated by intervention on ecto-5'-nucleotidase and adenosine deaminase activities.
Asunto(s)
Inhibidores de la Adenosina Desaminasa/uso terapéutico , Adenosina Difosfato/análogos & derivados , Adenosina/metabolismo , Trastorno de Personalidad Antisocial/tratamiento farmacológico , Etanol/efectos adversos , Efectos Tardíos de la Exposición Prenatal/tratamiento farmacológico , 5'-Nucleotidasa/antagonistas & inhibidores , Adenina/análogos & derivados , Adenina/farmacología , Adenina/uso terapéutico , Inhibidores de la Adenosina Desaminasa/farmacología , Adenosina Difosfato/farmacología , Adenosina Difosfato/uso terapéutico , Animales , Trastorno de Personalidad Antisocial/etiología , Conducta Animal , Modelos Animales de Enfermedad , Etanol/administración & dosificación , Femenino , Humanos , Embarazo , Interacción Social/efectos de los fármacos , Pez CebraRESUMEN
Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.
Asunto(s)
Agonistas del Receptor de Adenosina A1/farmacocinética , Agonistas del Receptor de Adenosina A3/farmacocinética , Profármacos/farmacocinética , Agonistas del Receptor Purinérgico P2Y/farmacocinética , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacocinética , Animales , Nucleótidos de Desoxiadenina/farmacocinética , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Antagonistas del Receptor Purinérgico P2Y/farmacocinética , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A3/metabolismo , Receptores Purinérgicos P2Y1/metabolismoRESUMEN
Purpose: Adenosine triphosphate (ATP) is involved in the diameter regulation of retinal vessels. The compound has been shown to induce both constriction and dilatation, but the detailed mechanisms underlying these effects and the site of action of the compound are not known in detail. Therefore, the purpose of the present study was to investigate whether the vasoactive effects of ATP on retinal vessels depend on intra- and extravascular application, and to study whether the effects differ at different vascular branching levels. Methods: Diameter changes in arterioles, pre-capillary arterioles, and capillaries were studied in perfused porcine hemiretinas (n = 48) ex vivo after intra- and extravascular application of the nondegradable ATP analogue ATP-γ-S or ATP in the presence or not of antagonists to the CD73/ecto-5'-nucleotidase (AOPCP), the P2-purinergic receptor (PPADS), the A3-adenosine receptor (MRS1523), and the synthesis of cyclooxygenase products (ibuprofen). Results: Intravascular ATP-induced constriction and extravascular ATP-induced dilatation of retinal arterioles, pre-capillary arterioles and capillaries, and dilatation was inhibited by ibuprofen. Both constriction and dilatation of arterioles were inhibited by antagonizing ATP degradation. Furthermore, constriction at all three branching levels was antagonized by blocking the A3 purinoceptor, whereas constriction in arterioles and pre-capillary arterioles was antagonized by blocking the P2 purinoceptor. Conclusions: ATP affects the diameter of retinal arterioles, pre-capillary arterioles, and capillaries through different pathways, and the effects depend on whether the compound is administered intravascularly or extravascularly. This may form the basis for selective interventions on retinal vascular disease with differential involvement of vessels at different branching levels.
Asunto(s)
Adenosina Trifosfato/farmacología , Arteria Retiniana/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , 5'-Nucleotidasa/farmacología , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Adenosina Trifosfato/análogos & derivados , Marcadores de Afinidad , Animales , Arteriolas/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Procedimientos Endovasculares , Inhibidores Enzimáticos/farmacología , Ibuprofeno/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Sus scrofaRESUMEN
OBJECTIVES: To determine whether ecto-5'-nucleotidase (e5NT) contributes to the release of adenosine and uridine and whether is establishes the role of e5NT in acute restraint stress-induced depression and anxiety-like behaviours in mice. METHODS: Acute restraint stress was induced to detect the level of nucleoside in the hippocampus. Mouse hippocampal brain proteins were isolated and subjected to Western blotting (WB) experiments to examine the protein expression levels of proteins that affect nucleoside release. Adenosine 5'-(α,ß-methylene)diphosphate (APCP), an e5NT inhibitor, was intraventricularly injected to investigate the regulatory effect of e5NT on nucleoside levels and behavioural changes caused by acute restraint stress in mice. KEY FINDINGS: Acute restraint stress increased the level of extracellular adenosine and uridine levels in the hippocampus of mice and significantly increased the expression of extracellular nucleoside-metabolizing enzymes were significantly increased. By administering APCP, the increase in adenosine and uridine levels caused by acute restraint stress could be suppressed. APCP inhibited behavioural changes, which were induced by acute restraint stress. CONCLUSIONS: These data suggest that acute restraint stress may alter extracellular adenosine and uridine levels content in the hippocampus of mice via e5NT, and thus, the inhibition of e5NT may improve the anxiety behaviour in mice. Therefore, e5NT may therefore be a potential therapeutic target for the treatment of anxiety in mice.
Asunto(s)
5'-Nucleotidasa/antagonistas & inhibidores , Adenosina Difosfato/análogos & derivados , Antidepresivos/farmacología , Conducta Animal/efectos de los fármacos , Depresión/prevención & control , Inhibidores Enzimáticos/farmacología , Hipocampo/efectos de los fármacos , Estrés Psicológico/tratamiento farmacológico , 5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Adenosina Difosfato/farmacología , Animales , Depresión/enzimología , Depresión/etiología , Depresión/psicología , Modelos Animales de Enfermedad , Prueba de Laberinto Elevado , Conducta Exploratoria/efectos de los fármacos , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Hipocampo/enzimología , Hipocampo/fisiopatología , Masculino , Ratones , Prueba de Campo Abierto/efectos de los fármacos , Restricción Física , Estrés Psicológico/enzimología , Estrés Psicológico/etiología , Estrés Psicológico/psicología , Uridina/metabolismoRESUMEN
AIMS: As PDGFRα (+) cells appear not to suppress the excitability of detrusor smooth muscle by generating SK3-dependent hyperpolarising as proposed in the gastrointestinal tract, we further explored the functional roles of PDGFRα (+) cells in regulating the spontaneous activity of urogenital tissues. METHODS: Using PDGFRα-eGFP mice, intracellular Ca2+ signaling in PDGFRα (+) cells of the bladder lamina propria, renal pelvis, and seminal vesicle were visualized using Cal-590 fluorescence. The distribution and SK3 expression of PDGFRα (+) cells were also examined by immunohistochemistry. RESULTS: In the bladder lamina propria, SK3 (-) PDGFRα (+) cells exhibited spontaneous Ca2+ transients and responded to stimulation of P2Y1 purinoceptors with MRS2365 (100 nM) or adenosine diphosphate (ADP) (100 µM) by developing Ca2+ transients. In the proximal renal pelvis, PDGFRα (+) cells were distributed in the mucosal, muscular and serosal layers but did not express SK3 immunoreactivity. PDGFRα (+) cells in the musculature resembling atypical smooth muscle cells generated spontaneous Ca2+ transients that were partially suppressed upon P2Y1-stimulation, while vigorously responding to human angiotensin II (100 nM). In the seminal vesicle, PDGFRα (+) cells in the musculature but not mucosa expressed SK3 immunoreactivity. In the mucosa, the P2Y1 stimulation evoked Ca2+ transients in both PDGFRα (+) cells and PDGFRα (-) cells. CONCLUSION: PDGFRα (+) cells in spontaneously active urogenital tissues display heterogeneity in terms of their SK3 expression and P2Y1-induced Ca2+ responses. Muscular PDGFRα (+) cells in the renal pelvis and mucosal PDGFRα (+) cells in the seminal vesicle may generate depolarizing signals to drive smooth muscle cells.
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Músculo Liso/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Vejiga Urinaria/metabolismo , Adenosina Difosfato/análogos & derivados , Animales , Masculino , Ratones , Ratones Transgénicos , Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Vejiga Urinaria/diagnóstico por imagenRESUMEN
A new approach to improve the effectiveness of acute myeloid leukemia (AML) treatment is to use the properties of purinergic signaling molecules secreted into the bone marrow milieu in response to leukemic cell growth. Therefore, our study aimed to evaluate the effects of extracellular adenine nucleotides and adenosine on the growth and death parameters in the leukemic THP-1 cell line. Cells were exposed to ATP, ADP, AMP, adenosine and nonhydrolyzable analogues of ATP and ADP (ATPγS and ADPßS) in a 1-1000 µM broad concentration range. The basal mRNA expression of the P1 and P2 receptors was evaluated by real-time PCR. Changes in the processes of cell growth and death were assessed by flow cytometry analysis of proliferation, cell cycle and apoptosis. Chemotaxis toward stromal cell-derived factor-1 (SDF-1) was performed using the modified Boyden chamber assay, and chemokine receptor type 4 (CXCR4) surface expression was quantified by flow cytometry. We indicated several antileukemic actions. High micromolar concentrations (100-1000 µM) of extracellular adenine nucleotides and adenosine inhibit the growth of cells by arresting the cell cycle and/or inducing apoptosis. ATP is characterized by the highest potency and widest range of effects, and is responsible for the cell cycle arrest and the apoptosis induction. Compared to ATP, the effect of ADP is slightly weaker. Adenosine mostly has a cytotoxic effect, with the induction of apoptosis. The last studied nucleotide, AMP, demonstrated only a weak cytotoxic effect without affecting the cell cycle. In addition, cell migration towards SDF-1 was inhibited by low micromolar concentrations (10 µM). One of the reasons for this action of ATPγS and adenosine was a reduction in CXCR4 surface expression, but this only partially explains the mechanism of antimigratory action. In summary, extracellular adenine nucleotides and adenosine inhibit THP-1 cell growth, cause death of cells and modulate the functioning of the SDF-1/CXCR4 axis. Thus, they negatively affect the processes that are responsible for the progression of AML and the difficulties in AML treatment.
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Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Adenosina/farmacología , Leucemia Mieloide Aguda/patología , Tionucleótidos/farmacología , Marcadores de Afinidad , Apoptosis , Ciclo Celular , Movimiento Celular , Proliferación Celular , Matriz Extracelular/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Células Tumorales CultivadasRESUMEN
Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and -granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6-/- platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6-/- platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled 2A adrenergic receptors, respectively, was not affected in GRK6-/- platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6-/- platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKC) phosphorylation were significantly potentiated in GRK6-/- platelets. Finally, GRK6-/- mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6-/- mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.
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Plaquetas/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Regulación de la Expresión Génica , Activación Plaquetaria , Receptores Acoplados a Proteínas G/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Animales , Femenino , Hemostáticos , Masculino , Ratones , Ratones Noqueados , Oligopéptidos/farmacología , Fosforilación , Agregación Plaquetaria , Tionucleótidos/farmacología , Trombina/metabolismo , Tromboxano A2/metabolismoRESUMEN
INTRODUCTION: Spinal cord injury (SCI) causes most severe motor and sensory dysfunctions. In Chinese traditional medicine, the agonist of a purinergic receptor is believed to have a positive effect on SCIs, and 2-Methylthio-adenosine-5'-diphosphate (2-MesADP) is a selective agonist of the P2Y purinergic receptor. METHODS: To investigate its therapeutic function and molecular mechanism in SCI, transcriptome analysis associated with weighted gene co-expression network analysis (WGCNA) was carried out at various time points after T9 crush injury. RESULTS: 2-MesADP demonstrated recovery of limb motor function at the 6 weeks after injury, accompanied by neuronal regeneration and axon remyelination at 2 and 6 weeks. Furthermore, gene profiling revealed alternated gene expression with the treatment of 2-MesADP. These genes were assigned to a total of 38 modules, followed by gene ontology analysis; of these, 18 represented neuronal apoptosis and regeneration, immune response, synaptic transmission, cell cycle, and angiogenesis. In the neuronal apoptosis and regeneration module, Nefh, NeuroD6, and Dcx in the 2-MesADP group were noticed due to their interesting expression pattern. The gene expression patterns of Mag, Mog, and Cnp, which played key roles in myelination, were significantly changed with the treatment of 2-MesADP. Wnt signal pathway was the most important pathway in 2-MesADP treatment for acute SCI. CONCLUSION: 2-MesADP enhanced locomotor recovery in mouse SCI by altering the expression of neuronal apoptosis and remyelination-related genes and Wnt signaling pathways.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Locomoción/fisiología , Agonistas Purinérgicos/farmacología , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal , Tionucleótidos/farmacología , Adenosina Difosfato/farmacología , Animales , Proteína Doblecortina , Humanos , Ratones , Regeneración Nerviosa/efectos de los fármacos , Recuperación de la Función/fisiología , Remielinización/efectos de los fármacos , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
CD73 inhibitors are promising drugs for the (immuno)therapy of cancer. Here, we present the synthesis, structure-activity relationships, and cocrystal structures of novel derivatives of the competitive CD73 inhibitor α,ß-methylene-ADP (AOPCP) substituted in the 2-position. Small polar or lipophilic residues increased potency, 2-iodo- and 2-chloro-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (15, 16) being the most potent inhibitors with Ki values toward human CD73 of 3-6 nM. Subject to the size and nature of the 2-substituent, variable binding modes were observed by X-ray crystallography. Depending on the binding mode, large species differences were found, e.g., 2-piperazinyl-AOPCP (21) was >12-fold less potent against rat CD73 compared to human CD73. This study shows that high CD73 inhibitory potency can be achieved by simply introducing a small substituent into the 2-position of AOPCP without the necessity of additional bulky N6-substituents. Moreover, it provides valuable insights into the binding modes of competitive CD73 inhibitors, representing an excellent basis for drug development.