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1.
EMBO Rep ; 11(6): 473-8, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20467438

RESUMO

Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino-acid substitution in the polyproline-binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol-3-kinase-C2alpha to the SH3-binding site of PSD95. In wild-type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.


Assuntos
Inflamação/complicações , Inflamação/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dor/complicações , Dor/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Domínios de Homologia de src , Animais , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases , Hipocampo/enzimologia , Hipocampo/patologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Plasticidade Neuronal , Mutação Puntual/genética , Ligação Proteica , Relação Estrutura-Atividade , Sinapses/enzimologia
2.
Biochem J ; 436(3): 651-60, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21410433

RESUMO

The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF-PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Fosfolipase D/fisiologia , Receptor 5-HT2A de Serotonina/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Fosfolipase D/química
3.
Curr Biol ; 16(16): 1591-605, 2006 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-16920620

RESUMO

BACKGROUND: Chronic established pain, especially that following nerve injury, is difficult to treat and represents a largely unmet therapeutic need. New insights are urgently required, and we reasoned that endogenous processes such as cooling-induced analgesia may point the way to novel strategies for intervention. Molecular receptors for cooling have been identified in sensory nerves, and we demonstrate here how activation of one of these, TRPM8, produces profound, mechanistically novel analgesia in chronic pain states. RESULTS: We show that activation of TRPM8 in a subpopulation of sensory afferents (by either cutaneous or intrathecal application of specific pharmacological agents or by modest cooling) elicits analgesia in neuropathic and other chronic pain models in rats, thereby inhibiting the characteristic sensitization of dorsal-horn neurons and behavioral-reflex facilitation. TRPM8 expression was increased in a subset of sensory neurons after nerve injury. The essential role of TRPM8 in suppression of sensitized pain responses was corroborated by specific knockdown of its expression after intrathecal application of an antisense oligonucleotide. We further show that the analgesic effect of TRPM8 activation is centrally mediated and relies on Group II/III metabotropic glutamate receptors (mGluRs), but not opioid receptors. We propose a scheme in which Group II/III mGluRs would respond to glutamate released from TRPM8-containing afferents to exert an inhibitory gate control over nociceptive inputs. CONCLUSIONS: TRPM8 and its central downstream mediators, as elements of endogenous-cooling-induced analgesia, represent a novel analgesic axis that can be exploited in chronic sensitized pain states.


Assuntos
Analgesia/métodos , Temperatura Baixa , Neuralgia/metabolismo , Neuralgia/terapia , Canais de Cátion TRPM/metabolismo , Acroleína/análogos & derivados , Acroleína/farmacologia , Aminoácidos/farmacologia , Análise de Variância , Animais , Western Blotting , Relação Dose-Resposta a Droga , Eletrofisiologia , Imuno-Histoquímica , Masculino , Mentol/farmacologia , Neuralgia/tratamento farmacológico , Oligonucleotídeos Antissenso , Pirimidinonas/farmacologia , Ratos , Ratos Wistar , Receptores de Glutamato/metabolismo , Reflexo/efeitos dos fármacos , Xantenos/farmacologia
4.
J Neurosci ; 22(4): 1363-72, 2002 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-11850463

RESUMO

Neuropathic pain (characterized by hyperalgesia and allodynia to mechanical and thermal stimuli) causes cellular changes in spinal dorsal horn neurons, some of which parallel those in synaptic plasticity associated with learning. Ubiquitin C-terminal hydrolase (UCH) appears to play a key role in long-term facilitation in Aplysia. The cooperation of UCH with the proteolytic enzyme complex known as the proteasome is required for the degradation of a number of signaling molecules within the cell that may remove normal restraints on synaptic plasticity. We have used electrophysiology, in situ hybridization histochemistry, semiquantitative RT-PCR, Western blotting, and in vivo behavioral reflex analysis to investigate the ubiquitin-proteasome system in a model of neuropathic pain. In neuropathic animals, ionophoretic application of selective proteasome inhibitors attenuated dorsal horn neuron firing evoked by normally innocuous brush or cold stimuli and by noxious mustard oil stimuli. In control animals, only mustard oil-evoked responses were inhibited. Intrathecal administration of proteasome inhibitors attenuated hyperalgesia and allodynia in neuropathic rats. Expression of UCH-L1 (a rat homolog of Aplysia neuronal UCH and of the human UCH-L1, also known as PGP 9.5) and its mRNA were selectively increased within the ipsilateral dorsal horn of neuropathic rats, supporting the idea of a role for the ubiquitin-proteasome system in nociceptive processing. Proteasome inhibitors selectively attenuate allodynic and hyperalgesic responses in neuropathic pain, with some reduction in normal nociceptive, but not non-nociceptive responses, and potentially represent a novel therapeutic strategy for neuropathic pain.


Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Dor/fisiopatologia , Neuropatia Ciática/fisiopatologia , Ubiquitina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Comportamento Animal , Modelos Animais de Doenças , Eletrofisiologia , Inibidores Enzimáticos/administração & dosagem , Hiperalgesia/etiologia , Hiperalgesia/fisiopatologia , Hibridização In Situ , Injeções Espinhais , Iontoforese , Ligadura , Masculino , Complexos Multienzimáticos/antagonistas & inibidores , Dor/tratamento farmacológico , Dor/etiologia , Medição da Dor , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/fisiopatologia , Complexo de Endopeptidases do Proteassoma , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reflexo/efeitos dos fármacos , Neuropatia Ciática/complicações , Neuropatia Ciática/tratamento farmacológico , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Ubiquitina Tiolesterase
5.
Pain ; 117(3): 421-432, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16150544

RESUMO

Spinal NMDA receptors (NMDA R) are important in neuropathic sensitisation and acute administration of antagonists can provide temporary attenuation of sensitisation. If establishment of the chronic pain state could be prevented by brief administration of such agents at or around the time of nerve injury (pre-emptive analgesia) it might be possible to avoid many of the unacceptable side effects associated with repeated administration of these or other antagonists. Several reports describe aspects of effective pre-emptive analgesia from NMDA R antagonists in animal models of neuropathic pain. The first aim of the present study was to make a direct comparison of changes in mechanical allodynia, cold allodynia and thermal hyperalgesia following nerve injury, demonstrating their increasing degree of susceptibility to pre-emptive NMDA R antagonist treatment. Secondly, we used immunoblotting and immunohistochemistry to investigate the effects of nerve injury on NMDA receptor subunit expression, revealing increased expression of NR2B, but not NR2A and reduced NR1 in the superficial dorsal horn. These changes were attenuated following NMDA receptor antagonist pre-treatment. Thirdly, we investigated the pharmacological properties of residual mechanical allodynia and cold allodynia that remained after pre-emptive treatment and revealed a greater sensitivity to NMDA R antagonists. These findings indicate that in addition to a marked suppression of thermal hyperalgesia and cold allodynia, pre-emptive treatment with NMDA R antagonist causes a lasting change in spinal NMDA R complexes such that remaining mechanical allodynia should be more effectively targeted by NMDA R antagonists.


Assuntos
Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Limiar da Dor/efeitos dos fármacos , Dor/prevenção & controle , Receptores de N-Metil-D-Aspartato/metabolismo , Medula Espinal/efeitos dos fármacos , Análise de Variância , Animais , Comportamento Animal , Western Blotting/métodos , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Lateralidade Funcional , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Dor/complicações , Medição da Dor , Ratos , Ratos Wistar , Tempo de Reação/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Medula Espinal/metabolismo , Medula Espinal/patologia , Fatores de Tempo , Traumatismos do Sistema Nervoso/complicações , Traumatismos do Sistema Nervoso/tratamento farmacológico
6.
Neuropharmacology ; 79: 136-51, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24269608

RESUMO

Effective relief from chronic hypersensitive pain states remains an unmet need. Here we report the discovery that the TRPM8 ion channel, co-operating with the 5-HT(1B) receptor (5-HT(1B)R) in a subset of sensory afferents, exerts an influence at the spinal cord level to suppress central hypersensitivity in pain processing throughout the central nervous system. Using cell line models, ex vivo rat neural tissue and in vivo pain models, we assessed functional Ca(2+) fluorometric responses, protein:protein interactions, immuno-localisation and reflex pain behaviours, with pharmacological and molecular interventions. We report 5-HT(1B)R expression in many TRPM8-containing afferents and direct interaction of these proteins in a novel multi-protein signalling complex, which includes phospholipase D1 (PLD1). We provide evidence that the 5-HT(1B)R activates PLD1 to subsequently activate PIP 5-kinase and generate PIP2, an allosteric enhancer of TRPM8, achieving a several-fold increase in potency of TRPM8 activation. The enhanced activation responses of synaptoneurosomes prepared from spinal cord and cortical regions of animals with a chronic inflammatory pain state are inhibited by TRPM8 activators that were applied in vivo topically to the skin, an effect potentiated by co-administered 5-HT(1B)R agonists and attenuated by 5-HT(1B)R antagonists, while 5-HT(1B)R agents alone had no detectable effect. Corresponding results are seen when assessing reflex behaviours in inflammatory and neuropathic pain models. Control experiments with alternative receptor/TRP channel combinations reveal no such synergy. Identification of this novel receptor/effector/channel complex and its impact on nociceptive processing give new insights into possible strategies for enhanced analgesia in chronic pain.


Assuntos
Dor/metabolismo , Fosfolipase D/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Canais de Cálcio/metabolismo , Células Cultivadas , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiopatologia , Células HEK293 , Humanos , Masculino , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/fisiopatologia , Dor/tratamento farmacológico , Ratos , Ratos Wistar , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiopatologia , Canal de Cátion TRPA1 , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo
7.
Cell Signal ; 25(4): 814-21, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23314176

RESUMO

The 5-HT2A receptor (5-HT2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT2AR.


Assuntos
Fosfolipase D/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Células MCF-7 , Fosfolipase D/antagonistas & inibidores , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/metabolismo , Receptor 5-HT2A de Serotonina/química , Receptor 5-HT2A de Serotonina/genética , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fosfolipases Tipo C/metabolismo
8.
Mol Cell Neurosci ; 24(1): 10-22, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14550765

RESUMO

Chronic pain states arise from peripheral nerve injury and are inadequately treated with current analgesics. Using intrathecal drug administration in a rat model of neuropathic pain, we demonstrate that AMPA receptors play a role in the central sensitisation that is thought to underpin chronic pain. The GluR2 subunit of the AMPA receptor binds to a number of intracellular adapter proteins including GRIP, PICK1 and NSF, which may link the receptor to proteins with signalling, scaffolding and other roles. We implicate for the first time a possible role for GRIP, PICK1 and NSF in neuropathic sensitisation from experiments with cell-permeable blocking peptides mimicking their GluR2 interaction motifs and also demonstrate differential changes in expression of these proteins following peripheral nerve injury. These studies suggest a critical involvement of protein:protein complexes associated with the AMPA receptor in neuropathic pain, and the possibility that they may have potential as novel therapeutic targets.


Assuntos
Proteínas de Transporte/metabolismo , Neuralgia/metabolismo , Proteínas Nucleares , Doenças do Sistema Nervoso Periférico/metabolismo , Receptores de AMPA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Transporte Vesicular , Animais , Proteínas de Transporte/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Doença Crônica , Proteínas do Citoesqueleto , Antagonistas de Aminoácidos Excitatórios/farmacologia , Lateralidade Funcional/efeitos dos fármacos , Lateralidade Funcional/fisiologia , Ácido Glutâmico/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Proteínas Sensíveis a N-Etilmaleimida , Neuralgia/fisiopatologia , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Peptídeos/farmacologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/genética , Reflexo/efeitos dos fármacos , Reflexo/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
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