RESUMEN
Growing evidences demonstrate that chemokines and chemokine receptors are up-regulated in resident central nervous system cells during Alzheimer's disease contributing to neuroinflammation and neurodegeneration. Prokineticin 2 belongs to a new family of chemokines which recently emerged as a critical player in immune system and inflammatory diseases. Since pharmacological blockade in vitro of the prokineticin system is able to antagonize Amyloid ß-induced neurotoxicity, the aim of the present study was to investigate in vivo effects of prokineticin receptor antagonist PC1 on memory impairment in a rodent model of Alzheimer's disease. Rats were intracerebroventricular infused with Aß1-42 and behavioral responses as well as the expression profile in hippocampus of prokineticin 2 and its receptors were investigated. Results demonstrated that Aß1-42-infused rats developed significant memory impairments together with a marked up-regulation of both prokineticin 2 and its receptors in hippocampal neurons and astrocytes. Treatment with PC1 significantly improved learning capability of Aß1-42-infused rats restoring the balance of prokineticin system. This study pointed to a neuroprotective role of PC1 on Aß1-42-induced memory deficits that could be ascribed to the ability of PC1 to modulate rat hippocampal prokineticin system and to recover the impaired Aß1-42-induced neurogenesis. This suggests that prokineticin system antagonism could be considered as a new approach for the study of AD etiopathology.
Asunto(s)
Enfermedad de Alzheimer , Hipocampo/efectos de los fármacos , Aprendizaje/efectos de los fármacos , Neuropéptidos/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Péptidos/antagonistas & inhibidores , Péptidos beta-Amiloides/toxicidad , Animales , Modelos Animales de Enfermedad , Hormonas Gastrointestinales/genética , Hormonas Gastrointestinales/metabolismo , Gliosis , Guanidinas/farmacología , Hipocampo/metabolismo , Infusiones Intraventriculares , Masculino , Aprendizaje por Laberinto , Memoria/efectos de los fármacos , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Neurogénesis/efectos de los fármacos , Neuropéptidos/genética , Neuropéptidos/metabolismo , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fragmentos de Péptidos/toxicidad , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Aprendizaje Espacial/efectos de los fármacos , Triazinas/farmacologíaRESUMEN
Prokineticin-2 (PK2) is a secreted bioactive peptide that signals through two GPCRs, the prokineticin receptors (PKRs), and regulates a variety of biological processes including angiogenesis, immunity and nociception. The PK2 primary transcript has two alternative splice variants, PK2 and PK2L (a Long form) which is cleaved in an active peptide, named PK2ß that preferentially binds to PKR1 receptor. The aim of this study was to characterize the PK2ß. Using different Saccharomyces cerevisiae strains, we examined the specificity of PKR1 and PKR2 G-protein coupling following PK2ß binding. Data obtained in yeast confirmed that PK2 binds both receptors, inducing a comparable response throughout a promiscuous coupling of G protein subtypes. Conversely, we demonstrated, for the first time, that PK2ß preferentially binding to PKR1, activates a signaling cascade that not depends on Gαi/o coupling. The binding specificity of PK2ß for PKR1 was evaluated by the analysis of PKR mutant in yeast and GST pull-down experiments, suggesting an important role of PKR1 amino-terminal region. We also evaluated the ability of PK2ß to differentially activate PKR1 and/or PKR2 by in vivo nociceptive experiments and we showed that PK2ß induces intense sensitization of peripheral nociceptors to painful stimuli through the activation of PKR1. To analyze PK2ß-induced signal transduction, we demonstrated the inability of PK2ß to induce STAT3 protein phosphorylation in organotypic primary explants from mice Dorsal Root Ganglion (DRG), an important pain station. The control of the concentration ratio between PK2ß and PK2 could be one of the keys to allow the specificity of the cell response of prokineticin signaling pathway.
Asunto(s)
Hormonas Gastrointestinales/metabolismo , Neuropéptidos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Células Cultivadas , Ganglios Espinales/metabolismo , Masculino , Ratones Endogámicos C57BL , Isoformas de Proteínas , Factor de Transcripción STAT3/metabolismo , Saccharomyces cerevisiae , Transducción de SeñalRESUMEN
Pain, particularly chronic pain, is still an unsolved medical condition. Central goals in pain control are to provide analgesia of adequate efficacy and to reduce complications associated with the currently available drugs. Opioids are the mainstay for the treatment of moderate to severe pain. However, opioid pain medications also cause detrimental side effects, thus highlighting the need of innovative and safer analgesics. Opioids mediate their actions via the activation of opioid receptors, with the mu-opioid receptor as the primary target for analgesia, but also for side effects. One long-standing focus of drug discovery is the pursuit for new opioids exhibiting a favorable dissociation between analgesia and adverse effects. In this study, we describe the in vitro and in vivo pharmacological profiles of the 14-O-phenylpropyl substituted analog of the mu-opioid agonist 14-O-methyloxymorphone (14-OMO). The consequence of the substitution of the 14-O-methyl in 14-OMO with a 14-O-phenylpropyl group on in vitro binding and functional activity, and in vivo behavioral properties (nociception and gastrointestinal motility) was investigated. In binding studies, 14-O-phenylpropyloxymorphone (POMO) displayed very high affinity at mu-, delta-, and kappa-opioid receptors (Ki values in nM, mu:delta:kappa = 0.073:0.13:0.30) in rodent brain membranes, with complete loss of mu-receptor selectivity compared to 14-OMO. In guinea-pig ileum and mouse vas deferens bioassays, POMO was a highly efficacious and full agonist, being more potent than 14-OMO. In the [35S]GTPγS binding assays with membranes from CHO cells expressing human opioid receptors, POMO was a potent mu/delta-receptor full agonist and a kappa-receptor partial agonist. In vivo, POMO was highly effective in acute thermal nociception (hot-plate test, AD50 = 0.7 nmol/kg) in mice after subcutaneous administration, with over 70- and 9000-fold increased potency than 14-OMO and morphine, respectively. POMO-induced antinociception is mediated through the activation of the mu-opioid receptor, and it does not involve delta- and kappa-opioid receptors. In the charcoal test, POMO produced fourfold less inhibition of the gastrointestinal transit than 14-OMO and morphine. In summary, POMO emerges as a new potent mixed mu/delta/kappa-opioid receptor agonist with reduced liability to cause constipation at antinociceptive doses.
RESUMEN
The mammalian prokineticins family comprises two conserved proteins, EG-VEGF/PROK1 and Bv8/PROK2, and their two highly related G protein-coupled receptors, PKR1 and PKR2. This signaling system has been linked to several important biological functions, including gastrointestinal tract motility, regulation of circadian rhythms, neurogenesis, angiogenesis and cancer progression, hematopoiesis, and nociception. Mutations in PKR2 or Bv8/PROK2 have been associated with Kallmann syndrome, a developmental disorder characterized by defective olfactory bulb neurogenesis, impaired development of gonadotropin-releasing hormone neurons, and infertility. Also, Bv8/PROK2 is strongly upregulated in neutrophils and other inflammatory cells in response to granulocyte-colony stimulating factor or other myeloid growth factors and functions as a pronociceptive mediator in inflamed tissues as well as a regulator of myeloid cell-dependent tumor angiogenesis. Bv8/PROK2 has been also implicated in neuropathic pain. Anti-Bv8/PROK2 antibodies or small molecule PKR inhibitors ameliorate pain arising from tissue injury and inhibit angiogenesis and inflammation associated with tumors or some autoimmune disorders.
Asunto(s)
Mediadores de Inflamación/metabolismo , Inflamación/metabolismo , Células Mieloides/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Movimiento Celular/fisiología , Humanos , Neurotransmisores/metabolismoRESUMEN
BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder recognized as the most common cause of chronic dementia among the ageing population. AD is histopathologically characterized by progressive loss of neurons and deposits of insoluble proteins, primarily composed of amyloid-ß pelaques and neurofibrillary tangles (NFTs). METHODS: Several molecular processes contribute to the formation of AD cellular hallmarks. Among them, post-translational modifications (PTMs) represent an attractive mechanism underlying the formation of covalent bonds between chemical groups/peptides to target proteins, which ultimately result modified in their function. Most of the proteins related to AD undergo PTMs. Several recent studies show that AD-related proteins like APP, Aß, tau, BACE1 undergo post-translational modifications. The effect of PTMs contributes to the normal function of cells, although aberrant protein modification, which may depend on many factors, can drive the onset or support the development of AD. RESULTS: Here we will discuss the effect of several PTMs on the functionality of AD-related proteins potentially contributing to the development of AD pathology. CONCLUSION: We will consider the role of Ubiquitination, Phosphorylation, SUMOylation, Acetylation and Nitrosylation on specific AD-related proteins and, more interestingly, the possible interactions that may occur between such different PTMs.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Procesamiento Proteico-Postraduccional/fisiología , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Humanos , Ovillos Neurofibrilares/fisiología , Fosforilación , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Prokineticin1 and prokineticin2 belong to a new family of chemokines identified in several species including mammals and characterized by the presence of five disulfide bridges. These proteins signal through two G-coupled receptors (prokineticin-receptor1 and prokineticin- receptor2) widely expressed in all tissues and involved in a large spectrum of biological activities, including angiogenesis, hematopoiesis, immune processes, inflammation and nociceptive transmission. Prokineticin2 is overexpressed in inflamed tissues and has a crucial role in neutrophil dependent inflammation and hypernociception. Following tissue inflammation, peripheral nerve injury, cancer, bone metastasis the expression of prokineticin2 and of the prokineticin-receptor2 is increased also within dorsal root ganglia and spinal cord. Prokineticin receptors, highly expressed in nociceptor endings and dorsal root ganglia, exert a tonic activation of TRPV1 and TRPA1 contributing to peripheral sensitization. Prokineticin2-induces activation of the prokineticin receptors in the spinal dorsal horn and in activated astrocytes contributes to central sensitization and maintains chronic and neuropathic pain. Prokineticin2, acting on prokineticin receptors on monocytes, macrophages and dendritic cells, induces chemotaxis and release of inflammatory and pronociceptive cytokines. Hence, the prokineticin system represents a novel therapeutic target in chronic pain conditions. Evaluation of the mechanism of action of prokineticin2 and the potential effectiveness of its inhibition is discussed.
Asunto(s)
Dolor Crónico/fisiopatología , Citocinas/fisiología , Hormonas Gastrointestinales/fisiología , Inflamación/fisiopatología , Neuropéptidos/fisiología , Receptores Acoplados a Proteínas G/fisiología , Animales , Dolor Crónico/metabolismo , Citocinas/química , Citocinas/genética , Citocinas/metabolismo , Hormonas Gastrointestinales/química , Hormonas Gastrointestinales/genética , Hormonas Gastrointestinales/metabolismo , Humanos , Inflamación/metabolismo , Neuralgia/metabolismo , Neuralgia/fisiopatología , Neuropéptidos/química , Neuropéptidos/genética , Neuropéptidos/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/química , Receptores de Péptidos/metabolismo , Transducción de Señal/fisiología , Regulación hacia Arriba , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/química , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismoRESUMEN
The accumulation of ß-amyloid (Aß) is one of the hallmarks of Alzheimer disease (AD). Beyond the inflammatory reactions promoted by Aß, it has been demonstrated that the prokineticin (PK) system, composed of the chemokine prokineticin 2 (PK2) and its receptors, is involved in Aß toxicity. In this study we have analyzed how the Aß chronic treatment affects the glutamatergic transmission on neurons from primary cortical cultures, clearly demonstrating the PK system involvement on its action mechanism. In fact, we have observed a significant increase of the ionic current through the AMPA receptors in primary cortical neurons and an up-regulation of the PK system in cultures chronically treated with Aß. All effects were nullified by the prokineticin antagonist PC-1. Moreover, we have herein firstly demonstrated that the incubation of primary cortical culture with Bv8, the amphibian homologue of PK2, was able to increase in neurons the AMPA currents at specific doses and exposure times, measured both as evoked and as spontaneous currents. This effect was not due to a modification of the AMPA receptor subunit expression. In contrast, the up-modulation of AMPA currents were blocked by PC-1 and were mediated by the activation of the intracellular protein kinase C (PKC) transduction pathways because Gö6983, the PKC inhibitor added in the medium, nullified the effect. Finally, cellular death induced by kainate was also reduced following treatment with PC1. In conclusion, our results show that the prokineticin system may be a key mediator in the Aß-induced neuronal damage, suggesting PK antagonists as new therapeutic compounds to ameliorate the AD progression.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Proteínas Anfibias/uso terapéutico , Péptidos beta-Amiloides/toxicidad , Ácido Glutámico/metabolismo , Neuropéptidos/uso terapéutico , Fármacos Neuroprotectores/farmacología , Enfermedad de Alzheimer/patología , Animales , Anuros , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Hormonas Gastrointestinales/metabolismo , Indoles/farmacología , Maleimidas/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Neuropéptidos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas Wistar , Receptores AMPA/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Bv8/prokineticin 2 (PK2) is a member of a bioactive family of peptides that regulate multiple functions in the CNS including hyperalgesia, neurogenesis, neuronal survival and inflammation. Recent studies have associated PK2 and prokineticin receptors (PKR) with human diseases, but because their role in neuropathology is still debated we examined whether prokineticins exert a protective or deleterious role in models of cerebral ischemia and ischemic tolerance in vitro. In order to mimic cerebral ischemia, we exposed primary murine cortical cell cultures or rat organotypic hippocampal slices to appropriate periods of oxygen-glucose deprivation (OGD), which leads to neuronal damage 24 h later. Ischemic tolerance was induced by exposing hippocampal slices to a preconditioning subtoxic pharmacological stimulus (3 µM NMDA for 1 h) 24 h before the exposure to OGD. Bv8 (10-100 nM) attenuated OGD injury in cortical cultures and hippocampal slices, and the effect was prevented by the PKR antagonist PC7. The development of OGD tolerance was associated with an increase in the expression of PK2, PKR1 and PKR2 mRNA and proteins and was prevented by addition of the antagonist PC7 into the medium during preconditioning. Both Bv8 at protective concentrations and the NMDA preconditioning stimulus promoted the phosphorylation of ERK1/2 and Akt. These findings indicate that the prokineticin system can be up-regulated by a defensive preconditioning subtoxic NMDA stimulus and that PK2 may act as an endogenous neuroprotective factor through the activation of the ERK1/2 and Akt transduction pathways.
Asunto(s)
Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Hormonas Gastrointestinales/biosíntesis , Hipocampo/metabolismo , Neuropéptidos/biosíntesis , Fármacos Neuroprotectores/metabolismo , Animales , Anuros , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Isquemia Encefálica/prevención & control , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hormonas Gastrointestinales/farmacología , Hormonas Gastrointestinales/uso terapéutico , Hipocampo/efectos de los fármacos , Ratones , Neuropéptidos/farmacología , Neuropéptidos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Técnicas de Cultivo de Órganos , RatasRESUMEN
Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease.
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Diabetes Mellitus Experimental/metabolismo , Hormonas Gastrointestinales/metabolismo , Hiperalgesia/metabolismo , Inflamación/metabolismo , Neuropéptidos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Western Blotting , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus Experimental/genética , Modelos Animales de Enfermedad , Hormonas Gastrointestinales/genética , Expresión Génica , Hiperalgesia/genética , Hiperalgesia/prevención & control , Inflamación/genética , Inflamación/prevención & control , Masculino , Ratones Endogámicos C57BL , Neuralgia/genética , Neuralgia/metabolismo , Neuralgia/prevención & control , Neuropéptidos/genética , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nervio Ciático/metabolismo , Médula Espinal/metabolismo , Triazinas/farmacologíaRESUMEN
Bv8/Prokineticin 2 (PROK2) is a bioactive peptide initially discovered as a regulator of gastrointestinal motility. Among multiple biological roles demonstrated for PROK2, it was recently established that PROK2 is an insult-inducible endangering mediator for cerebral damage. Aim of the present study was to evaluate the PROK2 and its receptors' potential involvement in amyloid beta (Aß) neurotoxicity, a hallmark of Alzheimer's disease (AD) and various forms of traumatic brain injury (TBI). Analyzing primary cortical cultures (CNs) and cortex and hippocampus from Aß treated rats, we found that PROK2 and its receptors PKR1 and PKR2 mRNA are up-regulated by Aß, suggesting their potential involvement in AD. Hence we evaluated if impairing the prokineticin system activation might have protective effect against neuronal death induced by Aß. We found that a PKR antagonist concentration-dependently protects CNs against Aß(1-42)-induced neurotoxicity, by reducing the Aß-induced PROK2 neuronal up-regulation. Moreover, the antagonist completely rescued LTP impairment in hippocampal slices from 6 month-old Tg2576 AD mice without affecting basal synaptic transmission and paired pulse-facilitation paradigms. These results indicate that PROK2 plays a role in cerebral amyloidosis and that PROK2 antagonists may represent a new approach for ameliorating the defining pathology of AD.
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Péptidos beta-Amiloides/metabolismo , Hormonas Gastrointestinales/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Animales , Apoptosis/efectos de los fármacos , Hormonas Gastrointestinales/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/genética , Masculino , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuropéptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Transporte de Proteínas , Ratas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVE: To investigate the potential role of prokineticin 2 (PK2), a bioactive peptide involved in multiple biological functions including immune modulation, in CNS autoimmune demyelinating disease. METHODS: We investigated the expression of PK2 in mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), and in patients with relapsing-remitting MS. We evaluated the biological effects of PK2 on expression of EAE and on development of T-cell response against myelin by blocking PK2 in vivo with PK2 receptor antagonists. We treated with PK2 immune cells activated against myelin antigen to explore the immune-modulating effects of this peptide in vitro. RESULTS: Pk2 messenger RNA was upregulated in spinal cord and lymph node cells (LNCs) of mice with EAE. PK2 protein was expressed in EAE inflammatory infiltrates and was increased in sera during EAE. In patients with relapsing-remitting MS, transcripts for PK2 were significantly increased in peripheral blood mononuclear cells compared with healthy controls, and PK2 serum concentrations were significantly higher. A PK2 receptor antagonist prevented or attenuated established EAE in chronic and relapsing-remitting models, reduced CNS inflammation and demyelination, and decreased the production of interferon (IFN)-γ and interleukin (IL)-17A cytokines in LNCs while increasing IL-10. PK2 in vitro increased IFN-γ and IL-17A and reduced IL-10 in splenocytes activated against myelin antigen. CONCLUSION: These data suggest that PK2 is a critical immune regulator in CNS autoimmune demyelination and may represent a new target for therapy.
RESUMEN
The new chemokine Prokineticin 2 (PROK2) and its receptors (PKR1 and PKR2) have a role in inflammatory pain and immunomodulation. Here we identified PROK2 as a critical mediator of neuropathic pain in the chronic constriction injury (CCI) of the sciatic nerve in mice and demonstrated that blocking the prokineticin receptors with two PKR1-preferring antagonists (PC1 and PC7) reduces pain and nerve damage. PROK2 mRNA expression was upregulated in the injured nerve since day 3 post injury (dpi) and in the ipsilateral DRG since 6 dpi. PROK2 protein overexpression was evident in Schwann Cells, infiltrating macrophages and axons in the peripheral nerve and in the neuronal bodies and some satellite cells in the DRG. Therapeutic treatment of neuropathic mice with the PKR-antagonist, PC1, impaired the PROK2 upregulation and signalling. This fact, besides alleviating pain, brought down the burden of proinflammatory cytokines in the damaged nerve and prompted an anti-inflammatory repair program. Such a treatment also reduced intraneural oedema and axon degeneration as demonstrated by the physiological skin innervation and thickness conserved in CCI-PC1 mice. These findings suggest that PROK2 plays a crucial role in neuropathic pain and might represent a novel target of treatment for this disease.
Asunto(s)
Hormonas Gastrointestinales/biosíntesis , Neuralgia/metabolismo , Neuropéptidos/biosíntesis , Células de Schwann/metabolismo , Nervio Ciático/metabolismo , Transducción de Señal , Regulación hacia Arriba , Animales , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Neuralgia/patología , Receptores Acoplados a Proteínas G/metabolismo , Células de Schwann/patología , Nervio Ciático/lesiones , Nervio Ciático/patología , Subtilisinas/metabolismoRESUMEN
A new efficient synthetic method to obtain prokineticin receptor antagonists based on the triazinedione scaffold is described. In this procedure the overall yield improves from 13% to about 54%, essentially for two factors: 1) N-(chlorocarbonyl) isocyanate is no more used, it represents the yield limiting step with an average yield not exceeding 30%. 2) The Mitsunobu reaction is not involved in the new synthetic scheme avoiding the use of time and solvent consuming column chromatography. All synthesized triazinediones were preliminary pharmacologically screened in vivo for their ability to reduce the Bv8-induced thermal hyperalgesia. In this assay all compounds displayed EC50 values in the picomolar-subpicomolar range, some triazinediones containing a 4-halogen substituted benzyl group in position 5 showed the best activity. The analogues containing a 4-fluorine atom (PC-7) and a 4-bromobenzyl group (PC-25) resulted 10 times more potent than the reference PC-1 that bears a 4-ethylbenzyl group. While the 4-trifluoromethylbenzyl substituted analog (PC-27) was 100 times more potent as compared to PC1.
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Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Triazinas/farmacología , Animales , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Relación Estructura-Actividad , Triazinas/administración & dosificación , Triazinas/síntesis químicaRESUMEN
Alzheimer's disease (AD) is a complex disorder that affects the central nervous system causing a severe neurodegeneration. This pathology affects an increasing number of people worldwide due to the overall aging of the human population. In recent years SUMO protein modification has emerged as a possible cellular mechanism involved in AD. Some of the proteins engaged in the physiopathological process of AD, like BACE1, GSK3-ß tau, AßPP, and JNK, are in fact subject to protein SUMO modifications or interactions. Here, we have investigated the SUMO/deSUMOylation balance and SUMO-related proteins during the onset and progression of the pathology in the Tg2576 mouse model of AD. We examined four age-stages (1.5, 3, 6, 17 months old) and observed shows an increase in SUMO-1 protein conjugation at 3 and 6 months in transgenic mice with respect to WT in both cortex and hippocampus. Interestingly this is paralleled by increased expression levels of Ubc9 and SENP1 in both brain regions. At 6 months of age also the SUMO-1 mRNA resulted augmented. SUMO-2-ylation was surprisingly decreased in old transgenic mice and was unaltered in the other time windows. The fact that alterations in SUMO/deSUMOylation equilibrium occur from the early phases of AD suggests that global posttranslational modifications may play an important role in the mechanisms underlying disease pathogenesis, thus providing potential targets for pharmacological interventions.
RESUMEN
Bv8 is a pronociceptive peptide that binds to two G-protein coupled prokineticin receptors, PK-R1 and PK-R2. These receptors are localized in the dorsal horn of the spinal cord and dorsal root ganglia (DRG) of nociceptive neurons in rodents. Systemic administration of Bv8 elicits a biphasic reduction in nociceptive thresholds to thermal and mechanical stimuli. Here, the possibility that Bv8 might directly modulate the expression and release of excitatory transmitters within the early and late phases of hyperalgesia was evaluated. Administration of Bv8 to mouse lumbar spinal cord sections produced a direct, significant and concentration-related release of CGRP. Bv8- or capsaicin-stimulated CGRP release was markedly enhanced in tissues taken from Bv8-pretreated mice during the late, but not the early, phase of hyperalgesia. Pretreatment of rats with protein synthesis inhibitors blocked the expression of the late, but not early, phase of Bv8-induced hyperalgesia. Finally, during the late-phase of hyperalgesia, there was an upregulation of CGRP and substance P immunoreactivity in the rat lumbar dorsal horn and DRG. Such upregulation was prevented by pretreatment with protein synthesis inhibitors. These data suggest that Bv8 induces hyperalgesia by direct release of excitatory transmitters in the spinal cord, consistent with the first phase of hyperalgesia. Additionally, Bv8 elicits a subsequent, protein-synthesis dependent increase in expression and release of excitatory transmitters that may underlie the long-lasting second phase of hyperalgesia. Activation of prokineticin receptors may therefore contribute to persistent hyperalgesia occurring as a consequence of tissue injury further suggesting that these receptors are attractive targets for development of therapeutics for pain treatment.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Hiperalgesia/metabolismo , Neuropéptidos/fisiología , Médula Espinal/metabolismo , Animales , Anisomicina/farmacología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Sustancia P/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Mammalian Bv8 (also called prokineticin 2) is a secreted protein that regulates diverse biological processes including pain perception. It belongs to a new family of chemokines, which activate two G-protein linked receptors (prokineticin receptor 1 and 2, PKR1 and PKR2) expressed in regions of the nervous system associated with pain and in cells participating to immuno-inflammatory responses. Primary sensitive neurons co-express PKRs and the transient potential receptor vanilloid 1, cooperating in nociceptor sensitization. Bv8, strongly upregulated in neutrophils and other inflammatory cells, is a main pronociceptive mediator in inflamed tissues, where it sensitizes peripheral nociceptors, stimulates neutrophil chemotaxis and modulates the release of inflammatory and pronociceptive cytokines. Availability of a nonpeptide PKR antagonist, leading to blockade of the Bv8/PKR system, ameliorates pain arising from tissue injury and reduces the time required for recovery from injury.
Asunto(s)
Analgésicos/farmacología , Hormonas Gastrointestinales/metabolismo , Neuropéptidos/metabolismo , Dolor/tratamiento farmacológico , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Animales , Humanos , Dolor/metabolismoRESUMEN
Bv8 is a small protein secreted by frog skin. Mammalian homologues of Bv8, the prokineticins PK1 and PK2, and their G-protein coupled receptors PKR1 and PKR2 have been identified and linked to several biological effects as gut motility, circadian rhythms, neurogenesis, angiogenesis and cancer progression, haematopoiesis and nociception. In rodents, administration of amphibian Bv8 lowers nociceptor thresholds to a broad spectrum of physical and chemical stimuli. The prokineticin receptors are present in regions of the nervous system associated with pain; primary sensitive neurons expressing PKRs also express the vanilloid receptor TRPV1, providing an anatomical basis for PKR1/TRPV1 cooperative interaction in nociceptor sensitization. Bv8/PK2, strongly up-regulated in neutrophils and other inflammatory cells, is a main pronociceptive mediator in inflamed tissues. Indeed Bv8/PK2 produced by inflammatory cells is released at the site of inflammation where it sensitizes peripheral nociceptors, stimulates chemotaxis and modulates the release of inflammatory and pronociceptive cytokines. Availability of a non-peptide PKR antagonist, leading to blockade the PK/PKR system, ameliorates pain arising from tissue injury and, additionally, reduces the time required for recovery from injury.
Asunto(s)
Hormonas Gastrointestinales/metabolismo , Neuropéptidos/metabolismo , Dolor/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Animales , Hematopoyesis/fisiología , Humanos , Inflamación/metabolismo , Sistema Nervioso/metabolismoRESUMEN
Bv8 is an amphibian peptide belonging to the widely distributed AVIT protein family. The mammalian orthologues of Bv8 were named prokineticin 1 and prokineticin 2. Two G-protein-coupled receptors for Bv8-prokineticins have been identified. The biological activities of Bv8/PK proteins range from angiogenesis and involvement in reproduction and cancer, to neuronal survival and neurogenesis, hypothalamic hormone secretion, circadian rhythm control and immunomodulatory processes. Identifying the structural determinants required for receptor binding of Bv8-PKs is mandatory for the design of PKR antagonists, which may be useful in the treatment and prevention of various disease states. Here we describe a procedure for the production in Pichia pastoris of Bv8 and 3 mutants: W24A-Bv8, in which the tryptophan in position 24 is substituted by alanine, the double mutant M1-W24A-Bv8, that contains an additional methionine at the N-terminus and Bv8-TyrTyr that includes two additional tyrosines at the C-terminus. The results evidence a relevant role of tryptophan 24 in Bv8-PKRs interaction.
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Proteínas Anfibias/biosíntesis , Anuros/genética , Neuropéptidos/biosíntesis , Pichia/genética , Proteínas Recombinantes/metabolismo , Proteínas Anfibias/química , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Cinética , Modelos Moleculares , Mutación , Neuropéptidos/química , Neuropéptidos/genética , Neuropéptidos/metabolismo , Pichia/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , TriptófanoRESUMEN
An efficient route was developed for the synthesis of the Fmoc-protected dipeptide 4, isostere of Gly-Gly containing an alpha-methylene beta-amino acid; the conformationally restricted analogues of Leu-enkephalin, 3a, and Met-enkephalin, 3b, respectively, were prepared by changing 4 for Gly(2)-Gly(3) in the native compounds 3a and 3b whose biological activities were significantly lower than the parent compounds.
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Dipéptidos/química , Dipéptidos/síntesis química , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacología , Encefalina Metionina/análogos & derivados , Encefalina Metionina/farmacología , Neurotransmisores/síntesis química , Neurotransmisores/farmacología , Secuencia de Aminoácidos , Animales , Diseño de Fármacos , Estimulación Eléctrica , Encefalina Leucina/química , Encefalina Metionina/química , Glicilglicina/análogos & derivados , Glicilglicina/síntesis química , Glicilglicina/química , Cobayas , Íleon , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Imitación Molecular , Estructura Molecular , Plexo Mientérico/efectos de los fármacos , Plexo Mientérico/fisiología , Neurotransmisores/química , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Estereoisomerismo , Conducto DeferenteRESUMEN
Neutrophil migration into injured tissues is invariably accompanied by pain. Bv8/prokineticin 2 (PK2), a chemokine characterized by a unique structural motif comprising five disulfide bonds, is highly expressed in inflamed tissues associated to infiltrating cells. Here, we demonstrate the fundamental role of granulocyte-derived PK2 (GrPK2) in initiating inflammatory pain and driving peripheral sensitization. In animal models of complete Freund's adjuvant-induced paw inflammation the development and duration of pain temporally correlated with the expression levels of PK2 in the inflamed sites. Such an increase in PK2 mRNA depends mainly on a marked up-regulation of PK2 gene transcription in granulocytes. A substantially lower up-regulation was also detected in macrophages. From a pool of peritoneal granulocytes, elicited in rats by oyster glycogen, we purified the GrPK2 protein, which displayed high affinity for the prokineticin receptors (PKRs) and, when injected into the rat paw, induced hypersensitivity to noxious stimuli as the amphibian prokineticin Bv8 did. Mice lacking PKR1 or PKR2 developed significantly less inflammation-induced hyperalgesia in comparison with WT mice, confirming the involvement of both PKRs in inflammatory pain. The inflammation-induced up-regulation of PK2 was significantly less in pkr1 null mice than in WT and pkr2 null mice, demonstrating a role of PKR1 in setting PK2 levels during inflammation. Pretreatment with a nonpeptide PKR antagonist, which preferentially binds PKR1, dose-dependently reduced and eventually abolished both prokineticin-induced hypernociception and inflammatory hyperalgesia. Inhibiting PK2 formation or antagonizing PKRs may represent another therapeutic approach for controlling inflammatory pain.