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1.
Cell ; 187(8): 1874-1888.e14, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38518773

RESUMO

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Pulmão , Polissacarídeos Bacterianos , Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Feminino , Masculino , Camundongos , Biofilmes , Escherichia coli/fisiologia , Hipotermia/metabolismo , Hipotermia/patologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/microbiologia , Pulmão/patologia , Pneumonia/microbiologia , Pneumonia/patologia , Pseudomonas aeruginosa/fisiologia , Células Receptoras Sensoriais , Polissacarídeos Bacterianos/metabolismo , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Nociceptores/metabolismo
2.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35046040

RESUMO

Inflammatory pain, such as hypersensitivity resulting from surgical tissue injury, occurs as a result of interactions between the immune and nervous systems with the orchestrated recruitment and activation of tissue-resident and circulating immune cells to the site of injury. Our previous studies identified a central role for Ly6Clow myeloid cells in the pathogenesis of postoperative pain. We now show that the chemokines CCL17 and CCL22, with their cognate receptor CCR4, are key mediators of this response. Both chemokines are up-regulated early after tissue injury by skin-resident dendritic and Langerhans cells to act on peripheral sensory neurons that express CCR4. CCL22, and to a lesser extent CCL17, elicit acute mechanical and thermal hypersensitivity when administered subcutaneously; this response abrogated by pharmacological blockade or genetic silencing of CCR4. Electrophysiological assessment of dissociated sensory neurons from naïve and postoperative mice showed that CCL22 was able to directly activate neurons and enhance their excitability after injury. These responses were blocked using C 021 and small interfering RNA (siRNA)-targeting CCR4. Finally, our data show that acute postoperative pain is significantly reduced in mice lacking CCR4, wild-type animals treated with CCR4 antagonist/siRNA, as well as transgenic mice depleted of dendritic cells. Together, these results suggest an essential role for the peripheral CCL17/22:CCR4 axis in the genesis of inflammatory pain via direct communication between skin-resident dendritic cells and sensory neurons, opening therapeutic avenues for its control.


Assuntos
Células de Langerhans/metabolismo , Dor Pós-Operatória/etiologia , Dor Pós-Operatória/metabolismo , Receptores CCR4/metabolismo , Células Receptoras Sensoriais/metabolismo , Potenciais de Ação , Animais , Biomarcadores , Quimiocina CCL17/genética , Quimiocina CCL17/metabolismo , Quimiocina CCL22/genética , Quimiocina CCL22/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Células de Langerhans/imunologia , Camundongos , Dor Pós-Operatória/diagnóstico , Transdução de Sinais
3.
Int J Mol Sci ; 23(5)2022 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-35269644

RESUMO

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca2+ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gαq led to a Gαq-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT1R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT1R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gαq-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Técnicas Biossensoriais , Animais , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Células HEK293 , Humanos , Ratos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo
4.
J Physiol ; 599(4): 1335-1354, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33180962

RESUMO

KEY POINTS: We have previously shown that carotid body stimulation by lysophosphatidic acid elicits a reflex stimulation of vagal efferent activity sufficient to cause bronchoconstriction in asthmatic rats. Here, we show that pathophysiological concentrations of asthma-associated prototypical Th2 cytokines also stimulate the carotid bodies. Stimulation of the carotid bodies by these asthmakines involves a PKCε-transient receptor potential vanilloid 1 (TRPV1) signalling mechanism likely dependent on TRPV1 S502 and T704 phosphorylation sites. As the carotid bodies' oxygen sensitivity is independent of PKCε-TRPV1 signalling, systemic blockade of PKCε may provide a novel therapeutic target to reduce allergen-induced asthmatic bronchoconstriction. Consistent with the therapeutic potential of blocking the PKCε-TRPV1 pathway, systemic delivery of a PKCε-blocking peptide suppresses asthmatic respiratory distress in response to allergen and reduces airway hyperresponsiveness to bradykinin. ABSTRACT: The autonomic nervous system orchestrates organ-specific, systemic and behavioural responses to inflammation. Recently, we demonstrated a vital role for lysophosphatidic acid in stimulating the primary autonomic oxygen chemoreceptors, the carotid bodies, in parasympathetic-mediated asthmatic airway hyperresponsiveness. However, the cacophony of stimulatory factors and cellular mechanisms of carotid body activation are unknown. Therefore, we set out to determine the intracellular signalling involved in carotid body-mediated sensing of asthmatic blood-borne inflammatory mediators. We employed a range of in vitro and rat in situ preparations, site-directed mutagenesis, patch-clamp, nerve recordings and pharmacological inhibition to assess cellular signalling. We show that the carotid bodies are also sensitive to asthma-associated prototypical Th2 cytokines which elicit sensory nerve excitation. This provides additional asthmatic ligands contributing to the previously established reflex arc resulting in efferent vagal activity and asthmatic bronchoconstriction. This novel sensing role for the carotid body is mediated by a PKCε-dependent stimulation of transient receptor potential vanilloid 1 (TRPV1), likely via TRPV1 phosphorylation at sites T704 and S502. Importantly, carotid body oxygen sensing was unaffected by blocking either PKCε or TRPV1. Further, we demonstrate that systemic PKCε blockade reduces asthmatic respiratory distress in response to allergen and airway hyperresponsiveness. These discoveries support an inflammation-dependent, oxygen-independent function for the carotid body and suggest that targeting PKCε provides a novel therapeutic option to abate allergic airway disease without altering life-saving autonomic hypoxic reflexes.


Assuntos
Asma , Corpo Carotídeo , Animais , Corpo Carotídeo/metabolismo , Fosforilação , Proteína Quinase C-épsilon , Ratos , Canais de Cátion TRPV/metabolismo
5.
Am J Physiol Gastrointest Liver Physiol ; 321(3): G280-G297, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34288735

RESUMO

Intestinal fibrosis is a common complication of the inflammatory bowel diseases (IBDs), contributing to tissue stiffening and luminal narrowing. Human nuclear receptor 4A 1 (NR4A1) was previously reported to regulate mesenchymal cell function and dampen fibrogenic signaling. NR4A1 gene variants are associated with IBD risk, and it has been shown to regulate intestinal inflammation. Here, we tested the hypothesis that NR4A1 acts as a negative regulator of intestinal fibrosis through regulating myofibroblast function. Using the SAMP1/YitFc mouse, we tested whether two pharmacological agents known to enhance NR4A1 signaling, cytosporone B (Csn-B) or 6-mercaptopurine (6-MP), could reduce fibrosis. We also used the dextran sulfate sodium (DSS) model of colitis and assessed the magnitude of colonic fibrosis in mouse nuclear receptor 4A 1 (Nr4a1-/-) and their wild-type littermates (Nr4a1+/+). Lastly, intestinal myofibroblasts isolated from Nr4a1-/- and Nr4a1+/+ mice or primary human intestinal myofibroblasts were stimulated with transforming growth factor-ß1 (TGF-ß1), in the presence or absence of Csn-B or 6-MP, and proliferation and ECM gene expression assessed. Csn-B or 6-MP treatment significantly reduced ileal thickness, collagen, and overall ECM content in SAMP1/YitFc mice. This was associated with a reduction in proliferative markers within the mesenchymal compartment. Nr4a1-/- mice exposed to DSS exhibited increased colonic thickening and ECM content. Nr4a1-/- myofibroblasts displayed enhanced TGF-ß1-induced proliferation. Furthermore, Csn-B or 6-MP treatment was antiproliferative in Nr4a1+/+ but not Nr4a1-/- cells. Lastly, activating NR4A1 in human myofibroblasts reduced TGF-ß1-induced collagen deposition and fibrosis-related gene expression. Our data suggest that NR4A1 can attenuate fibrotic processes in intestinal myofibroblasts and could provide a valuable clinical target to treat inflammation-associated intestinal fibrosis.NEW & NOTEWORTHY Fibrosis and increased muscle thickening contribute to stricture formation and intestinal obstruction, a complication that occurs in 30%-50% of patients with CD within 10 yr of disease onset. More than 50% of those who undergo surgery to remove the obstructed bowel will experience stricture recurrence. To date, there are no drug-based approaches approved to treat intestinal strictures. In the current submission, we identify NR4A1 as a novel target to treat inflammation-associated intestinal fibrosis.


Assuntos
Fibrose/metabolismo , Inflamação/metabolismo , Miofibroblastos/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Animais , Células Cultivadas , Humanos , Intestinos/patologia , Camundongos , Transdução de Sinais/fisiologia
6.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668926

RESUMO

Transient receptor potential vanilloids (TRPV1) are non-selective cation channels that sense and transduce inflammatory pain signals. We previously reported that activation of TRPV1 induced the translocation of ß-arrestin2 (ARRB2) from the cytoplasm to the nucleus, raising questions about the functional role of ARRB2 in the nucleus. Here, we determined the ARRB2 nuclear signalosome by conducting a quantitative proteomic analysis of the nucleus-sequestered L395Q ARRB2 mutant, compared to the cytosolic wild-type ARRB2 (WT ARRB2), in a heterologous expression system. We identified clusters of proteins that localize to the nucleolus and are involved in ribosomal biogenesis. Accordingly, L395Q ARRB2 or WT ARRB2 after capsaicin treatment were found to co-localize and interact with the nucleolar marker nucleophosmin (NPM1), treacle protein (TCOF1) and RNA polymerase I (POL I). We further investigated the role of nuclear ARRB2 signaling in regulating neuroplasticity. Using neuroblastoma (neuro2a) cells and dorsal root ganglia (DRG) neurons, we found that L395Q ARRB2 mutant increased POL I activity, inhibited the tumor suppressorp53 (p53) level and caused a decrease in the outgrowth of neurites. Together, our results suggest that the activation of TRPV1 promotes the ARRB2-mediated regulation of ribosomal biogenesis in the nucleolus. The ARRB2-TCOF1-p53 checkpoint signaling pathway might be involved in regulating neurite outgrowth associated with pathological pain conditions.


Assuntos
Nucléolo Celular/metabolismo , Crescimento Neuronal , Ribossomos/metabolismo , Canais de Cátion TRPV/metabolismo , Proteína Supressora de Tumor p53/metabolismo , beta-Arrestina 2/metabolismo , Animais , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Nucleofosmina , Ligação Proteica , Transporte Proteico , Proteômica , RNA Polimerase I/metabolismo
7.
Proc Natl Acad Sci U S A ; 114(42): 11235-11240, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-28973941

RESUMO

Pain is a main symptom of inflammatory diseases and often persists beyond clinical remission. Although we have a good understanding of the mechanisms of sensitization at the periphery during inflammation, little is known about the mediators that drive central sensitization. Recent reports have identified hematopoietic colony-stimulating factors as important regulators of tumor- and nerve injury-associated pain. Using a mouse model of colitis, we identify the proinflammatory cytokine granulocyte-colony-stimulating factor (G-CSF or Csf-3) as a key mediator of visceral sensitization. We report that G-CSF is specifically up-regulated in the thoracolumbar spinal cord of colitis-affected mice. Our results show that resident spinal microglia express the G-CSF receptor and that G-CSF signaling mediates microglial activation following colitis. Furthermore, healthy mice subjected to intrathecal injection of G-CSF exhibit pronounced visceral hypersensitivity, an effect that is abolished by microglial depletion. Mechanistically, we demonstrate that G-CSF injection increases Cathepsin S activity in spinal cord tissues. When cocultured with microglia BV-2 cells exposed to G-CSF, dorsal root ganglion (DRG) nociceptors become hyperexcitable. Blocking CX3CR1 or nitric oxide production during G-CSF treatment reduces excitability and G-CSF-induced visceral pain in vivo. Finally, administration of G-CSF-neutralizing antibody can prevent the establishment of persistent visceral pain postcolitis. Overall, our work uncovers a DRG neuron-microglia interaction that responds to G-CSF by engaging Cathepsin S-CX3CR1-inducible NOS signaling. This interaction represents a central step in visceral sensitization following colonic inflammation, thereby identifying spinal G-CSF as a target for treating chronic abdominal pain.


Assuntos
Colite/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Microglia/metabolismo , Medula Espinal/metabolismo , Dor Visceral/etiologia , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Catepsinas/metabolismo , Linhagem Celular , Colite/induzido quimicamente , Sulfato de Dextrana , Gânglios Espinais/metabolismo , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Dor Visceral/metabolismo
8.
Emerg Infect Dis ; 23(2): 280-283, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28098530

RESUMO

Murray Valley encephalitis virus (MVEV), a flavivirus belonging to the Japanese encephalitis serogroup, can cause severe clinical manifestations in humans. We report a fatal case of MVEV infection in a young woman who returned from Australia to Canada. The differential diagnosis for travel-associated encephalitis should include MVEV, particularly during outbreak years.


Assuntos
Doenças Transmissíveis Importadas , Vírus da Encefalite do Vale de Murray , Encefalite por Arbovirus/diagnóstico , Encefalite por Arbovirus/virologia , Viagem , Austrália/epidemiologia , Autopsia , Biomarcadores , Encéfalo/patologia , Canadá/epidemiologia , Surtos de Doenças , Vírus da Encefalite do Vale de Murray/classificação , Vírus da Encefalite do Vale de Murray/genética , Encefalite por Arbovirus/epidemiologia , Evolução Fatal , Feminino , Humanos , Imageamento por Ressonância Magnética , Adulto Jovem
9.
Mol Pain ; 122016.
Artigo em Inglês | MEDLINE | ID: mdl-27558883

RESUMO

BACKGROUND: Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. RESULTS: We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. CONCLUSIONS: Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function and points to the role of this stress protein in pain associated with neurodegenerative and/or metabolic disorders, including aging.


Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Neurônios/metabolismo , Canais de Cátion TRPV/metabolismo , Alcaloides/farmacologia , Animais , Anti-Helmínticos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Capsaicina/farmacologia , Células Cultivadas , Césio/farmacologia , Cloretos/farmacologia , Inibidores Enzimáticos/farmacologia , Adjuvante de Freund/toxicidade , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Guanidinas/farmacologia , Células HEK293 , Proteínas de Choque Térmico HSC70/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Camundongos , Neurônios/efeitos dos fármacos , Dor/etiologia , Dor/metabolismo , Dor/patologia , Bloqueadores dos Canais de Potássio/farmacologia , Quinolizinas/farmacologia , Ratos , Canais de Cátion TRPV/genética , Matrinas
10.
J Biol Chem ; 289(24): 16675-87, 2014 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-24808184

RESUMO

The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief.


Assuntos
Hiperalgesia/metabolismo , Multimerização Proteica , Canais de Cátion TRPV/metabolismo , Motivos de Aminoácidos , Animais , Artrite Experimental/metabolismo , Artrite Experimental/fisiopatologia , Sítios de Ligação , Deleção de Genes , Células HEK293 , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Ratos , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética
11.
Am J Physiol Gastrointest Liver Physiol ; 309(2): G87-99, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26021808

RESUMO

Quiescent phases of inflammatory bowel disease (IBD) are often accompanied by chronic abdominal pain. Although the transient receptor potential vanilloid 1 (TRPV1) ion channel has been postulated as an important mediator of visceral hypersensitivity, its functional role in postinflammatory pain remains elusive. This study aimed at establishing the role of TRPV1 in the peripheral sensitization underlying chronic visceral pain in the context of colitis. Wild-type and TRPV1-deficient mice were separated into three groups (control, acute colitis, and recovery), and experimental colitis was induced by oral administration of dextran sulfate sodium (DSS). Recovery mice showed increased chemically and mechanically evoked visceral hypersensitivity 5 wk post-DSS discontinuation, at which point inflammation had completely resolved. Significant changes in nonevoked pain-related behaviors could also be observed in these animals, indicative of persistent discomfort. These behavioral changes correlated with elevated colonic levels of substance P (SP) and TRPV1 in recovery mice, thus leading to the hypothesis that SP could sensitize TRPV1 function. In vitro experiments revealed that prolonged exposure to SP could indeed sensitize capsaicin-evoked currents in both cultured neurons and TRPV1-transfected human embryonic kidney (HEK) cells, a mechanism that involved TRPV1 ubiquitination and subsequent accumulation at the plasma membrane. Importantly, although TRPV1-deficient animals experienced similar disease severity and pain as wild-type mice in the acute phase of colitis, TRPV1 deletion prevented the development of postinflammatory visceral hypersensitivity and pain-associated behaviors. Collectively, our results suggest that chronic exposure of colon-innervating primary afferents to SP could sensitize TRPV1 and thus participate in the establishment of persistent abdominal pain following acute inflammation.


Assuntos
Dor Abdominal/metabolismo , Colite/metabolismo , Colo/inervação , Hiperalgesia/metabolismo , Limiar da Dor , Canais de Cátion TRPV/metabolismo , Dor Visceral/metabolismo , Dor Abdominal/induzido quimicamente , Dor Abdominal/genética , Dor Abdominal/fisiopatologia , Doença Aguda , Animais , Comportamento Animal , Colite/induzido quimicamente , Colite/genética , Colite/fisiopatologia , Sulfato de Dextrana , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Células HEK293 , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/fisiopatologia , Hiperalgesia/prevenção & controle , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Aferentes/metabolismo , Medição da Dor , Transdução de Sinais , Substância P/metabolismo , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Fatores de Tempo , Transfecção , Dor Visceral/induzido quimicamente , Dor Visceral/genética , Dor Visceral/fisiopatologia
12.
J Clin Invest ; 134(9)2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38690737

RESUMO

Inflammation and pain are intertwined responses to injury, infection, or chronic diseases. While acute inflammation is essential in determining pain resolution and opioid analgesia, maladaptive processes occurring during resolution can lead to the transition to chronic pain. Here we found that inflammation activates the cytosolic DNA-sensing protein stimulator of IFN genes (STING) in dorsal root ganglion nociceptors. Neuronal activation of STING promotes signaling through TANK-binding kinase 1 (TBK1) and triggers an IFN-ß response that mediates pain resolution. Notably, we found that mice expressing a nociceptor-specific gain-of-function mutation in STING exhibited an IFN gene signature that reduced nociceptor excitability and inflammatory hyperalgesia through a KChIP1-Kv4.3 regulation. Our findings reveal a role of IFN-regulated genes and KChIP1 downstream of STING in the resolution of inflammatory pain.


Assuntos
Proteínas de Membrana , Nociceptores , Animais , Camundongos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nociceptores/metabolismo , Gânglios Espinais/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Inflamação/genética , Inflamação/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Dor/metabolismo , Dor/genética , Transdução de Sinais , Masculino
13.
Neuropathology ; 33(1): 87-92, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22640269

RESUMO

We report a rare case of a 33-year-old man with a lipidized glioblastoma multiforme (GBM) in the right posterior frontal region. Histologically the tumor had all the typical features of a GBM but with the rare observation of lipidized differentiation. There were multiple mitoses, extensive vascular proliferation, focal necrosis and the tumor cells had abundant xanthomatous cytoplasm and marked nuclear pleomorphism. The tumor showed immunoreactivity with GFAP. The O(6) - methylguanine methyltransferase (MGMT) promoter was methylated and there were no isocitrate dehydrogenase (IDH)1 and IDH2 mutations. To the best of our knowledge, this is the first time MGMT promoter status and IDH mutation assessment have been reported in a case of lipidized GBM.


Assuntos
Neoplasias Encefálicas/patologia , Lobo Frontal/patologia , Glioblastoma/patologia , Adulto , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Lobo Frontal/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Masculino , Regiões Promotoras Genéticas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
14.
Cell Mol Gastroenterol Hepatol ; 13(4): 977-999, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34954381

RESUMO

BACKGROUND & AIMS: Chronic abdominal pain is a common symptom of inflammatory bowel diseases (IBDs). Peripheral and central mechanisms contribute to the transition from acute to chronic pain during active disease and clinical remission. Lower mechanical threshold and hyperexcitability of visceral afferents induce gliosis in central pain circuits, leading to persistent visceral hypersensitivity (VHS). In the spinal cord, microglia, the immune sentinels of the central nervous system, undergo activation in multiple models of VHS. Here, we investigated the mechanisms of microglia activation to identify centrally acting analgesics for chronic IBD pain. METHODS: Using Designer Receptors Exclusively Activated by Designer Drugs (DREADD) expressed in transient receptor potential vanilloid member 1-expressing visceral neurons that sense colonic inflammation, we tested whether neuronal activity was indispensable to control microglia activation and VHS. We then investigated the neuron-microglia signaling system involved in visceral pain chronification. RESULTS: We found that chemogenetic inhibition of transient receptor potential vanilloid member 1+ visceral afferents prevents microglial activation in the spinal cord and subsequent VHS in colitis mice. In contrast, chemogenetic activation, in the absence of colitis, enhanced microglial activation associated with VHS. We identified a purinergic signaling mechanism mediated by neuronal adenosine triphosphate (ATP) and microglial P2Y12 receptor, triggering VHS in colitis. Inhibition of P2RY12 prevented microglial reactivity and chronic VHS post-colitis. CONCLUSIONS: Overall, these data provide novel insights into the central mechanisms of chronic visceral pain and suggest that targeting microglial P2RY12 signaling could be harnessed to relieve pain in patients with IBD who are in remission.


Assuntos
Dor Crônica , Colite , Doenças Inflamatórias Intestinais , Dor Visceral , Animais , Humanos , Camundongos , Microglia , Neurônios , Antagonistas do Receptor Purinérgico P2Y , Canais de Cátion TRPV
15.
J Clin Invest ; 132(12)2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35608912

RESUMO

The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase known for its oncogenic potential that is involved in the development of the peripheral and central nervous system. ALK receptor ligands ALKAL1 and ALKAL2 were recently found to promote neuronal differentiation and survival. Here, we show that inflammation or injury enhanced ALKAL2 expression in a subset of TRPV1+ sensory neurons. Notably, ALKAL2 was particularly enriched in both mouse and human peptidergic nociceptors, yet weakly expressed in nonpeptidergic, large-diameter myelinated neurons or in the brain. Using a coculture expression system, we found that nociceptors exposed to ALKAL2 exhibited heightened excitability and neurite outgrowth. Intraplantar CFA or intrathecal infusion of recombinant ALKAL2 led to ALK phosphorylation in the lumbar dorsal horn of the spinal cord. Finally, depletion of ALKAL2 in dorsal root ganglia or blocking ALK with clinically available compounds crizotinib or lorlatinib reversed thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury, respectively. Overall, our work uncovers the ALKAL2/ALK signaling axis as a central regulator of nociceptor-induced sensitization. We propose that clinically approved ALK inhibitors used for non-small cell lung cancer and neuroblastomas could be repurposed to treat persistent pain conditions.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Citocinas/metabolismo , Neoplasias Pulmonares , Animais , Humanos , Hiperalgesia/metabolismo , Inflamação/patologia , Ligantes , Camundongos , Dor/tratamento farmacológico , Receptores Proteína Tirosina Quinases , Células Receptoras Sensoriais/metabolismo , Corno Dorsal da Medula Espinal/patologia
16.
Nat Neurosci ; 10(7): 854-60, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17558400

RESUMO

We investigated the regulation of T-type channels by Rho-associated kinase (ROCK). Activation of ROCK via the endogenous ligand lysophosphatidic acid (LPA) reversibly inhibited the peak current amplitudes of rat Ca(v)3.1 and Ca(v)3.3 channels without affecting the voltage dependence of activation or inactivation, whereas Ca(v)3.2 currents showed depolarizing shifts in these parameters. LPA-induced inhibition of Ca(v)3.1 was dependent on intracellular GTP, and was antagonized by treatment with ROCK and RhoA inhibitors, LPA receptor antagonists or GDPssS. Site-directed mutagenesis of the Ca(v)3.1 alpha1 subunit revealed that the ROCK-mediated effects involve two distinct phosphorylation consensus sites in the domain II-III linker. ROCK activation by LPA reduced native T-type currents in Y79 retinoblastoma and in lateral habenular neurons, and upregulated native Ca(v)3.2 current in dorsal root ganglion neurons. Our data suggest that ROCK is an important regulator of T-type calcium channels, with potentially far-reaching implications for multiple cell functions modulated by LPA.


Assuntos
Canais de Cálcio Tipo T/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Western Blotting , Canais de Cálcio Tipo T/metabolismo , Eletrofisiologia , Gânglios Espinais/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Imuno-Histoquímica , Lisofosfolipídeos/farmacologia , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Ratos , Retinoblastoma/metabolismo , Tionucleotídeos/metabolismo , Quinases Associadas a rho
17.
Drugs ; 81(1): 7-27, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33165872

RESUMO

The transient receptor potential vanilloid-1 (TRPV1) is a non-specific cation channel known for its sensitivity to pungent vanilloid compound (i.e. capsaicin) and noxious stimuli, including heat, low pH or inflammatory mediators. TRPV1 is found in the somatosensory system, particularly primary afferent neurons that respond to damaging or potentially damaging stimuli (nociceptors). Stimulation of TRPV1 evokes a burning sensation, reflecting a central role of the channel in pain. Pharmacological and genetic studies have validated TRPV1 as a therapeutic target in several preclinical models of chronic pain, including cancer, neuropathic, postoperative and musculoskeletal pain. While antagonists of TRPV1 were found to be a valuable addition to the pain therapeutic toolbox, their clinical use has been limited by detrimental side effects, such as hyperthermia. In contrast, capsaicin induces a prolonged defunctionalisation of nociceptors and thus opened the door to the development of a new class of therapeutics with long-lasting pain-relieving effects. Here we review the list of TRPV1 agonists undergoing clinical trials for chronic pain management, and discuss new indications, formulations or combination therapies being explored for capsaicin. While the analgesic pharmacopeia for chronic pain patients is ancient and poorly effective, modern TRPV1-targeted drugs could rapidly become available as the next generation of analgesics for a broad spectrum of pain conditions.


Assuntos
Analgésicos/farmacologia , Desenvolvimento de Medicamentos , Dor/tratamento farmacológico , Canais de Cátion TRPV/antagonistas & inibidores , Humanos , Dor/metabolismo , Canais de Cátion TRPV/metabolismo
18.
Mol Brain ; 14(1): 115, 2021 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-34274007

RESUMO

T-type calcium channels are important regulators of neuronal excitability. The mammalian brain expresses three T-type channel isoforms (Cav3.1, Cav3.2 and Cav3.3) with distinct biophysical properties that are critically regulated by temperature. Here, we test the effects of how temperature affects spike output in a reduced firing neuron model expressing specific Cav3 channel isoforms. The modeling data revealed only a minimal effect on baseline spontaneous firing near rest, but a dramatic increase in rebound burst discharge frequency for Cav3.1 compared to Cav3.2 or Cav3.3 due to differences in window current or activation/recovery time constants. The reduced response by Cav3.2 could optimize its activity where it is expressed in peripheral tissues more subject to temperature variations than Cav3.1 or Cav3.3 channels expressed prominently in the brain. These tests thus reveal that aspects of neuronal firing behavior are critically dependent on both temperature and T-type calcium channel subtype.


Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo T/metabolismo , Modelos Neurológicos , Temperatura , Células HEK293 , Humanos , Neurônios/metabolismo
19.
Trends Pharmacol Sci ; 30(1): 32-40, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19042038

RESUMO

T-type calcium channels are critically important for regulating neuronal excitability, both in the central and peripheral nervous system, and are essential mediators of hormone secretion. Conversely, T-type channel hyperactivity has been linked to neurological disorders such as absence epilepsy and neuropathic pain. Hence, it is critical to understand the cellular mechanisms that control T-type channel activity, including means of altering expression patterns of the channels, activation of intracellular messenger cascades that directly affect channel activity, and the regulation of alternate splicing of T-type channel genes. Although there is substantial literature dealing with regulation of native T-type channels, the underlying molecular mechanism have only recently been addressed. Here, we highlight recent advances in our understanding of T-type channel regulation, and their implications for brain function.


Assuntos
Canais de Cálcio Tipo T/fisiologia , Neurônios/fisiologia , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Canais de Cálcio Tipo T/química , Sinalização do Cálcio/fisiologia , Humanos , Modelos Teóricos , Neurônios/química , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia
20.
Channels (Austin) ; 14(1): 413-420, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33147416

RESUMO

Transient receptor potential melastatin 8 (TRPM8) channels play a central role in the detection of environmental cold temperatures in the somatosensory system. TRPM8 is found in a subset of unmyelinated (C-type) afferents located in the dorsal root (DRG) and trigeminal ganglion (TG). Cold hypersensitivity is a common symptom of neuropathic pain conditions caused by cancer therapy, spinal cord injury, viral infection, multiple sclerosis, diabetes, or withdrawal symptoms associated with chronic morphine treatment. Therefore, TRPM8 has received great attention as a therapeutic target. However, as the activity of TRPM8 is unique in sensing cool temperature as well as warming, it is critical to understand the signaling transduction pathways that control modality-specific activity of TRPM8 in healthy versus pathological settings. This review summarizes recent advances in our understanding of the mechanisms involved in the regulation of the TRPM8 activity.


Assuntos
Canais de Cátion TRPM/metabolismo , Animais , Proteínas de Ligação ao GTP/metabolismo , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilação
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