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1.
J Neurochem ; 123(4): 589-601, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22891703

RESUMO

Many extracellular factors sensitize nociceptors. Often they act simultaneously and/or sequentially on nociceptive neurons. We investigated if stimulation of the protein kinase C epsilon (PKCε) signaling pathway influences the signaling of a subsequent sensitizing stimulus. Central in activation of PKCs is their transient translocation to cellular membranes. We found in cultured nociceptive neurons that only a first stimulation of the PKCε signaling pathway resulted in PKCε translocation. We identified a novel inhibitory cascade to branch off upstream of PKCε, but downstream of Epac via IP3-induced calcium release. This signaling branch actively inhibited subsequent translocation and even attenuated ongoing translocation. A second 'sensitizing' stimulus was rerouted from the sensitizing to the inhibitory branch of the signaling cascade. Central for the rerouting was cytoplasmic calcium increase and CaMKII activation. Accordingly, in behavioral experiments, activation of calcium stores switched sensitizing substances into desensitizing substances in a CaMKII-dependent manner. This mechanism was also observed by in vivo C-fiber electrophysiology corroborating the peripheral location of the switch. Thus, we conclude that the net effect of signaling in nociceptors is defined by the context of the individual cell's signaling history.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Neurônios/metabolismo , Nociceptores/fisiologia , Limiar da Dor/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Análise de Variância , Animais , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/citologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Inositol 1,4,5-Trifosfato/farmacologia , Isoproterenol/farmacologia , Masculino , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/fisiologia , Neurônios/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Proteína Quinase C-épsilon/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Rianodina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Canais de Cátion TRPV/metabolismo , Tionucleotídeos/farmacologia , Uridina Trifosfato/farmacologia
2.
J Neurochem ; 117(6): 995-1008, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21480900

RESUMO

Recently, we described estrogen and agonists of the G-protein coupled estrogen receptor GPR30 to induce protein kinase C (PKC)ε-dependent pain sensitization. PKCε phosphorylates the ion channel transient receptor potential, vanilloid subclass I (TRPV1) close to a novel microtubule-TRPV1 binding site. We now modeled the binding of tubulin to the TRPV1 C-terminus. The model suggests PKCε phosphorylation of TRPV1-S800 to abolish the tubulin-TRPV1 interaction. Indeed, in vitro PKCε phosphorylation of TRPV1 hindered tubulin-binding to TRPV1. In vivo, treatment of sensory neurons and F-11 cells with estrogen and the GPR30 agonist, G-1, resulted in microtubule destabilization and retraction of microtubules from filopodial structures. We found estrogen and G-1 to regulate the stability of the microtubular network via PKC phosphorylation of the PKCε-phosphorylation site TRPV1-S800. Microtubule disassembly was not, however, dependent on TRPV1 ion conductivity. TRPV1 knock-down in rats inverted the effect of the microtubule-modulating drugs, Taxol and Nocodazole, on estrogen-induced and PKCε-dependent mechanical pain sensitization. Thus, we suggest the C-terminus of TRPV1 to be a signaling intermediate downstream of estrogen and PKCε, regulating microtubule-stability and microtubule-dependent pain sensitization.


Assuntos
Estradiol/farmacologia , Estrogênios/farmacologia , Microtúbulos/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Animais , Ligação Competitiva , Linhagem Celular , Ciclopentanos/farmacologia , Estrogênios/fisiologia , Gânglios Espinais/citologia , Técnicas de Silenciamento de Genes , Ativação do Canal Iônico , Ligantes , Masculino , Microtúbulos/ultraestrutura , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Dor/fisiopatologia , Fosforilação , Ligação Proteica , Proteína Quinase C-épsilon/fisiologia , Pseudópodes/ultraestrutura , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Transdução de Sinais , Canais de Cátion TRPV/genética , Tubulina (Proteína)/metabolismo
3.
Eur J Pain ; 15(8): 796-800, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21419675

RESUMO

Stress exacerbates both experimental and clinical pain, most well-characterized in irritable bowel and fibromyalgia syndromes. Since it has been hypothesized that cytokines play an etiopathogenic role in fibromyalgia and other chronic widespread pain conditions, we investigated the relationship between stress and cytokines in a model of stress-induced chronic somatic pain. A series of experiments were performed to evaluate the impact of stress on the hyperalgesia-induced by endotoxin (lipopolysaccharide, LPS) and the role of two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis α (TNFα). Fourteen days after exposure to a 4-day protocol of unpredictable sound stress, the ability of systemic LPS (100 µg/kg, i.p) to elicit cytokine-mediated mechanical hyperalgesia was measured in gastrocnemius muscle. LPS-induced hyperalgesia was significantly greater in stressed rats, but when rats were treated intrathecally with antisense oligodeoxynucleotide (ODN), to decrease either the gp130 subunit of the IL-6 receptor or the TNFα receptor, in nociceptors, skeletal muscle hyperalgesia in sound stressed, but not control, rats was prevented. These data suggest that chronic stress alters signaling in the primary afferent nociceptor for the hyperalgesia induced by endogenously produced pro-inflammatory cytokines.


Assuntos
Citocinas/fisiologia , Hiperalgesia/metabolismo , Músculo Esquelético/metabolismo , Nociceptores/fisiologia , Som/efeitos adversos , Estresse Psicológico/metabolismo , Estimulação Acústica/efeitos adversos , Animais , Modelos Animais de Doenças , Hiperalgesia/fisiopatologia , Masculino , Músculo Esquelético/inervação , Músculo Esquelético/fisiopatologia , Ratos , Ratos Sprague-Dawley , Estresse Psicológico/etiologia , Estresse Psicológico/fisiopatologia
4.
Mol Pain ; 6: 98, 2010 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-21187008

RESUMO

BACKGROUND: Dorsal root ganglia (DRG)-neurons are commonly characterized immunocytochemically. Cells are mostly grouped by the experimenter's eye as "marker-positive" and "marker-negative" according to their immunofluorescence intensity. Classification criteria remain largely undefined. Overcoming this shortfall, we established a quantitative automated microscopy (QuAM) for a defined and multiparametric analysis of adherent heterogeneous primary neurons on a single cell base.The growth factors NGF, GDNF and EGF activate the MAP-kinase Erk1/2 via receptor tyrosine kinase signalling. NGF and GDNF are established factors in regeneration and sensitization of nociceptive neurons. If also the tissue regenerating growth factor, EGF, influences nociceptors is so far unknown. We asked, if EGF can act on nociceptors, and if QuAM can elucidate differences between NGF, GDNF and EGF induced Erk1/2 activation kinetics. Finally, we evaluated, if the investigation of one signalling component allows prediction of the behavioral response to a reagent not tested on nociceptors such as EGF. RESULTS: We established a software-based neuron identification, described quantitatively DRG-neuron heterogeneity and correlated measured sample sizes and corresponding assay sensitivity. Analysing more than 70,000 individual neurons we defined neuronal subgroups based on differential Erk1/2 activation status in sensory neurons. Baseline activity levels varied strongly already in untreated neurons. NGF and GDNF subgroup responsiveness correlated with their subgroup specificity on IB4(+)- and IB4(-)-neurons, respectively. We confirmed expression of EGF-receptors in all sensory neurons. EGF treatment induced STAT3 translocation into the nucleus. Nevertheless, we could not detect any EGF induced Erk1/2 phosphorylation. Accordingly, intradermal injection of EGF resulted in a fundamentally different outcome than NGF/GDNF. EGF did not induce mechanical hyperalgesia, but blocked PGE2-induced sensitization. CONCLUSIONS: QuAM is a suitable if not necessary tool to analyze activation of endogenous signalling in heterogeneous cultures. NGF, GDNF and EGF stimulation of DRG-neurons shows differential Erk1/2 activation responses and a corresponding differential behavioral phenotype. Thus, in addition to expression-markers also signalling-activity can be taken for functional subgroup differentiation and as predictor of behavioral outcome. The anti-nociceptive function of EGF is an intriguing result in the context of tissue damage but also for understanding pain resulting from EGF-receptor block during cancer therapy.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Microscopia/métodos , Dor/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Gânglios Espinais , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Masculino , Fator de Crescimento Neural/farmacologia , Dor/fisiopatologia , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/metabolismo , Células Receptoras Sensoriais , Software
5.
J Pain ; 11(4): 369-77, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19962353

RESUMO

UNLABELLED: While occupational exposure to vibration is a common cause of acute and chronic musculoskeletal pain, eliminating exposure produces limited symptomatic improvement, and reexposure precipitates rapid recurrence or exacerbation. To evaluate mechanisms underlying these pain syndromes, we have developed a model in the rat, in which exposure to vibration (60-80Hz) induces, in skeletal muscle, both acute mechanical hyperalgesia as well as long-term changes characterized by enhanced hyperalgesia to a proinflammatory cytokine or reexposure to vibration. Exposure of a hind limb to vibration-produced mechanical hyperalgesia measured in the gastrocnemius muscle of the exposed hind limb, which persisted for approximately 2 weeks. When nociceptive thresholds had returned to baseline, exposure to a proinflammatory cytokine or reexposure to vibration produced markedly prolonged hyperalgesia. The chronic prolongation of vibration- and cytokine-hyperalgesia was prevented by spinal intrathecal injection of oligodeoxynucleotide (ODN) antisense to protein kinase Cepsilon, a second messenger in nociceptors implicated in the induction and maintenance of chronic pain. Vibration-induced hyperalgesia was inhibited by spinal intrathecal administration of ODN antisense to receptors for the type-1 tumor necrosis factor-alpha (TNFalpha) receptor. Finally, in TNFalpha-pretreated muscle, subsequent vibration-induced hyperalgesia was markedly prolonged. PERSPECTIVE: These studies establish a model of vibration-induced acute and chronic musculoskeletal pain, and identify the proinflammatory cytokine TNFalpha and the second messenger protein kinase Cepsilon as targets against which therapies might be directed to prevent and/or treat this common and very debilitating chronic pain syndrome.


Assuntos
Doenças Musculoesqueléticas/etiologia , Doenças Musculoesqueléticas/fisiopatologia , Exposição Ocupacional/efeitos adversos , Dor/etiologia , Dor/fisiopatologia , Vibração/efeitos adversos , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Hiperalgesia/etiologia , Hiperalgesia/fisiopatologia , Inflamação/etiologia , Inflamação/fisiopatologia , Mediadores da Inflamação/farmacologia , Injeções Espinhais , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Limiar da Dor/fisiologia , Proteína Quinase C-épsilon/antagonistas & inibidores , Proteína Quinase C-épsilon/genética , Ratos , Ratos Sprague-Dawley , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia , Fator de Necrose Tumoral alfa/farmacologia
6.
J Pain ; 10(10): 1073-7, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19576859

RESUMO

UNLABELLED: Although stress plays an important role in chronic widespread pain syndromes, such as fibromyalgia, the underlying mechanism has remained elusive. We have recently demonstrated, in a model of chronic widespread pain, that prolonged enhancement of immune mediator hyperalgesia, induced by unpredictable sound stress, requires a contribution of both the sympathoadrenal (epinephrine) and the hypothalamic-pituitary adrenal (corticosterone) neuroendocrine stress axes. Because this stress protocol produced sustained elevation of plasma epinephrine, in the current study we tested the hypothesis that the sympathoadrenal axis also plays a role in maintenance of symptoms in this model of chronic widespread pain. After establishment, adrenal medullectomy abolished the enhancement of epinephrine-induced cutaneous and muscle hyperalgesia. Administration of stress levels of epinephrine to adrenal medullectomized rats reconstituted the pain phenotype. These observations suggest that the sympathoadrenal stress axis plays a major role in the induction as well as maintenance of stress-induced enhancement of mechanical hyperalgesia, mediated by prolonged elevation of circulating epinephrine. PERSPECTIVE: We present data showing mechanical hyperalgesia persisting for up to 28 days after exposure to sound stress, with evidence that the sympathoadrenal axis mediator epinephrine plays a major role. These findings could have clinical implications with regard to novel potential treatments for chronic widespread pain syndromes, such as fibromyalgia.


Assuntos
Medula Suprarrenal/metabolismo , Catecolaminas/metabolismo , Hiperalgesia/fisiopatologia , Som/efeitos adversos , Estresse Psicológico/fisiopatologia , Sistema Nervoso Simpático/metabolismo , Estimulação Acústica/efeitos adversos , Animais , Catecolaminas/sangue , Catecolaminas/farmacologia , Doença Crônica , Modelos Animais de Doenças , Epinefrina/sangue , Epinefrina/metabolismo , Epinefrina/farmacologia , Hiperalgesia/complicações , Hiperalgesia/metabolismo , Injeções Intramusculares , Injeções Subcutâneas , Masculino , Testes Neuropsicológicos , Nociceptores/efeitos dos fármacos , Nociceptores/fisiologia , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Estimulação Física/efeitos adversos , Ratos , Ratos Sprague-Dawley , Estresse Psicológico/complicações , Estresse Psicológico/metabolismo , Fatores de Tempo , Tato/fisiologia
7.
J Neurosci ; 29(19): 6217-28, 2009 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-19439599

RESUMO

The transient receptor potential vanilloid 4 (TRPV4) contributes to mechanical hyperalgesia of diverse etiologies, presumably as part of a mechanoreceptor signaling complex (Alessandri-Haber et al., 2008). To investigate the hypothesis that a functional interaction between TRPV4 and stretch-activated ion channels (SACs) is involved in this mechanical transduction mechanism, we used a selective SACs inhibitor, GsMTx-4. Intradermal injection of GsMTx-4 in the rat hindpaw reversed the mechanical hyperalgesia induced by intradermal injection of inflammatory mediators. In vivo single fiber recordings showed that GsMTx-4 reversed inflammatory mediator-induced decrease in mechanical threshold in half of sensitized C-fibers. Furthermore, GsMTx-4 reduced hyperalgesia to both mechanical and hypotonic stimuli in different models of inflammatory and neuropathic pain, although it had no effect on baseline mechanical nociceptive thresholds. TRPC1 and TRPC6, two GsMTx-4-sensitive SACs, are expressed in dorsal root ganglion (DRG) neurons. Single-cell reverse transcription-PCR showed that messenger RNAs for TRPV4, TRPC1, and TRPC6 are frequently coexpressed in DRG neurons. Spinal intrathecal administration of oligodeoxynucleotides antisense to TRPC1 and TRPC6, like that to TRPV4, reversed the hyperalgesia to mechanical and hypotonic stimuli induced by inflammatory mediators without affecting baseline mechanical nociceptive threshold. However, antisense to TRPC6, but not to TRPC1, reversed the mechanical hyperalgesia induced by a thermal injury or the TRPV4-selective agonist 4alpha-PDD (4 alpha-phorbol 12,13-didecanoate). We conclude that TRPC1 and TRPC6 channels cooperate with TRPV4 channels to mediate mechanical hyperalgesia and primary afferent nociceptor sensitization, although they may have distinctive roles.


Assuntos
Gânglios Espinais/fisiologia , Hiperalgesia/fisiopatologia , Neurônios/fisiologia , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , , Expressão Gênica , Membro Posterior , Hiperalgesia/induzido quimicamente , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Limiar da Dor/fisiologia , Peptídeos/farmacologia , Ésteres de Forbol , Estimulação Física , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Venenos de Aranha/farmacologia , Canal de Cátion TRPC6 , Canais de Cátion TRPV/genética
8.
J Neurosci ; 28(22): 5721-30, 2008 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-18509033

RESUMO

Stress dramatically exacerbates pain in diseases such as fibromyalgia and rheumatoid arthritis, but the underlying mechanisms are unknown. We tested the hypothesis that stress causes generalized hyperalgesia by enhancing pronociceptive effects of immune mediators. Rats exposed to nonhabituating sound stress exhibited no change in mechanical nociceptive threshold, but showed a marked increase in hyperalgesia evoked by local injections of prostaglandin E(2) or epinephrine. This enhancement, which developed more than a week after exposure to stress, required concerted action of glucocorticoids and catecholamines at receptors located in the periphery on sensory afferents. The altered response to pronociceptive mediators involved a switch in coupling of their receptors from predominantly stimulatory to inhibitory G-proteins (G(s) to G(i)), and for prostaglandin E(2), emergence of novel dependence on protein kinase C epsilon. Thus, an important mechanism in generalized pain syndromes may be stress-induced coactivation of the hypothalamo-pituitary-adrenal and sympathoadrenal axes, causing a long-lasting alteration in intracellular signaling pathways, enabling normally innocuous levels of immune mediators to produce chronic hyperalgesia.


Assuntos
Neurônios Aferentes/fisiologia , Dor/patologia , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiopatologia , Adrenalectomia/métodos , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Corticosterona/farmacologia , Dinoprostona , Modelos Animais de Doenças , Epinefrina/efeitos adversos , Epinefrina/sangue , Antagonistas de Hormônios/farmacologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Masculino , Mifepristona/farmacologia , Músculo Esquelético/inervação , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Ratos , Ratos Sprague-Dawley , Pele/inervação , Som/efeitos adversos , Estresse Fisiológico/etiologia , Fatores de Tempo
9.
Eur J Neurosci ; 27(7): 1700-9, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18371086

RESUMO

We evaluated the signalling pathway by which estrogen acts in peripheral tissue to produce protein kinase Cepsilon (PKCepsilon)-dependent mechanical hyperalgesia. Specific agonists for the classical estrogen receptors (ER), ERalpha and ERbeta, did not result in activation of PKCepsilon in neurons of dissociated rat dorsal root ganglia. In contrast, G-1, a specific agonist of the recently identified G-protein-coupled estrogen receptor, GPR30, induced PKCepsilon translocation. Involvement of GPR30 and independence of ERalpha and ERbeta was confirmed using the GPR30 agonist and simultaneous ERalpha and ERbeta antagonist ICI 182,780 (fulvestrant). The GPR30 transcript could be amplified from dorsal root ganglia tissue. We found estrogen-induced as well as GPR30-agonist-induced PKCepsilon translocation to be restricted to the subgroup of nociceptive neurons positive for isolectin IB4 from Bandeiraea simplicifolia. Corroborating the cellular results, both GPR30 agonists, G-1 as well as ICI 182,780, resulted in the onset of PKCepsilon-dependent mechanical hyperalgesia if injected into paws of adult rats. We therefore suggest that estrogen acts acutely at GPR30 in nociceptors to produce mechanical hyperalgesia.


Assuntos
Moduladores de Receptor Estrogênico/farmacologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/fisiologia , Animais , Células Cultivadas , Ciclopentanos/farmacologia , Estradiol/análogos & derivados , Estradiol/farmacologia , Fulvestranto , Masculino , Estimulação Física/métodos , Proteína Quinase C-épsilon/metabolismo , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley
10.
Pain ; 135(1-2): 98-107, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17590515

RESUMO

Thermal burns induce pain at the site of injury, mechanical hyperalgesia, associated with a complex time-dependent inflammatory response. To determine the contribution of inflammatory mediators to burn injury-induced mechanical hyperalgesia, we measured dynamic changes in the levels of three potent hyperalgesic cytokines, interleukin IL-1 beta, IL-6, and tumor necrosis factor-alpha (TNFalpha), in skin of the rat, following a partial-thickness burn injury. Only IL-6 demonstrated a sustained increase ipsilateral but not contralateral to the burn, correlating with the prolonged ipsilateral mechanical hyperalgesia. Spinal intrathecal injection of oligodeoxynucleotides antisense for gp130, a receptor subunit shared by members of the IL-6 family of cytokines, attenuated both burn- and intradermal IL-6-induced hyperalgesia, as did intradermal injection of anti-IL-6 function blocking antibodies. These studies suggest that IL-6 is an important mediator of burn-injury pain.


Assuntos
Queimaduras/complicações , Citocinas/metabolismo , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Análise de Variância , Animais , Modelos Animais de Doenças , Lateralidade Funcional , Hiperalgesia/tratamento farmacológico , Hiperalgesia/imunologia , Injeções Espinhais/métodos , Interleucina-6/metabolismo , Masculino , Morfolinas/química , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia
11.
J Pain ; 7(12): 884-91, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17157774

RESUMO

UNLABELLED: Although mechanical hyperalgesia associated with medical procedures is the major source of severe pain in burn-injured patients, little is known about its underlying mechanism. One reason for this has been the lack of a model for mechanical hyperalgesia at the site of injury. We have modified an established partial-thickness burn model in the rat to produce long-lasting primary mechanical hyperalgesia, which is present from the first measurement at 0.5 h, reaches a maximum at 3 days, and is still significant after 7 days. Because nerve growth factor (NGF), which is elevated in burn-injured tissue, produces mechanical hyperalgesia and activates protein kinase C (PKC)-epsilon, a key mediator in inflammatory and neuropathic pain, we used this model to evaluate the role of the NGF receptor, tyrosine-receptor kinase A (TrkA), and PKC-epsilon in burn-induced primary mechanical hyperalgesia. Intrathecal administration of antisense oligodeoxynucleotides to TrkA and PKC-epsilon, starting 3 days before inducing a burn injury, caused dose-related decrease of burn-induced primary mechanical hyperalgesia. In addition, intradermal injection of a PKC-epsilon-selective inhibitor eliminated hyperalgesia. Our model provides a method to elucidate the underlying mechanism of burn-injury pain as well as to screen for targets for novel analgesic treatments of this important clinical condition. PERSPECTIVE: This manuscript presents the first model of thermal injury-induced mechanical hyperalgesia which mimics prolonged duration of clinical burn injury pain. We also perform proof of concept experiments demonstrating that our model provides a method to elucidate the mechanism of this important clinical condition.


Assuntos
Queimaduras/complicações , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Proteína Quinase C-épsilon/metabolismo , Receptor trkA/metabolismo , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Hiperalgesia/tratamento farmacológico , Masculino , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Peptídeos/administração & dosagem , Proteína Quinase C-épsilon/genética , Ratos , Ratos Sprague-Dawley , Tempo de Reação/fisiologia , Receptor trkA/genética , Fatores de Tempo
12.
Eur J Neurosci ; 24(2): 527-34, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16836642

RESUMO

Protein kinase C epsilon (PKCepsilon) is an important intracellular signaling molecule in primary afferent nociceptors, implicated in acute and chronic inflammatory as well as neuropathic pain. In behavioral experiments inflammatory mediators produce PKCepsilon-dependent hyperalgesia only in male rats. The mechanism underlying this sexual dimorphism is unknown. We show that the hormone environment of female rats changes the nociceptive signaling in the peripheral sensory neuron. This change is maintained in culture also in the absence of a gender-simulating environment. Stimulation of beta(2)-adrenergic receptors (beta(2)-AR) leads to PKCepsilon activation in cultured dorsal root ganglia (DRG) neurons derived from male but not from female rats. Addition of estrogen to male DRG neurons produces a switch to the female phenotype, namely abrogation of beta(2)-AR-initiated activation of PKCepsilon. Estrogen interferes downstream of the beta(2)-AR with the signaling pathway leading from exchange protein activated by cAMP (Epac) to PKCepsilon. The interfering action is fast indicating a transcriptional-independent mechanism. Estrogen has a dual effect on PKCepsilon. If applied before beta(2)-AR or Epac stimulation, estrogen abrogates the activation of PKCepsilon. In contrast, estrogen applied alone leads to a brief translocation of PKCepsilon. Also in vivo the activity of estrogen depends on the stimulation context. In male rats, intradermal injection of an Epac activator or estrogen alone induces mechanical hyperalgesia through a PKCepsilon-dependent mechanism. In contrast, injection of estrogen preceding the activation of Epac completely abrogates the Epac-induced mechanical hyperalgesia. Our results suggest that gender differences in nociception do not reflect the use of generally different mechanisms. Instead, a common set of signaling pathways can be modulated by hormones.


Assuntos
Estrogênios/metabolismo , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Neurônios Aferentes/metabolismo , Nociceptores/metabolismo , Proteína Quinase C-épsilon/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Estrogênios/farmacologia , Feminino , Gânglios Espinais/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Caracteres Sexuais , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
13.
J Neurosci ; 25(26): 6119-26, 2005 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-15987941

RESUMO

The epsilon isoform of protein kinase C (PKCepsilon) has emerged as a critical second messenger in sensitization toward mechanical stimulation in models of neuropathic (diabetes, alcoholism, and cancer therapy) as well as acute and chronic inflammatory pain. Signaling pathways leading to activation of PKCepsilon remain unknown. Recent results indicate signaling from cAMP to PKC. A mechanism connecting cAMP and PKC, two ubiquitous, commonly considered separate pathways, remains elusive. We found that, in cultured DRG neurons, signaling from cAMP to PKCepsilon is not mediated by PKA but by the recently identified cAMP-activated guanine exchange factor Epac. Epac, in turn, was upstream of phospholipase C (PLC) and PLD, both of which were necessary for translocation and activation of PKCepsilon. This signaling pathway was specific to isolectin B4-positive [IB4(+)] nociceptors. Also, in a behavioral model, cAMP produced mechanical hyperalgesia (tenderness) through Epac, PLC/PLD, and PKCepsilon. By delineating this signaling pathway, we provide a mechanism for cAMP-to-PKC signaling, give proof of principle that the mitogen-activated protein kinase pathway-activating protein Epac also stimulates PKC, describe the first physiological function unique for the IB4(+) subpopulation of sensory neurons, and find proof of principle that G-protein-coupled receptors can activate PKC not only through the G-proteins alpha(q) and betagamma but also through alpha(s).


Assuntos
AMP Cíclico/fisiologia , Glicoproteínas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Inflamação/fisiopatologia , Lectinas/metabolismo , Neurônios/fisiologia , Dor/fisiopatologia , Proteína Quinase C/metabolismo , Animais , Gânglios Espinais/fisiologia , Masculino , Técnicas de Cultura de Órgãos , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Versicanas
14.
Eur J Neurosci ; 21(12): 3379-86, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16026475

RESUMO

Epinephrine produces sexually dimorphic beta(2)-adrenergic receptor-mediated mechanical hyperalgesia, with male rats exhibiting greater hyperalgesia. Because female rats have higher plasma epinephrine levels, and beta-adrenergic receptor sensitivity is affected by chronic exposure to agonists, we tested the hypothesis that this sexual dimorphism is due to epinephrine-induced desensitization of beta(2)-adrenergic receptors. Following gonadectomy, epinephrine hyperalgesia, as measured by the Randall-Selitto paw-withdrawal test, was unchanged in male rats while in females it was increased. Prepubertal male and female rats do not demonstrate sexual dimorphism in either plasma epinephrine level or epinephrine-induced hyperalgesia. Adrenal medullectomy and adrenal denervation both significantly enhanced epinephrine hyperalgesia, but only in females. In contrast, the sexually dimorphic hyperalgesia induced by prostaglandin E(2), another agent that acts directly to sensitize primary afferent nociceptors, was not enhanced by adrenal medullectomy or denervation. Chronic administration of epinephrine in male rats, to produce plasma levels similar to those of gonad-intact females, significantly attenuated epinephrine-induced hyperalgesia, making it similar to that in females. These results strongly support the suggestion that estrogen regulates plasma epinephrine in female rats and differential sensitivity to beta(2)-adrenergic agonists accounts for the sexual dimorphism in epinephrine-induced hyperalgesia. Unexpectedly, regulation of adrenal medullary function by estrogen was also found to modulate baseline nociceptive threshold such that females had a lower nociceptive threshold.


Assuntos
Medula Suprarrenal/efeitos dos fármacos , Estradiol/administração & dosagem , Hiperalgesia/fisiopatologia , Limiar da Dor/efeitos dos fármacos , Receptores Adrenérgicos beta/fisiologia , Caracteres Sexuais , Medula Suprarrenal/fisiologia , Adrenalectomia/métodos , Análise de Variância , Animais , Animais Recém-Nascidos , Castração/métodos , Cromatografia Líquida de Alta Pressão/métodos , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Epinefrina/administração & dosagem , Epinefrina/sangue , Feminino , Masculino , Medição da Dor , Limiar da Dor/fisiologia , Ratos , Fatores de Tempo
15.
Eur J Neurosci ; 21(12): 3387-94, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16026476

RESUMO

The underlying mechanism for nerve growth factor (NGF) evoked pain and long-lasting mechanical hyperalgesia remains poorly understood. Using intrathecal antisense against the NGF receptor, receptor tyrosine kinase (TrkA), we found NGF to act at the primary afferent nociceptor directly in the Sprague-Dawley rat. Inhibitors of the three major pathways for TrkA receptor signalling, extracellular signal-related kinase (ERK)/mitogen-activated protein kinase kinase (MEK) (ERK/MEK), phosphatidylinositol 3-kinase (PI3K), and phospholipase Cgamma (PLCgamma) all attenuate NGF-induced hyperalgesia. Although inhibitors of kinases downstream of PI3K and PLCgamma[glycogen synthetase kinase 3 (GSK3), calmodulin-dependent protein kinase II (CAMII-K) or protein kinase C (PKC)] do not reduce mechanical hyperalgesia, hyperalgesia induced by activation of PI3K was blocked by ERK/MEK inhibitors, suggesting cross-talk from the PI3K to the ERK/MEK signalling pathway. As integrins have been shown to modulate epinephrine and prostaglandin E(2)-induced hyperalgesia, we also evaluated a role for integrins in NGF-induced mechanical hyperalgesia using beta(1)-integrin-specific antisense or antibodies.


Assuntos
Vias Aferentes/fisiologia , Hiperalgesia/induzido quimicamente , Fatores de Crescimento Neural , Nociceptores/fisiologia , Limiar da Dor/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Western Blotting/métodos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Integrinas/imunologia , Masculino , Modelos Biológicos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Limiar da Dor/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor trkA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo
16.
J Neurosci ; 24(18): 4444-52, 2004 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-15128858

RESUMO

The development of treatments for neuropathic pain has been hindered by our limited understanding of the basic mechanisms underlying abnormalities in nociceptor hyperexcitability. We recently showed that the polymodal receptor transient receptor potential vanilloid 4 (TRPV4), a member of the transient receptor potential (TRP) family of ion channels, may play a role in inflammatory pain (Alessandri-Haber et al., 2003). The present study tested whether TRVP4 also contributes to neuropathic pain, using a rat model of Taxol-induced painful peripheral neuropathy. Taxol is the most widely used drug for the treatment of a variety of tumor types, but the dose of Taxol that can be tolerated is limited by the development of a small-fiber painful peripheral neuropathy. We found that Taxol treatment enhanced the nociceptive behavioral responses to both mechanical and hypotonic stimulation of the hind paw. Spinal administration of antisense oligodeoxynucleotides to TRPV4, which reduced the expression of TRPV4 in sensory nerve, abolished Taxol-induced mechanical hyperalgesia and attenuated hypotonic hyperalgesia by 42%. The enhancement of osmotic nociception involves sensitization of osmotransduction in primary afferents because osmotransduction was enhanced in cultured sensory neurons isolated from Taxol-treated rats. Taxol-induced TRPV4-mediated hyperalgesia and the enhanced osmotransduction in cultured nociceptors were dependent on integrin/Src tyrosine kinase signaling. These results suggest that TRPV4 plays a crucial role in a painful peripheral neuropathy, making it a very promising target for the development of a novel class of analgesics.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Canais Iônicos/metabolismo , Neuralgia/fisiopatologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Animais , Antineoplásicos Fitogênicos , Comportamento Animal/efeitos dos fármacos , Cálcio/metabolismo , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Células Cultivadas , Modelos Animais de Doenças , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Soluções Hipotônicas , Integrinas/metabolismo , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Masculino , Neuralgia/induzido quimicamente , Neuralgia/tratamento farmacológico , Nociceptores/citologia , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Paclitaxel , Medição da Dor , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Canais de Cátion TRPV , Quinases da Família src/metabolismo
17.
Eur J Neurosci ; 19(3): 634-42, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14984413

RESUMO

Many painful conditions are associated with alterations in the extracellular matrix (ECM) of affected tissues. While several integrins, the receptors for ECM proteins, are present on sensory neurons that mediate pain, the possible role of these cell adhesion molecules in inflammatory or neuropathic pain has not been explored. We found that the intradermal injection of peptide fragments of domains of laminin and fibronectin important for adhesive signaling selectively inhibited the hyperalgesia caused by prostaglandin E2 (PGE2) and epinephrine (EPI), respectively. The block of EPI hyperalgesia was mimicked by other peptides containing the RGD integrin-binding sequence. Monoclonal antibodies (mAbs) against the alpha1 or alpha3 integrin subunits, which participate in laminin binding, selectively blocked PGE2 hyperalgesia, while a mAb against the alpha5 subunit, which participates in fibronectin binding, blocked only EPI-induced hyperalgesia. A mAb against the beta1 integrin subunit, common to receptors for both laminin and fibronectin, inhibited hyperalgesia caused by both agents, as did the knockdown of beta1 integrin expression by intrathecal injection of antisense oligodeoxynucleotides. The laminin peptide, but not the fibronectin peptides, also reversibly abolished the longer lasting inflammatory hyperalgesia induced by carrageenan. Finally, the neuropathic hyperalgesia caused by systemic administration of the cancer chemotherapy agent taxol was reversibly inhibited by antisense knockdown of beta1 integrin. These results strongly implicate specific integrins in the maintenance of inflammatory and neuropathic hyperalgesia.


Assuntos
Integrinas/metabolismo , Dor/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Anticorpos Monoclonais/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Western Blotting/métodos , Carragenina , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Epinefrina , Proteínas da Matriz Extracelular/farmacologia , Fibronectinas/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Fibras Nervosas Amielínicas/efeitos dos fármacos , Fibras Nervosas Amielínicas/fisiologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Paclitaxel/farmacologia , Dor/induzido quimicamente , Medição da Dor/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Peptídeos , Proteína Quinase C/metabolismo , Proteína Quinase C-épsilon , Subunidades Proteicas/imunologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
18.
Neuron ; 39(4): 613-24, 2003 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-12925276

RESUMO

Prostaglandin E(2) (PGE(2)) and epinephrine act directly on nociceptors to produce mechanical hyperalgesia through protein kinase A (PKA) alone or through a combination of PKA, protein kinase C epsilon (PKCepsilon), and extracellular signal-regulated kinase (ERK), respectively. Disruptors of the cytoskeleton (microfilaments, microtubules, and intermediate filaments) markedly attenuated the hyperalgesia in rat paws caused by injection of epinephrine or its downstream mediators. In contrast, the hyperalgesia induced by PGE(2) or its mediators was not affected by any of the cytoskeletal disruptors. These effects were mimicked in vitro, as measured by enhancement of the tetrodotoxin-resistant sodium current. When PGE(2) hyperalgesia was shifted to dependence on PKCepsilon and ERK as well as PKA, as when the tissue is "primed" by prior treatment with carrageenan, it too became dependent on an intact cytoskeleton. Thus, inflammatory mediator-induced mechanical hyperalgesia was differentially dependent on the cytoskeleton such that cytoskeletal dependence correlated with mediation by PKCepsilon and ERK.


Assuntos
Citoesqueleto/fisiologia , Neurônios Aferentes/fisiologia , Dor/fisiopatologia , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico , Dinoprostona/metabolismo , Eletrofisiologia , Epinefrina/metabolismo , Hiperalgesia/induzido quimicamente , Imuno-Histoquímica , Inflamação/fisiopatologia , MAP Quinase Quinase Quinase 3 , MAP Quinase Quinase Quinases/metabolismo , Masculino , Microscopia Confocal , Nociceptores/fisiologia , Técnicas de Patch-Clamp , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Quimiocinas/fisiologia
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