RESUMO
Medical treatments for corticotrophinomas are limited, and we therefore investigated the effects of epigenetic modulators, a new class of anti-tumour drugs, on the murine adrenocorticotropic hormone (ACTH)-secreting corticotrophinoma cell line AtT20. We found that AtT20 cells express members of the bromo and extra-terminal (BET) protein family, which bind acetylated histones, and therefore, studied the anti-proliferative and pro-apoptotic effects of two BET inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, using CellTiter Blue and Caspase Glo assays, respectively. JQ1 and PFI-1 significantly decreased proliferation by 95% (P < 0.0005) and 43% (P < 0.0005), respectively, but only JQ1 significantly increased apoptosis by >50-fold (P < 0.0005), when compared to untreated control cells. The anti-proliferative effects of JQ1 and PFI-1 remained for 96 h after removal of the respective compound. JQ1, but not PFI-1, affected the cell cycle, as assessed by propidium iodide staining and flow cytometry, and resulted in a higher number of AtT20 cells in the sub G1 phase. RNA-sequence analysis, which was confirmed by qRT-PCR and Western blot analyses, revealed that JQ1 treatment significantly altered expression of genes involved in apoptosis, such as NFκB, and the somatostatin receptor 2 (SSTR2) anti-proliferative signalling pathway, including SSTR2. JQ1 treatment also significantly reduced transcription and protein expression of the ACTH precursor pro-opiomelanocortin (POMC) and ACTH secretion by AtT20 cells. Thus, JQ1 treatment has anti-proliferative and pro-apoptotic effects on AtT20 cells and reduces ACTH secretion, thereby indicating that BET inhibition may provide a novel approach for treatment of corticotrophinomas.
Assuntos
Adenoma Hipofisário Secretor de ACT/tratamento farmacológico , Adenoma/tratamento farmacológico , Epigênese Genética/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Adenoma Hipofisário Secretor de ACT/genética , Adenoma Hipofisário Secretor de ACT/patologia , Adenoma/genética , Adenoma/patologia , Hormônio Adrenocorticotrópico/biossíntese , Animais , Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Camundongos , Triazóis/farmacologiaRESUMO
Cancer is associated with alterations in epigenetic mechanisms such as histone modifications and methylation of DNA, and inhibitors targeting epigenetic mechanisms represent a novel class of anti-cancer drugs. Neuroendocrine tumors (NETs) of the pancreas (PNETs) and bronchus (BNETs), which may have 5-year survivals of <50% and as low as 5%, respectively, represent targets for such drugs, as >40% of PNETs and ~35% of BNETs have mutations of the multiple endocrine neoplasia type 1 (MEN1) gene, which encodes menin that modifies histones by interacting with histone methyltransferases. We assessed 9 inhibitors of epigenetic pathways, for their effects on proliferation, by CellTiter Blue assay, and apoptosis, by CaspaseGlo assay, using 1 PNET and 2 BNET cell lines. Two inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, targeting the bromo and extra terminal (BET) protein family which bind acetylated histone residues, were most effective in decreasing proliferation (by 40-85%, P<0.001) and increasing apoptosis (by 2-3.6 fold, P<0.001) in all 3 NET cell lines. The anti-proliferative effects of JQ1 and PFI-1 remained present for at least 48 hours after removal of the compound. JQ1, but not PFI-1, had cell cycle effects, assessed by propidium iodide staining and flow cytometry, resulting in increased and decreased proportions of NET cells in G1, and S and G2 phases, respectively. RNA Sequencing analysis revealed that these JQ1 effects were associated with increased histone 2B expression, and likely mediated through altered activity of bromodomain-containing (Brd) proteins. Assessment of JQ1 in vivo, using a pancreatic beta cell-specific conditional Men1 knockout mouse model that develops PNETs, revealed that JQ1 significantly reduced proliferation (by ~50%, P<0.0005), assessed by bromodeoxyuridine incorporation, and increased apoptosis (by ~3 fold, P<0.0005), assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling, of PNETs. Thus, our studies demonstrate that BET protein inhibitors may provide new treatments for NETs.
RESUMO
BACKGROUND: Mycolactone is a polyketide toxin secreted by the mycobacterium Mycobacterium ulcerans, responsible for the extensive hypoalgesic skin lesions characteristic of patients with Buruli ulcer. A recent pre-clinical study proposed that mycolactone may produce analgesia via activation of the angiotensin II type 2 receptor (AT2R). In contrast, AT2R antagonist EMA401 has shown analgesic efficacy in animal models and clinical trials for neuropathic pain. We therefore investigated the morphological and functional effects of mycolactone in cultured human and rat dorsal root ganglia (DRG) neurons and the role of AT2R using EMA401. Primary sensory neurons were prepared from avulsed cervical human DRG and rat DRG; 24 h after plating, neurons were incubated for 24 to 96 h with synthetic mycolactone A/B, followed by immunostaining with antibodies to PGP9.5, Gap43, ß tubulin, or Mitotracker dye staining. Acute functional effects were examined by measuring capsaicin responses with calcium imaging in DRG neuronal cultures treated with mycolactone. RESULTS: Morphological effects: Mycolactone-treated cultures showed dramatically reduced numbers of surviving neurons and non-neuronal cells, reduced Gap43 and ß tubulin expression, degenerating neurites and reduced cell body diameter, compared with controls. Dose-related reduction of neurite length was observed in mycolactone-treated cultures. Mitochondria were distributed throughout the length of neurites and soma of control neurons, but clustered in the neurites and soma of mycolactone-treated neurons. Functional effects: Mycolactone-treated human and rat DRG neurons showed dose-related inhibition of capsaicin responses, which were reversed by calcineurin inhibitor cyclosporine and phosphodiesterase inhibitor 3-isobutyl-1-Methylxanthine, indicating involvement of cAMP/ATP reduction. The morphological and functional effects of mycolactone were not altered by Angiotensin II or AT2R antagonist EMA401. CONCLUSION: Mycolactone induces toxic effects in DRG neurons, leading to impaired nociceptor function, neurite degeneration, and cell death, resembling the cutaneous hypoalgesia and nerve damage in individuals with M. Ulcerans infection.
Assuntos
Úlcera de Buruli/complicações , Úlcera de Buruli/patologia , Gânglios Espinais/patologia , Hipestesia/complicações , Hipestesia/patologia , Degeneração Neural/patologia , Neuritos/patologia , Animais , Úlcera de Buruli/fisiopatologia , Capsaicina , Células Cultivadas , Feminino , Imunofluorescência , Proteína GAP-43/metabolismo , Gânglios Espinais/fisiopatologia , Humanos , Hipestesia/fisiopatologia , Macrolídeos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Degeneração Neural/complicações , Degeneração Neural/fisiopatologia , Ratos , Ratos Wistar , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: The angiotensin II (AngII) receptor subtype 2 (AT2 R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. METHODS: We used immunostaining with characterized antibodies to study the localization of AT2 R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2 R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. RESULTS: AT2 R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2 R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1 R antagonist losartan had no effect on capsaicin responses. AT2 R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2 R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. CONCLUSIONS: AT2 R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.
Assuntos
Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Compostos Benzidrílicos/farmacologia , Capsaicina/farmacologia , Isoquinolinas/farmacologia , Neuritos/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Células Cultivadas , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Neuritos/metabolismo , Ratos , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/efeitos dos fármacosRESUMO
Despite considerable research, effective and safe treatments for human pain disorders remain elusive. Understanding the biology of different human pain conditions and researching effective treatments continue to be dominated by animal models, some of which are of limited value. British and European legislation demands that non-animal approaches should be considered before embarking on research using experimental animals. Recent scientific and technical developments, particularly in human neuroimaging, offer the potential to replace some animal procedures in the study of human pain. A group of pain research experts from academia and industry met with the aim of exploring creatively the tools, strategies and challenges of replacing some animal experiments in pain research with ethically conducted studies of human patients and healthy volunteers, in combination with in vitro methods. This report considers how a range of neuroimaging techniques including functional magnetic resonance imaging, magnetoencephalography and positron emission tomography, singly and combined, can address human pain conditions. In addition, microdialysis in human subjects; genome-wide association research, twin studies and other epidemiological approaches; and in vitro cell and tissue research, are examined for their replacement potential in combination with neuroimaging. Recommendations highlight further opportunities to advance the replacement of animal studies with robust methods of relevance to understanding and treating human pain.
Assuntos
Ensaios Clínicos como Assunto/métodos , Diagnóstico por Imagem/métodos , Manejo da Dor , Dor/diagnóstico , Experimentação Animal , Educação , Voluntários Saudáveis , Experimentação Humana , Humanos , Reino UnidoAssuntos
Doença de Alzheimer/terapia , Antipsicóticos/uso terapêutico , Doença de Alzheimer/complicações , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Amiloide/efeitos dos fármacos , Amiloide/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Sintomas Comportamentais/tratamento farmacológico , Sintomas Comportamentais/etiologia , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/etiologia , Humanos , ImunomodulaçãoRESUMO
Burning mouth syndrome (BMS) is often an idiopathic chronic and intractable pain condition, affecting 1.5-5.5% of middle-aged and elderly women. We have studied the heat and capsaicin receptor TRPV1, and its regulator nerve growth factor (NGF), in BMS. Patients with BMS (n=10) and controls (n=10) were assessed for baseline and post-topical capsaicin pain scores, and their tongue biopsies immunostained for TRPV1, NGF, and structural nerve markers neurofilament and peripherin. Nerve fibres penetrating the epithelium were less abundant in BMS (p<0.0001), indicating a small fibre neuropathy. TRPV1-positive fibres were overall significantly increased in BMS (p=0.0011), as were NGF fibres (p<0.0001) and basal epithelial cell NGF staining (p<0.0147). There was a significant correlation between the baseline pain score and TRPV1 (p=0.0143) and NGF fibres (p=0.0252). A significant correlation was observed between baseline and post-capsaicin pain (p=0.0006). Selective TRPV1 and NGF blockers may provide a new therapy for BMS.
Assuntos
Síndrome da Ardência Bucal , Regulação da Expressão Gênica/fisiologia , Fibras Nervosas/metabolismo , Medição da Dor , Canais de Cátion TRPV/metabolismo , Doenças do Nervo Trigêmeo/complicações , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Síndrome da Ardência Bucal/etiologia , Síndrome da Ardência Bucal/metabolismo , Síndrome da Ardência Bucal/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.8 , Fator de Crescimento Neural/metabolismo , Proteínas de Neurofilamentos/metabolismo , Medição da Dor/métodos , Canais de Sódio/metabolismo , Língua/patologia , Doenças do Nervo Trigêmeo/patologiaRESUMO
We have studied the effect of key neurotrophic factors (NTFs) on morphology, levels of the vanilloid receptor-1 (TRPV1) and responses to capsaicin in adult human sensory neurons in vitro. Avulsed dorsal root ganglia (DRG, n = 5) were cultured with or without a combination of nerve growth factor (NGF), glial cell (line)-derived growth factor (GDNF) and neurotrophin3 (NT3) for 5 days. In the absence of NTFs, the diameter of neurons ranged from 20 to 100 microm (mean 42 +/- 4 microm). Adding NTFs caused a significant increase in neuronal sizes, up to 120 microm (mean diameter 62 +/- 5 microm, P < 0.01, t-test), an overall 35% increase of TRPV1-positive neurons (P < 0.003), and notably of large TRPV1-positive neurons > 80 microm (P < 0.05). Responses to capsaicin were significantly enhanced with calcium ratiometry (P < 0.0001). Short duration (1h) exposure of dissociated sensory neurons to NTFs increased numbers of TRPV1-positive neurons, but not of TRPV3, Nav 1.8 and IK1 and the morphological size-distribution remained similar to intact post-mortem DRG neurons. NTFs thus increase size, elevate TRPV1 levels and enhance capsaicin responses in cultured human DRG neurons; these changes may relate to pathophysiology in disease states, and provide an in vitro model to study novel analgesics.
Assuntos
Capsaicina/farmacologia , Gânglios Espinais/citologia , Fatores de Crescimento Neural/metabolismo , Neurônios Aferentes/efeitos dos fármacos , Canais de Cátion TRPV/biossíntese , Adulto , Tamanho Celular , Células Cultivadas , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.8 , Fator de Crescimento Neural/metabolismo , Neurônios Aferentes/citologia , Neurônios Aferentes/metabolismo , Neurotrofina 3/metabolismo , Canais de Potássio Cálcio-Ativados/biossíntese , Canais de Sódio/biossínteseRESUMO
BACKGROUND: The tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 (SNS1/PN3) is expressed by nociceptors and may play a role in pain states. METHODS: Using specific antibodies for immunohistochemistry, we studied Nav1.8 immunoreactivity in human dental pulp in relation to the neuronal marker neurofilament. Human tooth pulp was extracted from teeth harvested from a total of twenty-two patients (fourteen without dental pain, eight patients with dental pain). RESULTS: Fibres immunoreactive for Nav1.8, were significantly increased on image analysis in the painful group: median (range) Nav1.8 to Neurofilament % area ratio, non-painful 0.059 (0.006-0.24), painful 0.265 (0.13-0.5), P = 0.0019. CONCLUSION: Nav1.8 sodium channels may thus represent a therapeutic target in trigeminal nerve pain states.
RESUMO
BACKGROUND: Faecal urgency and incontinence with rectal hypersensitivity is a distressing, unexplained disorder that is inadequately treated. We aimed to determine whether expression of the heat and capsaicin receptor vanilloid receptor 1 (TRPV1 or VR1) was changed in rectal sensory fibres, and to correlate nerve fibre density with sensory abnormalities. METHODS: We compared full-thickness rectal biopsy samples from nine patients with physiologically characterised rectal hypersensitivity with tissue samples from 12 controls. Sensory thresholds to rectal balloon distension and heating the rectal mucosa were measured before biopsy. We assessed specimens with immunohistochemistry and image analysis using specific antibodies to TRPV1; nerve growth factor (NGF) receptor tyrosine kinase A; glial cell line-derived neurotrophic factor (GDNF); neuropeptides calcitonin gene-related peptide (CGRP) and substance P; the related vanilloid receptor-like protein (VRL) 2; glial markers S-100 and glial fibrillary acid protein (GFAP); and the nerve structural marker peripherin. FINDINGS: In rectal hypersensitivity, nerve fibres immunoreactive to TRPV1 were increased in muscle, submucosal, and mucosal layers: in the mucosal layer, the median% area positive was 0.44 (range 0.30-0.59) in patients who were hypersensitive and 0.11 (0.00-0.21) in controls (p=0.0005). The numbers of peripherin-positive fibres also increased in the mucosal layer (hypersensitive 3.00 [1.80-6.50], controls 1.20 [0.39-2.10]: (p=0.0002). The increase in TRVP1 correlated significantly with the decrease in rectal heat (p=0.03) and the distension (p=0.02) sensory thresholds. The thresholds for heat and distension were also significantly correlated (p=0.0028). Expression of nerve fibres positive for GDNF (p=0.001) and tyrosine kinase A (p=0.002) was also increased, as were cell bodies of the submucosal ganglia immunoreactive to CGRP (p=0.0009). INTERPRETATION: Faecal urgency and rectal hypersensitivity could result from increased numbers of polymodal sensory nerve fibres expressing TRPV1. The triggering factor or factors remain uncertain, but drugs that target nerve terminals that express this receptor, such as topical resiniferatoxin, deserve consideration.
Assuntos
Vias Aferentes/química , Proteínas de Transporte de Cátions , Incontinência Fecal/etiologia , Incontinência Fecal/patologia , Hiperestesia/etiologia , Hiperestesia/patologia , Canais Iônicos , Glicoproteínas de Membrana , Fibras Nervosas Amielínicas/química , Receptores de Droga/análise , Doenças Retais/etiologia , Doenças Retais/patologia , Adulto , Biópsia , Temperatura Corporal , Estudos de Casos e Controles , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Proteína Glial Fibrilar Ácida/análise , Humanos , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/análise , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Neural/análise , Fatores de Crescimento Neural/análise , Proteínas do Tecido Nervoso/análise , Periferinas , Fatores Desencadeantes , Receptores Proteína Tirosina Quinases/análise , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/análise , Receptores de Droga/imunologia , Proteínas S100/análise , Limiar Sensorial , Substância P/análise , Canais de Cátion TRPVRESUMO
The cannabinoid agonist, HU210 has been evaluated in vivo in nociceptive and inflammatory pain models in the rat. The ED50 for the anti-nociceptive (increasing mechanical withdrawal threshold) effect was 0.1 mg/kg-1 i.p., and for anti-hypersensitivity and anti-inflammatory activity was 5 g/kg-1 i.p. (in the carrageenan model). The selective CB1 antagonist, AM281 (0.5 microg/kg-1 i.p.) reversed effects of HU210 (10 and 30 microg/kg-1 i.p.) in both nociceptive and inflammatory models of hypersensitivity. The selective CB2 antagonist, SR144528 (1 mg/kg-1 i.p.) antagonised effects of HU210 (30 microg/kg-1 i.p.) in the carrageenan induced inflammatory hypersensitivity. The CB2 agonist, 1-(2,3-Dichlorobenzoyl)-5-methoxy-2-methyl-(2-(morpholin-4-yl)ethyl)-1H-indole (GW405833) inhibited the hypersensitivity and was anti-inflammatory in vivo. These effects were blocked by SR144528. These findings suggest that CB1 receptors are involved in nociceptive pain and that both CB1 and CB2 receptors are involved in inflammatory hypersensitivity. Future studies will investigate effects on identified inflammatory cells within the inflamed tissue to further elucidate the role of cannabinoid receptors.
Assuntos
Dor/fisiopatologia , Receptor CB2 de Canabinoide , Receptores de Droga/fisiologia , Doença Aguda , Animais , Canfanos/farmacologia , Canabinoides/farmacologia , Carragenina , Dronabinol/análogos & derivados , Dronabinol/farmacologia , Hipersensibilidade/fisiopatologia , Indóis/farmacologia , Masculino , Morfolinas/farmacologia , Inflamação Neurogênica/induzido quimicamente , Inflamação Neurogênica/fisiopatologia , Nociceptores/efeitos dos fármacos , Nociceptores/fisiologia , Dor/induzido quimicamente , Pirazóis/farmacologia , Ratos , Ratos Endogâmicos , Receptores de Canabinoides , Receptores de Droga/agonistasRESUMO
Calcium-activated potassium ion channels SK and IK (small and intermediate conductance, respectively) may be important in the pathophysiology of pain following nerve injury, as SK channels are known to impose a period of reduced excitability after each action potential by afterhyperpolarization. We studied the presence and changes of human SK1 (hSK1)- and hIK1-like immunoreactivity in control and injured human dorsal root ganglia (DRG) and peripheral nerves and their regulation by key neurotrophic factors in cultured rat sensory neurones. Using specific antibodies, hSK-1 and hIK-1-like immunoreactivity was detected in a majority of large and small/medium-sized cell bodies of human DRG. hSK1 immunoreactivity was decreased significantly in cell bodies of avulsed human DRG (n = 8, surgery delay 8 h to 12 months). There was a decrease in hIK1-like immunoreactivity predominantly in large cells acutely (<3 weeks after injury), but also in small/medium cells of chronic cases. Twenty-three injured peripheral nerves were studied (surgery delay 8 h to 12 months); in five of these, hIK1-like immunoreactivity was detected proximally but not distally to injury, whereas neurofilament staining confirmed the presence of nerve fibres in both regions. These five nerves, unlike the others, had all undergone Wallerian degeneration previously and the loss of hIK1-like immunoreactivity may therefore reflect reduced axonal transport of this ion channel across the injury site in regenerated fibres, as well as decreased expression in the cell body. In vitro studies of neonatal rat DRG neurones showed that nerve growth factor (NGF) significantly increased the percentage of hSK1-positive cells, whereas neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) failed to show a significant effect. NT-3 stimulated hIK1 expression, while NGF and GDNF were ineffective. As expected, NGF increased expression of the voltage-gated sodium channel SNS1/PN3 in this system. Decreased retrograde transport of these neurotrophic factors in injured sensory neurones may thus reduce expression of these ion channels and increase excitability. Blockade of IK1-like and other potassium channels by aminopyridines (4-AP and 3,4-DAP) may also explain the paraesthesiae induced by these medications. Selective potassium channel openers are likely to represent novel therapies for pain following nerve injury.
Assuntos
Gânglios Espinais/metabolismo , Fator de Crescimento Neural/farmacologia , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Neurotrofina 3/farmacologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Recém-Nascidos , Anticorpos/metabolismo , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/lesões , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neurônios Aferentes/metabolismo , Fármacos Neuroprotetores/farmacologia , Traumatismos dos Nervos Periféricos , Nervos Periféricos/metabolismo , Ratos , Ratos Wistar , Canais de Potássio Ativados por Cálcio de Condutância BaixaRESUMO
The sodium channels SNS/PN3 and NaN/SNS2 are regulated by the neurotrophic factors-nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF), and may play an important role in the development of pain after nerve injury or inflammation. These key molecules have been studied in an amputated causalgic finger and control tissues by immunohistochemistry. There was a marked increase in the number and intensity of SNS/PN3-immunoreactive nerve terminals in the affected finger, while GDNF-immunoreactivity was not observed, in contrast to controls. No differences were observed for NGF, trk A, NT-3 or NaN/SNS2-immunoreactivity. While further studies are required, these findings suggest that accumulation of SNS/PN3 and/or loss of GDNF may contribute to pain in causalgia, and that selective blockers of SNS/PN3 and/or rhGDNF may provide effective novel treatments.
Assuntos
Causalgia/metabolismo , Dedos/fisiopatologia , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/deficiência , Neurônios Aferentes/metabolismo , Neuropeptídeos/metabolismo , Nociceptores/metabolismo , Nervo Radial/metabolismo , Canais de Sódio/metabolismo , Idoso , Cotos de Amputação/patologia , Cotos de Amputação/fisiopatologia , Causalgia/fisiopatologia , Feminino , Dedos/inervação , Dedos/cirurgia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Imuno-Histoquímica , Mecanorreceptores/metabolismo , Mecanorreceptores/patologia , Canal de Sódio Disparado por Voltagem NAV1.8 , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Nociceptores/fisiopatologia , Complicações Pós-Operatórias/metabolismo , Complicações Pós-Operatórias/fisiopatologia , Nervo Radial/fisiopatologiaRESUMO
Voltage-gated sodium channels consist of a pore-containing alpha-subunit and one or more auxiliary beta-subunits, which may modulate channel function. We previously demonstrated that sodium channel SNS/PN3 alpha-subunits were decreased in human sensory cell bodies after spinal root avulsion injury, and accumulated at injured nerve terminals in pain states. Using specific antibodies for immunohistochemistry, we have now detected sodium channel beta1 and beta2 subunits in sensory cell bodies within control human postmortem sensory ganglia (78% of small/medium (< or = 50 microm) and 68% of large (> or = 50 microm) cells); their changes in cervical sensory ganglia after avulsion injury paralleled those described for SNS/PN3 alpha-subunits. Our results suggest that alpha- and beta-subunits share common regulatory mechanisms, but present distinct targets for novel analgesics.
Assuntos
Gânglios Espinais/citologia , Gânglios Espinais/lesões , Neurônios Aferentes/metabolismo , Neuropeptídeos/metabolismo , Canais de Sódio/metabolismo , Adulto , Sequência de Aminoácidos , Especificidade de Anticorpos , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Rim/citologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.8 , Neuralgia/metabolismo , Neurônios Aferentes/química , Neuropeptídeos/análise , Neuropeptídeos/imunologia , Proteínas Recombinantes/genética , Canais de Sódio/análise , Canais de Sódio/imunologiaRESUMO
Sensory neurones co-express voltage-gated sodium channels that mediate TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) currents, which may contribute to chronic pain after nerve injury. We previously demonstrated that TTX-R channels were decreased acutely in human sensory cell bodies after central axotomy, but accumulated in nerve terminals after peripheral axotomy. We have now studied the TTX-S channels PN1 and Brain III, using specific antibodies for immunohistochemistry, in dorsal root ganglia (DRG) from 10 patients with traumatic central axotomy, nerves from 16 patients with peripheral axotomy, and controls. PN1 showed temporal changes similar to the TTX-R channels in sensory cell bodies of injured DRG. In contrast, Brain III was found only in injured nerves (not control nerves, or control/central axotomy DRG). PNI and Brain III are distinct targets for novel analgesics.
Assuntos
Gânglios Espinais/lesões , Gânglios Espinais/metabolismo , Plasticidade Neuronal/fisiologia , Neuropeptídeos/metabolismo , Canais de Sódio/metabolismo , Tetrodotoxina/farmacologia , Adulto , Idoso , Especificidade de Anticorpos , Plexo Braquial/citologia , Plexo Braquial/lesões , Plexo Braquial/metabolismo , Linhagem Celular , Feminino , Gânglios Espinais/citologia , Humanos , Rim/citologia , Masculino , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.7 , Neurônios Aferentes/química , Neurônios Aferentes/metabolismo , Neuropeptídeos/análise , Neuropeptídeos/imunologia , Canais de Sódio/análise , Canais de Sódio/imunologia , TransfecçãoRESUMO
The voltage-gated 'glial' sodium channel NaG belongs to a distinct molecular class within the multi-gene family of mammalian sodium channels. Originally found in central and peripheral glia, NaG has since been detected in neurons in rat dorsal root ganglia (DRG) and may play a role in Schwann cell-axon interactions. We have studied the presence of NaG-like immunoreactivity in the intact and injured human peripheral nervous system using a specific affinity-purified antibody. Nerve fibres in normal and injured peripheral nerves and normal skin exhibited intense NaG-immunoreactivity. Numerous NaG-immunoreactive nerve fibres surrounded neuronal cell bodies within postmortem control DRG, and in DRG avulsed from the spinal cord (i.e. after traumatic central axotomy). There were no significant differences in the pattern of NaG immunostaining between control and avulsed DRG, or with delay after injury. Generally, the neuronal cell bodies were only very weakly immunoreactive to NaG, indicating that the NaG immunoreactivity was predominantly in Schwann cells/myelin. In accord, we demonstrated NaG immunostaining in cultured human and rat Schwann cells, and in distal nerve after wallerian degeneration. NaG thus appears to be a useful new marker for Schwann cells in the human PNS, and a role in neuropathy deserves investigation.
Assuntos
Neuroglia/metabolismo , Traumatismos dos Nervos Periféricos , Nervos Periféricos/metabolismo , Canais de Sódio/análise , Animais , Animais Recém-Nascidos , Biomarcadores/análise , Células Cultivadas , Humanos , Imuno-Histoquímica/métodos , Neuroglia/patologia , Ratos , Ratos Endogâmicos Lew , Células de Schwann/químicaRESUMO
Mast cells (MC) release potent mediators which alter enteric nerve and smooth muscle function and may play a role in the pathogenesis of the irritable bowel syndrome (IBS). The aim of this study was to determine if MC were increased in the colon of IBS patients compared to controls. Biopsy specimens were obtained from the caecum, ascending colon, descending colon and rectum of 28 patients: 14 IBS (Rome criteria); seven normal; and seven inflammatory controls. Tissue was stained immunohistochemically using a monoclonal mouse antibody for human mast cell tryptase (AA1). Tissue area occupied by tryptase-positive MC (volume density of mast cells) was quantified by image analysis. The number of plasma cells, lymphocytes, eosinophils, neutrophils and macrophages were each graded semiquantitatively (0-4) in haematoxylin and eosin stained sections. Mast cell volume density was significantly (P < 0.05) higher in IBS (0.91 +/- 0.18; CI 0.79; 1.0) than normal controls (0.55 +/- 0.14; CI 0.40; 0.69) in the caecum but not at other sites. Apart from MC, there was no evidence of increased cellular infiltrate in the IBS group. MC were significantly increased in the caecum of IBS patients compared to controls. The multiple effects of the intestinal mast cell alone, or as a participant of a persistent inflammatory response, may be fundamental to the pathogenesis of IBS.
Assuntos
Colite/patologia , Doenças Funcionais do Colo/patologia , Mucosa Intestinal/patologia , Mastócitos/patologia , Adulto , Idoso , Análise de Variância , Contagem de Células , Distribuição de Qui-Quadrado , Colite/fisiopatologia , Doenças Funcionais do Colo/fisiopatologia , Colonoscopia , Feminino , Granulócitos/patologia , Humanos , Linfócitos/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
The effects of bradykinin and the bradykinin B(2) receptor antagonists D-Arg-[Hyp(3),Thi(5,8),D-Phe(7)]-bradykinin (NPC 349) and D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-bradykinin (Hoe 140) were examined in the electrically-stimulated rat vas deferens. Cumulative additions of bradykinin (1-3000 nM) produced two distinct responses: an enhancement in the magnitude of the basal electrically-induced twitch response (neurogenic response) and an increase in the baseline tension (musculotropic response). NPC 349 (10-100 microM) produced concentration-dependent surmountable rightward shifts of both the bradykinin neurogenic and musculotropic response curves. In contrast, while Hoe 140 (10-100 nM) caused an apparently surmountable antagonism of the bradykinin neurogenic response, it caused an apparent insurmountable antagonism of the bradykinin musculotropic response. Interestingly, co-incubation of Hoe 140 (30 nM) with NPC 349 (30 and 100 microM) resulted in a concentration-related upwards displacement of the Hoe 140-suppressed bradykinin musculotropic response curve. Thus, Hoe 140 can be described as a pseudo-irreversible antagonist against the bradykinin musculotropic response. No time-dependent changes were observed in the maximum bradykinin musculotropic response attainable when NPC 349 (100 microM) additions were made for the final 2 or 18 min of the Hoe 140 incubation (20 min). These findings indicate that slow reversibility of Hoe 140 from the bradykinin B(2) receptor is unlikely to be the mechanism responsible for the pseudo-irreversible antagonism of the bradykinin-induced musculotropic response. Instead, we propose an alternative explanation involving a third, unstable and inactive form of the bradykinin B(2) receptor.
Assuntos
Antagonistas dos Receptores da Bradicinina , Bradicinina/análogos & derivados , Ducto Deferente/efeitos dos fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Angiotensina II/farmacologia , Animais , Bradicinina/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/fisiologia , Norepinefrina/farmacologia , Ratos , Ratos Wistar , Ducto Deferente/inervação , Ducto Deferente/fisiologiaRESUMO
Certain dorsal horn neurones respond in a graded manner to noxious colorectal distension (CRD). Morphine inhibits these responses in the spinalized rat, but the role of excitatory amino acids in baseline visceral nociceptive transmission is less clear. This study examines the effect of the mu-opiate receptor agonist fentanyl, and the non-NMDA and NMDA antagonists DNQX and MK-801, respectively, on such responses to CRD in the sodium pentobarbitone-anaesthetized rat. Male rats were prepared for extracellular recording from the lumbosacral spinal cord. 90 neurones responsive to CRD, located throughout the dorsal horn, were classified according to their response duration and latency to 60 mmHg distension, as SL-A (short latency-abrupt; 59%), SL-S (short latency-sustained; 23%), L-L (long-latency; 10%) and Inhib (inhibited; 8%). Convergent cutaneous receptive fields were mapped for 79/90 neurones and classified as LT (low threshold), WDR (wide dynamic range) or HT (high threshold). CRD (20-100 mm Hg) elicited graded responses in most neurones. In 6/6 SL-S neurones, fentanyl (1-8 microg kg-1) dose-dependently inhibited the response to 60 mm Hg CRD, in a naloxone-sensitive manner, with an ID50 value (+/-95% confidence limits) of 2.48 (1.7-3. 7) microg kg-1. In 6/6 SL-A neurones, fentanyl had no significant effect on the response to CRD. DNQX (0.03-3 mg kg-1) produced a dose-dependent inhibition of the response to CRD in 5/5 SL-A neurones, with an ID50 value of 0.32 (0.01-41.1) mg kg-1. MK-801 (0. 03-0.3 mg kg-1) had no significant effect on responses to CRD in 6/6 SL-A neurones. The differential inhibitory effects of fentanyl on two neuronal subtypes may indicate functional differences. In SL-A neurones AMPA/kainate, but not NMDA receptors are involved in mediating baseline nociceptive neurotransmission.
Assuntos
Analgésicos Opioides/farmacologia , Colo/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Células do Corno Posterior/efeitos dos fármacos , Reto/efeitos dos fármacos , Animais , Colo/fisiologia , Maleato de Dizocilpina/farmacologia , Fentanila/farmacologia , Masculino , Células do Corno Posterior/fisiologia , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Reto/fisiologiaRESUMO
BACKGROUND: Noxious intestinal distention elicits a reflex depressor response in the sodium pentobarbitone anaesthetised rat, which can be used as an index of visceral nociception. 5-HT(3) receptor antagonists inhibit this reflex. Repeated colorectal distention (CRD) induces Fos like immunoreactivity (Fos-LI) in the rat spinal cord. AIMS: To examine the effect of the 5-HT(3) receptor antagonist alosetron on the depressor response to CRD, and on Fos expression in the lumbosacral spinal cord. METHODS: Male rats were anaesthetised with sodium pentobarbitone, and mean arterial blood pressure monitored during repeated colorectal balloon inflation before and after treatment with alosetron or saline. Rats anaesthetised with urethane and treated with alosetron or saline underwent a repeated CRD paradigm, after which the lumbosacral spinal cord was removed and processed for visualisation of Fos-LI. RESULTS: CRD elicited reproducible, volume dependent falls in arterial blood pressure, and repeated distention-effect curves were constructed. Alosetron (1-100 microg/kg intravenously) inhibited the depressor response to CRD in a dose related manner, with an ID(50) value of 3.0 microg/kg. Following repeated CRD, numbers of Fos-LI neurones were significantly increased to 1246 (total in 12 sections at 120 microm intervals from L6 to S1) compared with 49 in sham distended animals. Pretreatment with alosetron (100 microg/kg) significantly reduced numbers of Fos-LI neurones to 479.8. CONCLUSION: The 5-HT(3) receptor antagonist alosetron inhibits the depressor response to CRD in a potent and dose dependent manner. It also inhibits CRD induced Fos-LI in the spinal cord. These results suggest that 5-HT(3) receptors are involved in visceral nociceptive transmission, perhaps located on primary afferent or spinal neurones.