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2.
PLoS Biol ; 15(7): e2002457, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28719605

RESUMO

Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function.


Assuntos
Dor/metabolismo , Receptor EphB2/metabolismo , Receptores de N-Metil-D-Aspartato/análise , Animais , Células HEK293 , Humanos , Camundongos , Neurônios/metabolismo , Fosforilação , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Análise de Sequência de Proteína , Medula Espinal/metabolismo , Medula Espinal/patologia , Tirosina/metabolismo
3.
J Neurosci ; 35(16): 6307-17, 2015 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-25904784

RESUMO

The mechanisms that lead to the maintenance of chronic pain states are poorly understood, but their elucidation could lead to new insights into how pain becomes chronic and how it can potentially be reversed. We investigated the role of spinal dorsal horn neurons and descending circuitry in plasticity mediating a transition to pathological pain plasticity suggesting the presence of a chronic pain state using hyperalgesic priming. We found that when dorsal horn neurokinin 1 receptor-positive neurons or descending serotonergic neurons were ablated before hyperalgesic priming, IL-6- and carrageenan-induced mechanical hypersensitivity was impaired, and subsequent prostaglandin E2 (PGE2) response was blunted. However, when these neurons were lesioned after the induction of priming, they had no effect on the PGE2 response, reflecting differential mechanisms driving plasticity in a primed state. In stark contrast, animals with a spinally applied dopaminergic lesion showed intact IL-6- and carrageenan-induced mechanical hypersensitivity, but the subsequent PGE2 injection failed to cause mechanical hypersensitivity. Moreover, ablating spinally projecting dopaminergic neurons after the resolution of the IL-6- or carrageenan-induced response also reversed the maintenance of priming as assessed through mechanical hypersensitivity and the mouse grimace scale. Pharmacological antagonism of spinal dopamine D1/D5 receptors reversed priming, whereas D1/D5 agonists induced mechanical hypersensitivity exclusively in primed mice. Strikingly, engagement of D1/D5 coupled with anisomycin in primed animals reversed a chronic pain state, consistent with reconsolidation-like effects in the spinal dorsal horn. These findings demonstrate a novel role for descending dopaminergic neurons in the maintenance of pathological pain plasticity.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Células do Corno Posterior/fisiologia , Receptores de Dopamina D1/fisiologia , Receptores de Dopamina D5/fisiologia , Receptores da Neurocinina-1/fisiologia , Animais , Benzazepinas/farmacologia , Carragenina/farmacologia , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Hiperalgesia/induzido quimicamente , Interleucina-6/farmacologia , Masculino , Camundongos , Células do Corno Posterior/efeitos dos fármacos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D5/agonistas , Receptores de Dopamina D5/antagonistas & inibidores , Neurônios Serotoninérgicos/fisiologia , Sulpirida/farmacologia
4.
Mol Pain ; 10: 45, 2014 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-24993495

RESUMO

Transcriptional regulation of genes by cyclic AMP response element binding protein (CREB) is essential for the maintenance of long-term memory. Moreover, retrograde axonal trafficking of CREB in response to nerve growth factor (NGF) is critical for the survival of developing primary sensory neurons. We have previously demonstrated that hindpaw injection of interleukin-6 (IL-6) induces mechanical hypersensitivity and hyperalgesic priming that is prevented by the local injection of protein synthesis inhibitors. However, proteins that are locally synthesized that might lead to this effect have not been identified. We hypothesized that retrograde axonal trafficking of nascently synthesized CREB might link local, activity-dependent translation to nociceptive plasticity. To test this hypothesis, we determined if IL-6 enhances the expression of CREB and if it subsequently undergoes retrograde axonal transport. IL-6 treatment of sensory neurons in vitro caused an increase in CREB protein and in vivo treatment evoked an increase in CREB in the sciatic nerve consistent with retrograde transport. Importantly, co-injection of IL-6 with the methionine analogue azido-homoalanine (AHA), to assess nascently synthesized proteins, revealed an increase in CREB containing AHA in the sciatic nerve 2 hrs post injection, indicating retrograde transport of nascently synthesized CREB. Behaviorally, blockade of retrograde transport by disruption of microtubules or inhibition of dynein or intrathecal injection of cAMP response element (CRE) consensus sequence DNA oligonucleotides, which act as decoys for CREB DNA binding, prevented the development of IL-6-induced mechanical hypersensitivity and hyperalgesic priming. Consistent with previous studies in inflammatory models, intraplantar IL-6 enhanced the expression of BDNF in dorsal root ganglion (DRG). This effect was blocked by inhibition of retrograde axonal transport and by intrathecal CRE oligonucleotides. Collectively, these findings point to a novel mechanism of axonal translation and retrograde trafficking linking locally-generated signals to long-term nociceptive sensitization.


Assuntos
Transporte Axonal/efeitos dos fármacos , Proteína de Ligação a CREB/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Dor Nociceptiva/induzido quimicamente , Células Receptoras Sensoriais/efeitos dos fármacos , Animais , Transporte Axonal/fisiologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Colchicina/farmacologia , Modelos Animais de Doenças , Gânglios Espinais/patologia , Interleucina-6/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dor Nociceptiva/patologia , Nocodazol/farmacologia , Transporte Proteico/efeitos dos fármacos , Quinazolinonas/farmacologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Células Receptoras Sensoriais/metabolismo , Moduladores de Tubulina/farmacologia
5.
FASEB J ; 27(4): 1498-510, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23292071

RESUMO

Protease-activated receptor-2 (PAR2) is a G-protein coupled receptor (GPCR) associated with a variety of pathologies. However, the therapeutic potential of PAR2 is limited by a lack of potent and specific ligands. Following proteolytic cleavage, PAR2 is activated through a tethered ligand. Hence, we reasoned that lipidation of peptidomimetic ligands could promote membrane targeting and thus significantly improve potency and constructed a series of synthetic tethered ligands (STLs). STLs contained a peptidomimetic PAR2 agonist (2-aminothiazol-4-yl-LIGRL-NH2) bound to a palmitoyl group (Pam) via polyethylene glycol (PEG) linkers. In a high-throughput physiological assay, these STL agonists displayed EC50 values as low as 1.47 nM, representing a ∼200 fold improvement over the untethered parent ligand. Similarly, these STL agonists were potent activators of signaling pathways associated with PAR2: EC50 for Ca(2+) response as low as 3.95 nM; EC50 for MAPK response as low as 9.49 nM. Moreover, STLs demonstrated significant improvement in potency in vivo, evoking mechanical allodynia with an EC50 of 14.4 pmol. STLs failed to elicit responses in PAR2(-/-) cells at agonist concentrations of >300-fold their EC50 values. Our results demonstrate that the STL approach is a powerful tool for increasing ligand potency at PAR2 and represent opportunities for drug development at other protease activated receptors and across GPCRs.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Palmitatos/farmacologia , Peptidomiméticos/farmacologia , Receptor PAR-2/agonistas , Cálcio/metabolismo , Linhagem Celular/efeitos dos fármacos , Humanos , Hiperalgesia/tratamento farmacológico , Ligantes , Ornitina/análogos & derivados , Ornitina/farmacologia , Relação Estrutura-Atividade
6.
Mol Pain ; 9: 12, 2013 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-23510079

RESUMO

BACKGROUND: Chronic pain is an important medical problem affecting hundreds of millions of people worldwide. Mechanisms underlying the maintenance of chronic pain states are poorly understood but the elucidation of such mechanisms have the potential to reveal novel therapeutics capable of reversing a chronic pain state. We have recently shown that the maintenance of a chronic pain state is dependent on an atypical PKC, PKMζ, but the mechanisms involved in controlling PKMζ in chronic pain are completely unknown. Here we have tested the hypothesis that brain derived neurotrophic factor (BDNF) regulates PKMζ, and possibly other aPKCs, to maintain a centralized chronic pain state. RESULTS: We first demonstrate that although other kinases play a role in the initiation of persistent nociceptive sensitization, they are not involved in the maintenance of this chronic pain state indicating that a ZIP-reversible process is responsible for the maintenance of persistent sensitization. We further show that BDNF plays a critical role in initiating and maintaining persistent nociceptive sensitization and that this occurs via a ZIP-reversible process. Moreover, at spinal synapses, BDNF controls PKMζ and PKCλ nascent synthesis via mTORC1 and BDNF enhances PKMζ phosphorylaton. Finally, we show that BDNF signaling to PKMζ and PKCλ is conserved across CNS synapses demonstrating molecular links between pain and memory mechanisms. CONCLUSIONS: Hence, BDNF is a key regulator of aPKC synthesis and phosphorylation and an essential mediator of the maintenance of a centralized chronic pain state. These findings point to BDNF regulation of aPKC as a potential therapeutic target for the permanent reversal of a chronic pain state.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Dor Crônica/enzimologia , Proteína Quinase C/metabolismo , Sinapses/enzimologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/patologia , Dor Crônica/patologia , Fator de Iniciação 4F em Eucariotos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/enzimologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Transporte Proteico/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo
7.
J Neurosci ; 31(18): 6646-53, 2011 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-21543593

RESUMO

Sensitization of the pain pathway is believed to promote clinical pain disorders. We hypothesized that the persistence of a sensitized state in the spinal dorsal horn might depend on the activity of protein kinase M ζ (PKMζ), an essential mechanism of late long-term potentiation (LTP). To test this hypothesis, we used intraplantar injections of interleukin-6 (IL-6) in mice to elicit a transient allodynic state that endured ∼3 d. After the resolution of IL-6-induced allodynia, a subsequent intraplantar injection of prostaglandin E(2) (PGE(2)) or intrathecal injection of the metabotropic glutamate receptor 1/5 (mGluR1/5) agonist DHPG (dihydroxyphenylglycol) precipitated allodynia and/or nocifensive responses. Intraplantar injection of IL-6 followed immediately by intrathecal injection of a PKMζ inhibitor prevented the expression of subsequent PGE(2)-induced allodynia. Inhibitors of protein translation were effective in preventing PGE(2)-induced allodynia when given immediately after IL-6, but not after the initial allodynia had resolved. In contrast, spinal PKMζ inhibition completely abolished both prolonged allodynia to hindpaw PGE(2) and enhanced nocifensive behaviors evoked by intrathecal mGluR1/5 agonist injection after the resolution of IL-6-induced allodynia. Moreover, spinal PKMζ inhibition prevented the enhanced response to subsequent stimuli following resolution of hypersensitivity induced by plantar incision. The present findings demonstrate that the spinal cord encodes an engram for persistent nociceptive sensitization that is analogous to molecular mechanisms of late LTP and suggest that spinally directed PKMζ inhibitors may offer therapeutic benefit for injury-induced pain states.


Assuntos
Hiperalgesia/metabolismo , Percepção da Dor/fisiologia , Proteína Quinase C/metabolismo , Medula Espinal/metabolismo , Análise de Variância , Animais , Comportamento Animal , Dinoprostona/farmacologia , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Hiperalgesia/induzido quimicamente , Injeções Espinhais , Interleucina-6/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Mutantes Neurológicos , Medição da Dor/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Proteína Quinase C/antagonistas & inibidores , Medula Espinal/efeitos dos fármacos
8.
J Biol Chem ; 286(21): 19076-88, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21467041

RESUMO

Protease-activated receptor-2 (PAR(2)) is one of four protease-activated G-protein-coupled receptors. PAR(2) is expressed on multiple cell types where it contributes to cellular responses to endogenous and exogenous proteases. Proteolytic cleavage of PAR(2) reveals a tethered ligand that activates PAR(2) and two major downstream signaling pathways: mitogen-activated protein kinase (MAPK) and intracellular Ca(2+) signaling. Peptides or peptidomimetics can mimic binding of the tethered ligand to stimulate signaling without the nonspecific effects of proteases. The most commonly used peptide activators of PAR(2) (e.g. SLIGRL-NH(2) and SLIGKV-NH(2)) lack potency at the receptor. However, although the potency of 2-furoyl-LIGRLO-NH(2) (2-f-LIGRLO-NH(2)) underscores the use of peptidomimetic PAR(2) ligands as a mechanism to enhance pharmacological action at PAR(2), 2-f-LIGRLO-NH(2) has not been thoroughly evaluated. We evaluated the known agonist 2-f-LIGRLO-NH(2) and two recently described pentapeptidomimetic PAR(2)-specific agonists, 2-aminothiazol-4-yl-LIGRL-NH(2) (2-at-LIGRL-NH(2)) and 6-aminonicotinyl-LIGRL-NH(2) (6-an-LIGRL-NH(2)). All peptidomimetic agonists stimulated PAR(2)-dependent in vitro physiological responses, MAPK signaling, and Ca(2+) signaling with an overall rank order of potency of 2-f-LIGRLO-NH(2) ≈ 2-at-LIGRL-NH(2) > 6-an-LIGRL-NH(2) ≫ SLIGRL-NH(2). Because PAR(2) plays a major role in pathological pain conditions and to test potency of the peptidomimetic agonists in vivo, we evaluated these agonists in models relevant to nociception. All three agonists activated Ca(2+) signaling in nociceptors in vitro, and both 2-at-LIGRL-NH(2) and 2-f-LIGRLO-NH(2) stimulated PAR(2)-dependent thermal hyperalgesia in vivo. We have characterized three high potency ligands that can be used to explore the physiological role of PAR(2) in a variety of systems and pathologies.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Receptor PAR-2/agonistas , Cálcio/metabolismo , Sinalização do Cálcio/genética , Linhagem Celular Transformada , Humanos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/genética , Hiperalgesia/metabolismo , Ligantes , Sistema de Sinalização das MAP Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Peptidomiméticos/síntese química , Receptor PAR-2/genética , Receptor PAR-2/metabolismo
9.
Mol Pain ; 8: 5, 2012 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-22269797

RESUMO

BACKGROUND: Despite advances in our understanding of basic mechanisms driving post-surgical pain, treating incision-induced pain remains a major clinical challenge. Moreover, surgery has been implicated as a major cause of chronic pain conditions. Hence, more efficacious treatments are needed to inhibit incision-induced pain and prevent the transition to chronic pain following surgery. We reasoned that activators of AMP-activated protein kinase (AMPK) may represent a novel treatment avenue for the local treatment of incision-induced pain because AMPK activators inhibit ERK and mTOR signaling, two important pathways involved in the sensitization of peripheral nociceptors. RESULTS: To test this hypothesis we used a potent and efficacious activator of AMPK, resveratrol. Our results demonstrate that resveratrol profoundly inhibits ERK and mTOR signaling in sensory neurons in a time- and concentration-dependent fashion and that these effects are mediated by AMPK activation and independent of sirtuin activity. Interleukin-6 (IL-6) is thought to play an important role in incision-induced pain and resveratrol potently inhibited IL-6-mediated signaling to ERK in sensory neurons and blocked IL-6-mediated allodynia in vivo through a local mechanism of action. Using a model of incision-induced allodynia in mice, we further demonstrate that local injection of resveratrol around the surgical wound strongly attenuates incision-induced allodynia. Intraplantar IL-6 injection and plantar incision induces persistent nociceptive sensitization to PGE2 injection into the affected paw after the resolution of allodynia to the initial stimulus. We further show that resveratrol treatment at the time of IL-6 injection or plantar incision completely blocks the development of persistent nociceptive sensitization consistent with the blockade of a transition to a chronic pain state by resveratrol treatment. CONCLUSIONS: These results highlight the importance of signaling to translation control in peripheral sensitization of nociceptors and provide further evidence for activation of AMPK as a novel treatment avenue for acute and chronic pain states.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Dor Aguda/tratamento farmacológico , Dor Crônica/tratamento farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Receptoras Sensoriais/enzimologia , Estilbenos/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Dor Aguda/enzimologia , Dor Aguda/patologia , Animais , Dor Crônica/enzimologia , Dor Crônica/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fator de Iniciação 4F em Eucariotos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Hiperalgesia/complicações , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Interleucina-6/administração & dosagem , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dor Pós-Operatória/complicações , Dor Pós-Operatória/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Resveratrol , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/patologia , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fatores de Tempo , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/patologia
10.
Bioconjug Chem ; 23(10): 2098-104, 2012 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-22994402

RESUMO

Protease activated receptor-2 (PAR(2)) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR(2) can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR(2) to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR(2) peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH(2) (2-f-LIGRLO-dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR(2) agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH(2), 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH(2), 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH(2), 6-an-LIGRL) and PAR(2). A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH(2), 3-ia-LIGRL) that does not activate PAR(2) signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR(2) binding. In summary, we have developed a europium-containing PAR(2) agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR(2) ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR(2) ligands.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Európio/química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Receptor PAR-2/agonistas , Animais , Ligação Competitiva , Linhagem Celular , Humanos , Oligopeptídeos/síntese química , Ácido Pentético/química , Ratos , Receptor PAR-2/metabolismo , Espectrometria de Fluorescência , Coloração e Rotulagem , Fatores de Tempo
11.
J Neurosci ; 30(45): 15113-23, 2010 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-21068317

RESUMO

Despite the emergence of translational control pathways as mediators of nociceptive sensitization, effector molecules and mechanisms responsible for modulating activity in these pathways in pain conditions are largely unknown. We demonstrate that two major algogens, the cytokine interleukin 6 (IL-6) and the neurotrophin nerve growth factor (NGF), which are intimately linked to nociceptive plasticity across preclinical models and human pain conditions, signal primarily through two distinct pathways to enhance translation in sensory neurons by converging onto the eukaryotic initiation factor (eIF) eIF4F complex. We directly demonstrate that the net result of IL-6 and NGF signaling is an enhancement of eIF4F complex formation and an induction of nascent protein synthesis in primary afferent neurons and their axons. Moreover, IL-6- and NGF-induced mechanical nociceptive plasticity is blocked by inhibitors of general and cap-dependent protein synthesis. These results establish IL-6- and NGF-mediated cap-dependent translation of local proteins as a new model for nociceptive plasticity.


Assuntos
Fator de Iniciação 4F em Eucariotos/metabolismo , Interleucina-6/metabolismo , Fator de Crescimento Neural/metabolismo , Plasticidade Neuronal/fisiologia , Nociceptores/metabolismo , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Western Blotting , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imuno-Histoquímica , Interleucina-6/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fator de Crescimento Neural/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
12.
Mol Pain ; 7: 70, 2011 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-21936900

RESUMO

Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. We hypothesized that dysregulated translation regulation pathways may underlie neuropathic pain. Peripheral nerve injury induced reorganization of translation machinery in the peripheral nervous system of rats and mice, including enhanced mTOR and ERK activity, increased phosphorylation of mTOR and ERK downstream targets, augmented eIF4F complex formation and enhanced nascent protein synthesis. The AMP activated protein kinase (AMPK) activators, metformin and A769662, inhibited translation regulation signaling pathways, eIF4F complex formation, nascent protein synthesis in injured nerves and sodium channel-dependent excitability of sensory neurons resulting in a resolution of neuropathic allodynia. Therefore, injury-induced dysregulation of translation control underlies pathology leading to neuropathic pain and reveals AMPK as a novel therapeutic target for the potential treatment of neuropathic pain.


Assuntos
Terapia de Alvo Molecular , Neuralgia/tratamento farmacológico , Neuralgia/enzimologia , Proteínas Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Potenciais de Ação/efeitos dos fármacos , Animais , Western Blotting , Modelos Animais de Doenças , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/uso terapêutico , Fator de Iniciação 4F em Eucariotos/biossíntese , Hiperalgesia/complicações , Hiperalgesia/tratamento farmacológico , Hiperalgesia/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neuralgia/fisiopatologia , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Transdução de Sinais/efeitos dos fármacos
13.
Neurobiol Pain ; 5: 100024, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31194015

RESUMO

Brain-derived neurotrophic factor (BDNF) signaling through its cognate receptor, TrkB, is a well-known promoter of synaptic plasticity at nociceptive synapses in the dorsal horn of the spinal cord. Existing evidence suggests that BDNF/TrkB signaling in neuropathic pain is sex dependent. We tested the hypothesis that the effects of BDNF/TrkB signaling in hyperalgesic priming might also be sexually dimorphic. Using the incision postsurgical pain model in male mice, we show that BDNF sequestration with TrkB-Fc administered at the time of surgery blocks the initiation and maintenance of hyperalgesic priming. However, when BDNF signaling was blocked prior to the precipitation of hyperalgesic priming with prostaglandin E2 (PGE2), priming was not reversed. This result is in contrast to our findings in male mice with interleukin-6 (IL6) as the priming stimulus where TrkB-Fc was effective in reversing the maintenance of hyperalgesic priming. Furthermore, in IL6-induced hyperalgesic priming, the BDNF sequestering agent, TrkB-fc, was effective in reversing the maintenance of hyperalgesic priming in male mice; however, when this experiment was conducted in female mice, we did not observe any effect of TrkB-fc. This markedly sexual dimorphic effect in mice is consistent with recent studies showing a similar effect in neuropathic pain models. We tested whether the sexual dimorphic role for BDNF was consistent across species. Importantly, we find that this sexual dimorphism does not occur in rats where TrkB-fc reverses hyperalgesic priming fully in both sexes. Finally, to determine the source of BDNF in hyperalgesic priming in mice, we used transgenic mice (Cx3cr1CreER  × Bdnfflx/flx mice) with BDNF eliminated from microglia. From these experiments we conclude that BDNF from microglia does not contribute to hyperalgesic priming and that the key source of BDNF for hyperalgesic priming is likely nociceptors in the dorsal root ganglion. These experiments demonstrate the importance of testing mechanistic hypotheses in both sexes in multiple species to gain insight into complex biology underlying chronic pain.

14.
Neuroscience ; 359: 119-129, 2017 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-28729062

RESUMO

New therapeutics to manage post-surgical pain are needed to mitigate the liabilities of opioid and other analgesics. Our previous work shows that key modulators of excitability in peripheral nociceptors, such as extracellular signal-regulated kinases (ERK) are inhibited by activation of adenosine monophosphate activated protein kinase (AMPK). We hypothesized that AMPK activation would attenuate acute incision-evoked mechanical hypersensitivity and the development of hyperalgesic priming caused by surgery in mice. Here we have used a variety of administration routes and combinations of AMPK activators to test this hypothesis. Topical administration of a resveratrol-based cream inhibited acute mechanical hypersensitivity evoked by incision and blocked the development of hyperalgesic priming. We also observed that systemic administration of metformin dose-dependently inhibited incision-evoked mechanical hypersensitivity and hyperalgesic priming. Interestingly, low doses of systemic metformin and local resveratrol that had no acute effect were able to mitigate development of hyperalgesic priming. Combined treatment with doses of systemic metformin and local resveratrol that were not effective on their own enhanced the acute efficacy of the individual AMPK activators for post-surgical mechanical pain alleviation and blocked the development of hyperalgesic priming. Finally, we used dorsal root ganglion (DRG) neurons in culture to show that resveratrol and metformin given in combination shift the concentration-response curve for AMPK activation to the left and increase the magnitude of AMPK activation. Therefore, we find that topical administration is an effective treatment route of administration and combining systemic and local treatments led to anti-nociceptive efficacy in acute mechanical hypersensitivity at doses that were not effective alone. Collectively our work demonstrates a specific effect of AMPK activators on post-surgical pain and points to novel therapeutic opportunities with potential immediate impact in the clinical setting.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Analgésicos/administração & dosagem , Gânglios Espinais/efeitos dos fármacos , Hiperalgesia/metabolismo , Neurônios/efeitos dos fármacos , Dor/metabolismo , Animais , Células Cultivadas , Hiperalgesia/prevenção & controle , Masculino , Metformina/administração & dosagem , Camundongos Endogâmicos ICR , Dor/complicações , Dor/tratamento farmacológico , Limiar da Dor/efeitos dos fármacos , Complicações Pós-Operatórias/tratamento farmacológico , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Estilbenos/administração & dosagem
15.
Pain ; 156(5): 859-867, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25734998

RESUMO

Protease-activated receptor type 2 (PAR2) is known to play an important role in inflammatory, visceral, and cancer-evoked pain based on studies using PAR2 knockout (PAR2(-/-)) mice. We have tested the hypothesis that specific activation of PAR2 is sufficient to induce a chronic pain state through extracellular signal-regulated kinase (ERK) signaling to protein synthesis machinery. We have further tested whether the maintenance of this chronic pain state involves a brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (trkB)/atypical protein kinase C (aPKC) signaling axis. We observed that intraplantar injection of the novel highly specific PAR2 agonist, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at), evokes a long-lasting acute mechanical hypersensitivity (median effective dose ∼12 pmoles), facial grimacing, and causes robust hyperalgesic priming as revealed by a subsequent mechanical hypersensitivity and facial grimacing to prostaglandin E2 (PGE2) injection. The promechanical hypersensitivity effect of 2-at is completely absent in PAR2(-/-) mice as is hyperalgesic priming. Intraplantar injection of the upstream ERK inhibitor, U0126, and the eukaryotic initiation factor (eIF) 4F complex inhibitor, 4EGI-1, prevented the development of acute mechanical hypersensitivity and hyperalgesic priming after 2-at injection. Systemic injection of the trkB antagonist ANA-12 similarly inhibited PAR2-mediated mechanical hypersensitivity, grimacing, and hyperalgesic priming. Inhibition of aPKC (intrathecal delivery of ZIP) or trkB (systemic administration of ANA-12) after the resolution of 2-at-induced mechanical hypersensitivity reversed the maintenance of hyperalgesic priming. Hence, PAR2 activation is sufficient to induce neuronal plasticity leading to a chronic pain state, the maintenance of which is dependent on a BDNF/trkB/aPKC signaling axis.


Assuntos
Dor Crônica/metabolismo , Hiperalgesia/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/antagonistas & inibidores , Receptor PAR-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Azepinas/farmacologia , Comportamento Animal/efeitos dos fármacos , Benzamidas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Butadienos/farmacologia , Dor Crônica/induzido quimicamente , Dor Crônica/tratamento farmacológico , Dor Crônica/psicologia , Dinoprostona/farmacologia , Modelos Animais de Doenças , Expressão Facial , Hidrazonas/farmacologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Hiperalgesia/psicologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Nitrilas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Receptor PAR-2/deficiência , Receptor trkB/antagonistas & inibidores , Tiazóis/farmacologia
16.
Br J Pharmacol ; 172(18): 4535-4545, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26140338

RESUMO

BACKGROUND AND PURPOSE: Proteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described. EXPERIMENTAL APPROACH: We modified the peptidomimetic PAR2 agonist, 2-furoyl-LIGRLO-NH2 , to create a novel PAR2 peptidomimetic ligand, C391. C391 was evaluated for PAR2 agonist/antagonist activity to PAR2 across Gq signalling pathways using the naturally expressing PAR2 cell line 16HBE14o-. For antagonist studies, a highly potent and specific peptidomimetic agonist (2-aminothiazo-4-yl-LIGRL-NH2 ) and proteinase agonist (trypsin) were used to activate PAR2. C391 was also evaluated in vivo for reduction of thermal hyperalgesia, mediated by mast cell degranulation, in mice. KEY RESULTS: C391 is a potent and specific peptidomimetic antagonist, blocking multiple signalling pathways (Gq -dependent Ca2+ , MAPK) induced following peptidomimetic or proteinase activation of human PAR2. In a PAR2-dependent behavioural assay in mice, C391 dose-dependently (75 µg maximum effect) blocked the thermal hyperalgesia, mediated by mast cell degranulation. CONCLUSIONS AND IMPLICATIONS: C391 is the first low MW antagonist to block both PAR2 Ca2+ and MAPK signalling pathways activated by peptidomimetics and/or proteinase activation. C391 represents a new molecular structure for PAR2 antagonism and can serve as a basis for further development for this important therapeutic target.

17.
PLoS One ; 9(6): e99140, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24927179

RESUMO

Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2-2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG3-Hdc retained potency (EC50 = 2.1 nM; 1.3-3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20-100 nM). 2at-LI-PEG3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260-360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Animais , Células CHO , Células Cultivadas , Cricetulus , Masculino , Camundongos , Camundongos Endogâmicos ICR , Peptidomiméticos , Proto-Oncogene Mas , Relação Estrutura-Atividade
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