RESUMEN
Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.
Asunto(s)
Transferasas Alquil y Aril/genética , ARN de Transferencia/metabolismo , Selenoproteínas/metabolismo , Transferasas Alquil y Aril/metabolismo , Animales , Línea Celular , Cisteína/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Ratones , Neuronas/metabolismo , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Biosíntesis de Proteínas/genética , ARN de Transferencia/genética , Ribosomas/metabolismo , Selenio/metabolismo , Selenocisteína/genética , Selenoproteína P/genética , Selenoproteínas/genéticaRESUMEN
(E)-Methyl 2-((2S,3S,7aS,12bS)-3-ethyl-7a-hydroxy-8-methoxy-1,2,3,4,6,7,7a,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate (7-hydroxymitragynine), a main active constituent of the traditional herbal medicine Mitragyna speciosa, is an indole alkaloid that is structurally different from morphine. 7-Hydroxymitragynine induces a potent antinociceptive effect on mouse acute pain through µ-opioid receptors. In this study, we developed dual-acting µ- and δ-opioid agonists MGM-15 and MGM-16 from 7-hydroxymitragynine for the treatment of acute and chronic pain. MGM-16 showed a higher potency than that of 7-hydroxymitragynine and MGM-15 in in vitro and in vivo assays. MGM-16 exhibited a high affinity for µ- and δ-opioid receptors, with K(i) values of 2.1 and 7.0 nM, respectively. MGM-16 showed µ- and δ-opioid full agonistic effects in a guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding assay and in a functional test using electrically elicited guinea pig ileum and mouse vas deferens contractions. Systemic administration of MGM-16 produced antinociceptive effects in a mouse acute pain model and antiallodynic effects in a chronic pain model. The antinociceptive effect of MGM-16 was approximately 240 times more potent than that of morphine in a mouse tail-flick test, and its antiallodynic effect was approximately 100 times more potent than that of gabapentin in partial sciatic nerve-ligated mice, especially with oral administration. The antinociceptive effect of MGM-16 was completely and partially blocked by the µ-selective antagonist ß-funaltrexamine hydrochloride (ß-FNA) and by the δ-selective antagonist naltrindole, respectively, in a tail-flick test. The antiallodynic effect of MGM-16 was completely blocked by ß-FNA and naltrindole in a neuropathic pain model. These findings suggest that MGM-16 could become a class of a compound with potential therapeutic utility for treating neuropathic pain.
Asunto(s)
Hiperalgesia/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Alcaloides de Triptamina Secologanina/farmacología , Administración Oral , Animales , Células CHO , Cricetinae , Cricetulus , Hiperalgesia/fisiopatología , Íleon/efectos de los fármacos , Íleon/fisiopatología , Inyecciones Subcutáneas , Masculino , Ratones , Contracción Muscular , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Neuralgia/fisiopatología , Estimulación Física , Conejos , Ensayo de Unión Radioligante , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides mu/antagonistas & inhibidores , Neuropatía Ciática/tratamiento farmacológico , Neuropatía Ciática/fisiopatología , Alcaloides de Triptamina Secologanina/química , Alcaloides de Triptamina Secologanina/uso terapéutico , Estereoisomerismo , Tacto , Conducto Deferente/efectos de los fármacos , Conducto Deferente/fisiopatologíaAsunto(s)
Caquexia/metabolismo , Hipotálamo/metabolismo , Neoplasias/complicaciones , Receptor de Melanocortina Tipo 4/metabolismo , Receptores de Melanocortina/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Caquexia/etiología , Modelos Animales de Enfermedad , Hipotálamo/fisiopatología , Masculino , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , ARN Mensajero/biosíntesis , Ratas , Receptor de Melanocortina Tipo 3 , Receptor de Melanocortina Tipo 4/genética , Receptores de Melanocortina/genéticaRESUMEN
BACKGROUND: There is accumulating evidence that the activation of spinal glial cells, especially microglia, is a key event in the pathogenesis of neuropathic pain. However, the inhibition of microglial activation is often ineffective, especially for long-lasting persistent neuropathic pain. So far, neuropathic pain remains largely intractable and a new therapeutic strategy for the pain is still required. METHODS/PRINCIPAL FINDINGS: Using Seltzer model mice, we investigated the temporal aspect of two types of neuropathic pain behaviors, i.e., thermal hyperalgesia and mechanical allodynia, as well as that of morphological changes in spinal microglia and astrocytes by immunohistochemical studies. Firstly, we analyzed the pattern of progression in the pain behaviors, and found that the pain consisted of an "early induction phase" and subsequent "late maintenance phase". We next analyzed the temporal changes in spinal glial cells, and found that the induction and the maintenance phase of pain were associated with the activation of microglia and astrocytes, respectively. When Bushi, a Japanese herbal medicine often used for several types of persistent pain, was administered chronically, it inhibited the maintenance phase of pain without affecting the induction phase, which was in accordance with the inhibition of astrocytic activation in the spinal cord. These analgesic effects and the inhibition of astrocytic activation by Bushi were mimicked by the intrathecal injection of fluorocitrate, an inhibitor of astrocytic activation. Finally, we tested the direct effect of Bushi on astrocytic activation, and found that Bushi suppressed the IL-1ß- or IL-18-evoked ERK1/2-phosphorylation in cultured astrocytes but not the ATP-evoked p38- and ERK1/2-phosphorylation in microglia in vitro. CONCLUSIONS: Our results indicated that the activation of spinal astrocytes was responsible for the late maintenance phase of neuropathic pain in the Seltzer model mice and, therefore, the inhibition of astrocytic activation by Bushi could be a useful therapeutic strategy for treating neuropathic pain.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/patología , Neuralgia/tratamiento farmacológico , Neuralgia/patología , Fitoterapia , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Astrocitos/metabolismo , Conducta Animal/efectos de los fármacos , Células Cultivadas , Citratos/administración & dosificación , Citratos/farmacología , Citratos/uso terapéutico , Modelos Animales de Enfermedad , Hiperalgesia/complicaciones , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/patología , Inyecciones Intraperitoneales , Inyecciones Espinales , Ratones , Ratones Endogámicos ICR , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Minociclina/administración & dosificación , Minociclina/farmacología , Minociclina/uso terapéutico , Neuralgia/complicaciones , Dimensión del Dolor , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Factores de TiempoRESUMEN
Most reports in the literature have shown that the effects of opioid analgesics are primarily mediated by mu-opioid receptor (MOR), whereas other potential targets of opioid analgesics have not been thoroughly characterized. In this study, we found that extracellular application of morphine, fentanyl or oxycodone, which are all considered to be MOR agonists, at relatively high concentrations, but not endogenous mu-opioid peptides, produced a concentration-dependent suppression of sodium currents in cultured thalamic neurons. These effects of opioids were not affected by either a MOR antagonist naloxone or a deletion of MOR gene. Among these opioids, fentanyl strongly suppressed sodium currents to the same degree as lidocaine, and both morphine and oxycodone slightly but significantly reduced sodium currents when they were present extracellularly. In contrast, the intracellular application of morphine, but not oxycodone, fentanyl or lidocaine, reduced sodium currents. These results suggest that morphine, fentanyl and oxycodone each produce the MOR-independent suppression of sodium currents by distinct mechanisms in thalamic neurons.
Asunto(s)
Analgésicos Opioides/farmacología , Neuronas/metabolismo , Receptores Opioides mu/metabolismo , Sodio/metabolismo , Tálamo/metabolismo , Animales , Células Cultivadas , Fentanilo/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados , Morfina/farmacología , Neuronas/efectos de los fármacos , Péptidos Opioides/metabolismo , Oxicodona/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/agonistas , Receptores Opioides mu/genética , Tálamo/citología , Tálamo/efectos de los fármacosRESUMEN
Mitragynine is a major indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that has opium-like properties, although its chemical structure is quite different from that of morphine. We attempted to develop novel analgesics derived from mitragynine, and thus synthesized the ethylene glycol-bridged and C10-fluorinated derivative of mitragynine, MGM-9 [(E)-methyl 2-(3-ethyl-7a,12a-(epoxyethanoxy)-9-fluoro-1,2,3,4,6,7,12,12b-octahydro-8-methoxyindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate]. We hypothesized that a dual-acting mu- and kappa-opioid agonist could produce potent antinociceptive effects with fewer rewarding effects compared with mu agonists. In this study, MGM-9 exhibited high affinity for mu- and kappa-opioid receptors with Ki values of 7.3 and 18 nM, respectively. MGM-9 showed a potent opioid agonistic effect, and its effects were meditated by mu- and kappa-opioid receptor mechanisms in in vitro assays. Subcutaneous and oral administration of MGM-9 produced potent antinociceptive effects in mouse tail-flick, hot-plate, and writhing tests. When administered orally, the antinociceptive effect of MGM-9 was seven to 22 times more potent than that of morphine. The antinociceptive effects of MGM-9 were mediated by both mu- and kappa-opioid receptors. Subcutaneous administration of MGM-9 twice daily for 5 days led to antinociceptive tolerance. In the gastrointestinal transit study, MGM-9 inhibited gastrointestinal transit, but its effect was weaker than that of morphine at equi-antinociceptive doses. Furthermore, MGM-9 induced less hyperlocomotion and fewer rewarding effects than morphine. The rewarding effect of MGM-9 was blocked by a mu antagonist and enhanced by a kappa antagonist. Taken together, the results suggest that MGM-9 is a promising novel analgesic that has a stronger antinociceptive effect and weaker adverse effects than morphine.
Asunto(s)
Umbral del Dolor/efectos de los fármacos , Dolor/tratamiento farmacológico , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Recompensa , Alcaloides de Triptamina Secologanina/farmacología , Analgésicos Opioides/farmacología , Análisis de Varianza , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Tolerancia a Medicamentos , Tránsito Gastrointestinal/efectos de los fármacos , Cobayas , Masculino , Ratones , Morfina/uso terapéutico , Derivados de la Morfina/uso terapéutico , Dolor/clasificación , Dolor/etiología , Dimensión del Dolor/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Tiempo de Reacción/efectos de los fármacos , Alcaloides de Triptamina Secologanina/química , Alcaloides de Triptamina Secologanina/uso terapéutico , Factores de TiempoRESUMEN
Morphine is a prototypical mu-opioid receptor (MOR) agonist, and can directly inhibit pain transmission at both spinal and supraspinal levels. In the present study, we investigated the properties of thalamic neurons in an opioid-sensitive pain-modulating circuit. Application of morphine to cultured thalamic neurons evoked a potentiation of glutamate-induced peak currents, which was blocked by the MOR antagonist. Application of the protein kinase C inhibitor chelerythrine significantly inhibited the morphine-evoked enhancement of glutamate-induced currents. Immunoreactivity for MOR was observed with high density in the habenular nucleus (Hb) of the thalamus in rats, which was clearly co-localized with NMDA receptor subunit 1 (NRI). In this study, we show that microinjection of morphine into the Hb produced a dose-dependent increase in the tail-flick latency and enhanced the antinociceptive effect induced by the intra-Hb injection of glutamate. When fluoro-gold (FG) was used as a retrograde tracer, we found that FG-labeled neurons in the Hb after the microinjection of FG into the periaqueductal gray expressed both MOR and NR1. The present data suggest that the stimulation of MOR in the Hb may be involved in activation of the descending antinociceptive pathway through glutamatergic neurotransmission via the NMDA receptor.
Asunto(s)
Morfina/farmacología , Narcóticos/farmacología , Neuronas/citología , Dolor/tratamiento farmacológico , Transmisión Sináptica/efectos de los fármacos , Tálamo/citología , Animales , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Embrión de Mamíferos , Ácido Glutámico/farmacología , Inyecciones Espinales , Masculino , Microinyecciones , Neuroglía , Dolor/inducido químicamente , Dolor/fisiopatología , Dimensión del Dolor/métodos , Técnicas de Placa-Clamp/métodos , Sustancia Gris Periacueductal/efectos de los fármacos , Sustancia Gris Periacueductal/fisiopatología , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/metabolismo , Estilbamidinas/metabolismoRESUMEN
It has been clinically reported that chronic exposure to diphenylarsinic acid (DPAA) induced prominent cerebellar symptoms in apartment building residents in Kamisu, Japan. The aim of the present study was then to investigate the effect of chronic treatment with DPAA on the central motor impairment in mice. In the present study, we found that chronic in vivo exposure to a high dose of DPAA induced motor impairment in adult mice. This impairment was reversed by withdrawal following chronic DPAA treatment. The [35S]GTPgammaS binding assay showed the down-regulation of the dopamine receptor function in the striatum in adult mice treated with DPAA. We also found that neonatal exposure to a low dose of DPAA induced motor learning impairment in mice. Furthermore, treatment with an extremely low dose of DPAA caused the activation of caspase-3, the increase in glial fibrillary acidic protein-like immunoreactivity (IR) and the reduction in levels of myelin-associated glycoprotein-IR in mouse cerebellum neuron/glia co-cultures. In addition, we found that neonatal exposure to a low dose of DPAA induced anxiogenic behavior in a plus maze in mice. Taken together, these results suggest that chronic treatment with DPAA may induce motor impairment in adult mice. Moreover, neonatal exposure to DPAA leads to the irreversible motor impairment associated with abnormalities in the cerebellum.
Asunto(s)
Trastornos de Ansiedad/inducido químicamente , Intoxicación por Arsénico/etiología , Arsenicales/efectos adversos , Cerebelo/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Trastornos del Movimiento/etiología , Trastornos Psicomotores/inducido químicamente , Contaminantes Químicos del Agua/efectos adversos , Animales , Caspasa 3/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Exposición a Riesgos Ambientales/efectos adversos , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Neuroglía/efectos de los fármacos , Neuroglía/enzimología , Neuroglía/patología , EmbarazoRESUMEN
We previously reported that either (+)-matrine (matridin-15-one) or (+)-allomatrine (the C-6 epimer of matrine)-induced antinociceptive effect was attenuated by s.c. pretreatment with a kappa-opioid receptor (KOR) antagonist nor-binaltorphimine (nor-BNI), indicating the critical role of KORs in antinociceptive effects induced by these alkaloids. In the present study, we found that i.c.v. administration of either (+)-matrine- or (+)-allomatrine induced antinociceptive effects in the mouse tail-flick and warm-plate test, whereas these alkaloids when given spinally failed to induce antinociception. In the guanosine-5'-O-(3-[(35)S]thio)trisphosphate ([(35)S]GTPgammaS) binding assay, we demonstrated that neither (+)-matrine nor (+)-allomatrine produced the stimulation of [(35)S]GTPgammaS binding in the membranes of the spinal cord, indicating that (+)-matrine- and (+)-allomatrine-induced supraspinal antinociceptive actions was not due to a direct stimulation of KORs by these alkaloids. Therefore, we next investigated the involvement of dynorphin A (1-17) release at the spinal or supraspinal site in (+)-matrine- or (+)-allomatrine-induced antinociception. The i.c.v. pretreatment with an antiserum against dynorphin A (1-17) could not affect the antinociceptive effect induced by s.c. treatment of (+)-matrine. In contrast, the s.c.-administered (+)-matrine- and (+)-allomatrine-induced antinociceptive effect was significantly attenuated by i.t. pretreatment of an antiserum against dynorphin A (1-17). The present data suggest that either (+)-matrine or (+)-allomatrine when given i.c.v. may stimulate the descending dynorphinergic neuron, resulting in the stimulation of KORs in the spinal cord, and this phenomenon in turn produces the antinociception in mice.
Asunto(s)
Alcaloides/farmacología , Analgésicos/farmacología , Dinorfinas/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Alcaloides/aislamiento & purificación , Analgésicos/aislamiento & purificación , Animales , Dinorfinas/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Antagonistas de Narcóticos/farmacología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Neuronas/metabolismo , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Quinolizinas , Médula Espinal/metabolismo , MatrinasRESUMEN
The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride [(-)U-50,488H] on antinociception and G-protein activation induced by mu-opioid receptor agonists in mice. A single s.c. injection of (-)U-50,488H produced a dose-dependent antinociception, and this effect was reversed by a selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Furthermore, a single s.c. pre-treatment with (-)U-50,488H had no effect on the mu-opioid receptor agonist-induced antinociception. In contrast, repeated s.c. administration of (-)U-50,488H resulted in the development of tolerance to (-)U-50,488H-induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (-)U-50,488H significantly enhanced the antinociceptive effect of selective mu-opioid receptor agonists endomorphin-1, endomorphin-2 and [d-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO). Using the guanosine-5'-o-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding assay, we found that (-)U-50,488H was able to produce a nor-BNI-reversible increase in [35S]GTP gamma S binding to membranes of the mouse thalamus, which has a high level of kappa-opioid receptors. Repeated administration of (-)U-50,488H caused a significant reduction in the (-)U-50,488H-stimulated [35S]GTP gamma S binding in this region, whereas chronic treatment with (-)U-50,488H exhibited the increase in the endomorphin-1-, endomorphin-2- and DAMGO-stimulated [35S]GTP gamma S bindings in membranes of the thalamus and periaqueductal gray. These results suggest that repeated stimulation of kappa-opioid receptors leads to the heterologous up-regulation of mu-opioid receptor functions in the thalamus and periaqueductal gray regions, which may be associated with the supersensitivity of mu-opioid receptor-mediated antinociception.
Asunto(s)
Adaptación Fisiológica/fisiología , Proteínas de Unión al GTP/metabolismo , Receptores Opioides kappa/fisiología , Receptores Opioides mu/fisiología , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/farmacología , Analgésicos/farmacología , Analgésicos no Narcóticos/farmacología , Analgésicos Opioides/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Masculino , Bulbo Raquídeo/química , Ratones , Ratones Endogámicos ICR , Oligopéptidos/farmacología , Dimensión del Dolor/efectos de los fármacos , Puente/química , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Tálamo/química , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
The CCA-adding enzyme builds and repairs the 3' terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3'-terminal CCA, as do all mature tRNAs; the other 35% ends in 3' CC or possibly 3' C. The 3'-terminal A of U2 snRNA cannot be encoded because the 3' end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10-16 extra 3' nucleotides that are removed by one or more 3' exonucleases. Thus, if 3' exonuclease activity removes the encoded 3' CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3'-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3'-terminal A, CA, or CCA to human U2 snRNA lacking 3'-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with the Escherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3' stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun.