RESUMEN
Cannabinoid CB1 receptor, the molecular target of endocannabinoids and cannabis active components, is one of the most abundant metabotropic receptors in the brain. Cannabis is widely used for both recreational and medicinal purposes. Despite the ever-growing fundamental roles of microRNAs in the brain, the possible molecular connections between the CB1 receptor and microRNAs are surprisingly unknown. Here, by using reporter gene constructs that express interaction sequences for microRNAs in human SH-SY5Y neuroblastoma cells, we show that CB1 receptor activation enhances the expression of several microRNAs, including let-7d. This was confirmed by measuring hsa-let-7d expression levels. Accordingly, knocking-down CB1 receptor in zebrafish reduced dre-let-7d levels, and knocking-out CB1 receptor in mice decreased mmu-let-7d levels in the cortex, striatum and hippocampus. Conversely, knocking-down let-7d increased CB1 receptor mRNA expression in zebrafish, SH-SY5Y cells and primary striatal neurons. Likewise, in primary striatal neurons chronically exposed to a cannabinoid or opioid agonist, a let-7d-inhibiting sequence facilitated not only cannabinoid or opioid signaling but also cannabinoid/opioid cross-signaling. Taken together, these findings provide the first evidence for a bidirectional link between the CB1 receptor and a microRNA, namely let-7d, and thus unveil a new player in the complex process of cannabinoid action.
Asunto(s)
Cannabinoides/biosíntesis , MicroARNs/biosíntesis , Receptor Cannabinoide CB1/biosíntesis , Animales , Canfanos/farmacología , Línea Celular Tumoral , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Pirazoles/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Pez CebraRESUMEN
Serotonergic hallucinogens, such as lysergic acid diethylamide (LSD) and dimethoxy-bromoamphetamine (DOB), provoke stereotype-like shaking behaviour in rodents, which is hypothesised to engage frontocortical glutamate receptor activation secondary to serotonin2A (5-HT2A) related glutamate release. Challenging this hypothesis, we here investigate whether tolerance to LSD and DOB correlates with frontocortical adaptations of 5-HT2A and/or overall-glutamate binding sites. LSD and DOB (0.025 and 0.25 mg/kg, i.p.) induce a ketanserin-sensitive (0.5 mg/kg, i.p., 30-min pretreatment) increase in shaking behaviour (including head twitches and wet dog shakes), which with repeated application (7× in 4 ds) is undermined by tolerance. Tolerance to DOB, as indexed by DOB-sensitive [(3)H]spiroperidol and DOB induced [(35)S]GTP-gamma-S binding, is accompanied by a frontocortical decrease in 5-HT2A binding sites and 5-HT2 signalling, respectively; glutamate-sensitive [(3)H]glutamate binding sites, in contrast, remain unchanged. As to LSD, 5-HT2 signalling and 5-HT2A binding, respectively, are not or only marginally affected, yet [(3)H]glutamate binding is significantly decreased. Correlation analysis interrelates tolerance to DOB to the reduced 5-HT2A (r=.80) as well as the unchanged [(3)H]glutamate binding sites (r=.84); tolerance to LSD, as opposed, shares variance with the reduction in [(3)H]glutamate binding sites only (r=.86). Given that DOB and LSD both induce tolerance, one correlating with 5-HT2A, the other with glutamate receptor adaptations, it might be inferred that tolerance can arise at either level. That is, if a hallucinogen (like LSD in our study) fails to induce 5-HT2A (down-)regulation, glutamate receptors (activated postsynaptic to 5-HT2A related glutamate release) might instead adapt and thus prevent further overstimulation of the cortex.
Asunto(s)
2,5-Dimetoxi-4-Metilanfetamina/análogos & derivados , Lóbulo Frontal/metabolismo , Alucinógenos/farmacología , Dietilamida del Ácido Lisérgico/farmacología , Actividad Motora/efectos de los fármacos , Receptor de Serotonina 5-HT2A/efectos de los fármacos , Receptores de Glutamato/efectos de los fármacos , 2,5-Dimetoxi-4-Metilanfetamina/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Tolerancia a Medicamentos , Lóbulo Frontal/efectos de los fármacos , Ácido Glutámico/metabolismo , Ketanserina/farmacología , Masculino , Ratas , Antagonistas de la Serotonina/farmacologíaRESUMEN
A re-balance of postsynaptic serotonin (5-HT) receptor signalling, with an increase in 5-HT1A and a decrease in 5-HT2A signalling, is a final common pathway multiple antidepressants share. Given that the 5-HT1A/2A agonist lysergic acid diethylamide (LSD), when repeatedly applied, selectively downregulates 5-HT2A, but not 5-HT1A receptors, one might expect LSD to similarly re-balance the postsynaptic 5-HT signalling. Challenging this idea, we use an animal model of depression specifically responding to repeated antidepressant treatment (olfactory bulbectomy), and test the antidepressant-like properties of repeated LSD treatment (0.13 mg/kg/d, 11 d). In line with former findings, we observe that bulbectomised rats show marked deficits in active avoidance learning. These deficits, similarly as we earlier noted with imipramine, are largely reversed by repeated LSD administration. Additionally, bulbectomised rats exhibit distinct anomalies of monoamine receptor signalling in hippocampus and/or frontal cortex; from these, only the hippocampal decrease in 5-HT2 related [(35)S]-GTP-gamma-S binding is normalised by LSD. Importantly, the sham-operated rats do not profit from LSD, and exhibit reduced hippocampal 5-HT2 signalling. As behavioural deficits after bulbectomy respond to agents classified as antidepressants only, we conclude that the effect of LSD in this model can be considered antidepressant-like, and discuss it in terms of a re-balance of hippocampal 5-HT2/5-HT1A signalling.
Asunto(s)
Antidepresivos/administración & dosificación , Reacción de Prevención/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Depresión/tratamiento farmacológico , Hipocampo/efectos de los fármacos , Dietilamida del Ácido Lisérgico/administración & dosificación , Serotonina/metabolismo , Transmisión Sináptica/efectos de los fármacos , Animales , Depresión/metabolismo , Depresión/fisiopatología , Depresión/psicología , Modelos Animales de Enfermedad , Esquema de Medicación , Hipocampo/metabolismo , Hipocampo/fisiopatología , Masculino , Bulbo Olfatorio/cirugía , Ratas Wistar , Receptor de Serotonina 5-HT1A/metabolismo , Factores de TiempoRESUMEN
Preconditioning increases the neurons' resistance to subsequent hypoxia. An in vitro study was conducted to explore kinetic aspects of hypoxic preconditioning. Hippocampal slices were exposed to one single or repeated episodes of oxygen and glucose deprivation (OGD). The interval between OGD episodes varied between 30 min and 180 min. OGD led to a significant reduction in the population spike amplitude. Subsequent episodes of OGD did not result in a further reduction in the population spike amplitude if the interval between the episodes was ca. 60 min, which demonstrated that there were preconditioning effects. In the experiment using an interval of 30 min, population spike amplitude decreased after each OGD episode. The set-up described is useful for detecting damaging effects of OGD as well as preconditioning effects. A time window of ca. 60 min is required to induce protective mechanisms.
Asunto(s)
Hipocampo/fisiopatología , Hipoxia Encefálica/prevención & control , Potenciales de Acción , Animales , Potenciales Evocados , Glucosa/deficiencia , Hipocampo/metabolismo , Hipoxia Encefálica/metabolismo , Hipoxia Encefálica/fisiopatología , Técnicas In Vitro , Precondicionamiento Isquémico , Masculino , Oxígeno/metabolismo , Ratas Wistar , Factores de TiempoRESUMEN
OBJECTIVE: Mitragyna speciosa and its extracts are named kratom (dried leaves, extract). It contains several alkaloids and is used in traditional medicine to alleviate musculoskeletal pain, hypertension, coughing, diarrhea, and as an opiate substitute for addicts. Abuse and addiction to kratom is described, and kratom has attracted increasing interest in Western countries. Individual effects of kratom on opioidergic, adrenergic, serotonergic, and dopaminergic receptors are known, but not all of the effects have been explained. Pharmacokinetic and pharmacodynamic data are needed. METHODS: The effects of kratom extract on mice behavior were investigated following oral (po), intraperitoneal (ip), and intracerebroventricular (icv) application. Receptor-binding studies were performed. RESULTS: In µ opioid receptor knockout mice (-/-) and wild type (+/+) animals, the extract reduced locomotor activity after ip and low po doses in +/+ animals, but not after icv administration. The ip effect was counteracted by 0.3 mg/kg of apomorphine sc, suggesting dopaminergic presynaptic activity. An analgesic effect was only found in -/- mice after icv application. Norbinaltorphimine abolished the analgesic effect, but not the inhibitory effect, on locomotor activity, indicating that the analgesic effect is mediated via κ opioid receptors. Oral doses, which did not diminish locomotor activity, impaired the acquisition of shuttle box avoidance learning. There was no effect on consolidation. Binding studies showed affinity of kratom to µ, δ, and κ opioid receptors and to dopamine D1 receptors. CONCLUSIONS: The results obtained in drug-naïve mice demonstrate weak behavioral effects mediated via µ and κ opioid receptors.
Asunto(s)
Conducta Animal/efectos de los fármacos , Mitragyna/química , Extractos Vegetales/farmacología , Administración Oral , Animales , Ansiedad/psicología , Western Blotting , Interacciones Farmacológicas , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Células HEK293 , Calor , Humanos , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Ratones , Ratones Noqueados , Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Umbral del Dolor/efectos de los fármacos , Extractos Vegetales/administración & dosificación , Receptores de Dopamina D1/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismoRESUMEN
Opioids are irreplaceable for the treatment of severe pain. However, opioid-induced immunomodulation affects therapies. Here we report that treatment of human T lymphocytes with the opioids fentanyl, methadone, loperamide and beta-endorphin resulted in a strong induction of the cytokine interleukin-4. In contrast, morphine and buprenorphine induced markedly and significantly lower levels of interleukin-4 mRNA and protein. These findings suggest agonist-biased µ opioid receptor signaling in T cells. In the future, better knowledge about agonist-specific immunomodulatory effects of opioids offers the possibility to select drugs for a therapy with more favorable and/or less detrimental side effects in immune cells.
Asunto(s)
Analgésicos Opioides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Interleucina-4/biosíntesis , Receptores Opioides mu/agonistas , Subgrupos de Linfocitos T/efectos de los fármacos , Femenino , Humanos , Células Jurkat , Masculino , Receptores Opioides mu/fisiología , Subgrupos de Linfocitos T/metabolismoRESUMEN
We studied nociceptive behavior and the effects of analgesics in Wistar (Wist) and Sprague Dawley (SPD) rats and in CB1 receptor-deficient mice with neuropathic pain experimentally. Neuropathic pain was induced by loose ligation of the sciatic nerve (chronic constriction injury, CCI). In CCI rats from both strains, cold allodynia and a reduced thermal pain threshold were detected, whereas no effect was found in the hot plate test. Thermal pain threshold was used to study the antinociceptive effects of morphine, gabapentin, and parecoxib 5 days after surgery. Doses of gabapentin and morphine which had no effect on sham-operated animals provoked antinociceptive activity in CCI rats from both strains. An antinociceptive effect of parecoxib was only found in CCI Wist rats. No pharmacokinetic differences were detected between the two strains in parecoxib metabolism. Antinociceptive activity caused by parecoxib was attenuated by the CB1 antagonist rimonabant. To further clarify parecoxib-CB1 interaction, the effect of parecoxib was investigated in CB1-deficient mice and wild-type animals. CCI did not affect thermal pain threshold and mechanical pain threshold was decreased. Parecoxib normalized the altered mechanical pain threshold in CCI wild-type animals, whereas it had only a marginal effect in CB1 receptor deficient mice. Receptor binding experiments showed increased CB1 binding in parecoxib-treated CCI Wist rats. Levels of the CB1 receptor mRNA remained constant in both strains of rats 5 days after surgery. Differences in antinociceptive activity might be due to modification of the cannabinoid system.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Modelos Animales de Enfermedad , Isoxazoles/uso terapéutico , Neuralgia/metabolismo , Receptor Cannabinoide CB1/metabolismo , Neuropatía Ciática/metabolismo , Animales , Cannabinoides/metabolismo , Enfermedad Crónica , Constricción Patológica/tratamiento farmacológico , Constricción Patológica/genética , Constricción Patológica/metabolismo , Inhibidores de la Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Femenino , Isoxazoles/metabolismo , Isoxazoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuralgia/tratamiento farmacológico , Neuralgia/genética , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Neuropatía Ciática/tratamiento farmacológico , Neuropatía Ciática/genética , Especificidad de la Especie , Resultado del TratamientoRESUMEN
Opioids potently modulate neuronal functions, for example, by regulating the activity of transcription factors. Here, we investigated the effect of morphine on the activity of the transcription factor nuclear factor κB (NF-κB). Establishing cellular models for our investigations, we demonstrated that NF-κB mediated the tumor necrosis factor (TNF)-induced transcription of the cannabinoid receptor type 1 gene in primary fetal striatal neurons from rats and the human neuroblastoma cell line SH SY5Y. The activity of NF-κB in these models was strongly inhibited by morphine, which was achieved by a marked up-regulation of the inhibitor of nuclear factor-κB (IκB). The opioid-induced up-regulation of IκB was dependent on the transcription factors NF-κB itself and activator protein-1 (AP-1). In fact, stimulation of the cells with morphine resulted in a transient activation of NF-κB and a strong induction of c-Fos, one of the constituents of AP-1. This resulted in IκB levels significantly exceeding the basal, constitutive levels of IκB. These data, together with experiments in which AP-1 and IκB were down-regulated by decoy oligonucleotides and siRNA, suggest that the morphine-induced activation of AP-1 and the subsequent overexpression of IκB are key factors in the inhibition of NF-κB by the drug. In contrast, stimulation of primary neurons from rats and SH SY5Y cells with TNF, which is a classic activator of NF-κB, resulted in a resynthesis of IκB, in which the basal levels of IκB were restored only but did not result in an activation of AP-1 and overexpression of IκB.
Asunto(s)
Morfina/farmacología , FN-kappa B/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Animales , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Genes Reporteros , Humanos , FN-kappa B/fisiología , Neuronas/metabolismo , ARN Mensajero/genética , Ratas , Receptor Cannabinoide CB1/genética , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/fisiología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
OBJECTIVE: The aim of this study was to investigate the effect of the epigenetic modifiers trichostatin A and 5-aza-2'-deoxycytidine on the expression of the cannabinoid receptors CB1 and CB2 and µ-opioid receptors in human SH SY5Y neuroblastoma cells and human Jurkat T lymphocytes. METHODS: Using quantitative real-time RT-PCR, mRNA specific for the aforementioned receptors was determined. The functionality of the induced receptors was determined by analyzing the effect of the ligands to regulate intracellular cAMP. RESULTS: We demonstrated that treatment of SH SY5Y cells, which endogenously express µ-opioid receptors and CB1, but not CB2, resulted in de novo induction of CB2, while mRNA levels of CB1 and µ-opioid receptors were not significantly altered. In contrast, treatment of Jurkat lymphocytes, which endogenously express CB2, but not CB1 and µ-opioid receptors, resulted in de novo induction of CB1 and µ-opioid receptors, while mRNA levels of CB2 were not significantly altered. Furthermore, the functionality of the induced µ-opioid receptors and CB1 in the Jurkat cells was demonstrated. CONCLUSIONS: Our data suggest an epigenetically regulated expression of cannabinoid receptors and µ-opioid receptors. Their induction by epigenetic modifiers in distinct cells of the nervous and immune system might result in increased effects of the cognate drugs on neuronal and immune functions. Such modifications might be useful for novel therapies for various disorders, e.g. multiple sclerosis, where the elevated transmission of cannabinoid or opioid signals is beneficial.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Ácidos Hidroxámicos/farmacología , Neuroblastoma/genética , Receptor Cannabinoide CB1/efectos de los fármacos , Receptor Cannabinoide CB2/efectos de los fármacos , Receptores Opioides mu/efectos de los fármacos , Azacitidina/farmacología , Línea Celular Tumoral , Decitabina , Epigénesis Genética , Humanos , Células Jurkat , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Morphine is one of the most potent analgesic drugs. However, the utility of morphine in the management of chronic pain is limited by its rapid development of tolerance. Morphine exerts all of its pharmacological effects via the µ-opioid receptor. In many systems, tolerance is associated with phosphorylation and desensitization of G-protein-coupled receptors (GPCRs). In case of the µ-opioid receptor, phosphorylation occurs in an agonist-selective manner. High-efficacy agonists such as [d-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), fentanyl, or etonitazene stimulate the phosphorylation of both C-terminal threonine 370 (T370) and serine 375 (S375). In contrast, morphine promotes the phosphorylation of S375 but fails to stimulate T370 phosphorylation. Here, we have assessed the contribution of S375 phosphorylation to the development of antinociceptive tolerance to high- and low-efficacy µ agonists in vivo. We show that S375 phosphorylation of the µ-opioid receptor occurs in intact mouse brain in a dose-dependent manner after administration of morphine, fentanyl, or etonitazene. In knock-in mice expressing the phosphorylation-deficient S375A mutant of the µ-opioid receptor, morphine and fentanyl exhibited greater dose-dependent antinociceptive responses than in wild-type mice. However, acute and chronic tolerance to morphine was retained in S375A mutant mice. In contrast, antinociceptive tolerance after repeated subcutaneous application of etonitazene or repeated intracerebroventricular application of DAMGO was diminished. Thus, tolerance to µ agonists with different efficacies develops through distinct pathways. Whereas tolerance induced by DAMGO or etonitazene requires agonist-driven phosphorylation of S375, the development and maintenance of antinociceptive tolerance to morphine occurs independent of S375 phosphorylation.
Asunto(s)
Analgésicos Opioides/farmacología , Morfina/farmacología , Dimensión del Dolor/efectos de los fármacos , Receptores Opioides mu/agonistas , Receptores Opioides mu/biosíntesis , Alanina/genética , Animales , Tolerancia a Medicamentos/fisiología , Técnicas de Sustitución del Gen , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Dimensión del Dolor/métodos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Receptores Opioides mu/genética , Serina/genéticaRESUMEN
BACKGROUND AND PURPOSE: Morphine activates the µ-opioid receptor without causing its rapid endocytosis. In contrast, full agonists such as [d-Ala(2) -MePhe(4) -Gly-ol]enkephalin (DAMGO) or etonitazene stimulate a rapid and profound internalization. However, the detailed molecular events underlying the differential regulation of receptor trafficking by distinct opioid agonists remain incompletely understood. EXPERIMENTAL APPROACH: Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal residues serine 363 (Ser363), threonine 370 (Thr370) and serine 375 (Ser375), which enabled us to selectively detect either the Ser363-, Thr370- or Ser375-phosphorylated form of the receptor. KEY RESULTS: We showed that agonist-induced phosphorylation occurs at Thr370 and Ser375, whereas Ser363 is constitutively phosphorylated in the absence of agonist. We further demonstated that DAMGO and etonitazene stimulated the phosphorylation of both Thr370 and Ser375. In contrast, morphine promoted the phosphorylation of Ser375, but failed to stimulate Thr370 phosphorylation. In the presence of DAMGO, Ser375 phosphorylation occurred at a faster rate than phosphorylation of Thr370, indicating that Ser375 is the primary site of agonist-dependent phosphorylation. Activation of PKC by phorbol 12-myristate 13-acetate increased receptor phosphorylation only on Thr370, but not on Ser375, indicating that Thr370 can also undergo heterologous PKC-mediated phosphorylation. We also showed that µ receptor dephosphorylation can occur within minutes at or near the plasma membrane, and that agonist removal is a major prerequisite for Thr370 and Ser375 dephosphorylation. CONCLUSIONS AND IMPLICATIONS: Together, we showed for the first time that distinct agonists stimulate site-specific patterns of phosphorylation, which are intimately related to their ability to elicit µ-opioid receptor sequestration. LINKED ARTICLE: This article is commented on by Kelly, pp. 294-297 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01387.x.
Asunto(s)
Analgésicos Opioides/farmacología , Anticuerpos Fosfo-Específicos/metabolismo , Receptores Opioides mu/metabolismo , Secuencia de Aminoácidos , Animales , Bencimidazoles/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Células HEK293 , Humanos , Morfina/farmacología , Fosforilación , Conejos , Receptores Opioides mu/químicaRESUMEN
Cyclooxygenase 2 inhibitors (COX 2) such as parecoxib (par) and valdecoxib (val) are used in the treatment of neuropathic pain. Using the radioligand binding assay it was demonstrated that both the prodrug par as well as its active metabolite val have a specific affinity to the cannabinoid (CB) receptor measured in CB1-expressing HEK 293 cells and rat brain tissue. Agonist activity was detected by GTPγS assays, cAMP formation experiments and ex vivo modulation of glutamate and GABA release of the rat brain tissue. In comparison to the specific cannabinoid agonist, WIN 55,212-2, the two COX 2 inhibitors are about 2 orders of magnitude less potent. The data suggest that the analgesic effects of par and its metabolite val in Wistar rats may be at least partially mediated by a direct interaction with the CB1 receptors. The COX 2 inhibitors appear to be a hypothetically useful tool for add-on therapy of neuropathic pain.
Asunto(s)
Química Encefálica/efectos de los fármacos , Inhibidores de la Ciclooxigenasa 2/farmacología , Isoxazoles/farmacología , Receptor Cannabinoide CB1/efectos de los fármacos , Sulfonamidas/farmacología , Aminoácidos/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , AMP Cíclico/biosíntesis , Ciclohexanoles/metabolismo , Ácido Glutámico/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Masculino , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/genética , Transfección , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Various immunomodulatory effects of opioids are mediated by mu opioid receptors. While in resting T lymphocytes their expression is repressed, mu opioid receptors are induced by interleukin-4 via the transcription factor STAT6. Here we investigated mechanisms underlying this induction in human Jurkat T cells. Although interleukin-4 induced a rapid activation of STAT6 by phosphorylation within few minutes, chromatin-immune-precipitation analysis revealed that the binding of STAT6 to its regulatory DNA element on the mu opioid receptor promoter occurs later than 2h after interleukin-4-stimulation. Detectable amounts of the mu opioid receptor mRNA were observed later than 3h after stimulation. Preceding the binding of STAT6, several epigenetic mechanisms were observed that are known to modify the chromatin architecture of a gene. Thus, we detected by chromatin-immune-precipitation analysis transient association of the mu opioid receptor gene promoter with trimethylated histone H3 at lysine 4, phosphorylated (serine 10) plus acetylated (lysine 14) histone H3, and acetylated histone H4 at lysine 16. In addition, binding of the methyl-cytosine-guanine dinucleotide-binding protein MeCP2 to the mu opioid receptor promoter decreased during the interleukin-4 treatment of Jurkat cells. Furthermore, we detected a transient association of the mu opioid receptor promoter with Brg-1, which is a protein contained in ATP-dependent chromatin remodeling complexes and known to facilitate transcriptional activation of a gene. Together, these data suggest that epigenetic modifications of the chromatin of the mu opioid receptor gene are involved in the transcriptional activation of the gene in response to interleukin-4 in T cells.
Asunto(s)
Epigénesis Genética/inmunología , Interleucina-4/genética , Receptores Opioides mu/genética , Transducción de Señal/inmunología , Activación Transcripcional/inmunología , Western Blotting , Cromatina , Epigénesis Genética/genética , Expresión Génica , Humanos , Interleucina-4/metabolismo , Células Jurkat , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Receptores Opioides mu/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismoRESUMEN
We demonstrated recently that opioid-induced activation of phospholipase D2 (PLD2) enhances mu- (MOPr) and delta-opioid receptor endocytosis/recycling and thus reduces the development of opioid receptor desensitization and tolerance. However, the mechanistic basis for the PLD2-mediated induction of opioid receptor endocytosis is currently unknown. Here we show that PLD2-generated phosphatidic acid (PA) might play a key role in facilitating the endocytosis of opioid receptors. However, PLD2-derived PA is known to be further converted to diacylglycerol (DAG) by PA phosphohydrolase (PPAP2). In fact, blocking of PA phosphohydrolase activity by propranolol or PPAP2-short interfering RNA (siRNA) transfection significantly attenuated agonist-induced opioid receptor endocytosis. The primary importance of PA-derived DAG in the induction of opioid receptor endocytosis was further supported by the finding that increasing the DAG level by inhibiting the reconversion of DAG into PA with the DAG kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) or the addition of the synthetic cell-permeable DAG analog 1,2-dioctanoyl-sn-glycerol (DOG), further increased the agonist-induced opioid receptor endocytosis. Moreover, the addition of DOG bypasses the PLD2-siRNA- or PPAP2-siRNA-mediated impairment of DAG synthesis and resulted in a restoration of agonist-induced opioid receptor internalization. Further studies established a functional link between PA-derived DAG and the activation of p38 mitogen-activated protein kinase (MAPK) and the subsequent phosphorylation of the Rab5 effector early endosome antigen 1, which has been demonstrated recently to be required for the induction of MOPr endocytosis. Taken together, our results revealed that the regulation of opioid receptor endocytosis by PLD2 involves the conversion of its product PA to DAG resulting in an activation of the p38 MAPK pathway.
Asunto(s)
Endocitosis , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Transducción de Señal , Secuencia de Bases , Línea Celular , Cartilla de ADN , Endocitosis/efectos de los fármacos , Humanos , Piperidinas/farmacología , Quinazolinonas/farmacología , Ensayo de Unión RadioliganteRESUMEN
Pituitary adenylate cyclase activating peptide (PACAP) and the chemokine stromal cell-derived factor (SDF-1) have been implicated in neuroprotection, neurogenesis, and regeneration. Focal ischemia is associated with rapid upregulation of PACAP in perifocal neurons and delayed induction of SDF-1 in hypoxic/ischemic tissues, the latter process being involved in the recruitment of stem cells and inflammatory cells. Here, we studied mRNA patterns of PACAP, SDF-1 and the cognate receptors PAC1 and CXCR4 by in situ hybridization in the rat hippocampus after transient global ischemia, a rat model for programmed death of CA1 pyramidal neurons. Cell death in CA1 was not associated with local induction of PACAP and SDF-1 expression or recruitment of CXCR4-expressing infiltrates. However, there was a transient, almost complete loss of SDF-1 expression in microvessels in all hippocampal regions. Granule cells transiently showed a decrease of SDF-1 and an increase of PACAP expression. While PAC1 mRNA was moderately decreased throughout the hippocampus, CXCR4 expression was selectively increased in the subgranular layer. We propose that altered PACAP and SDF-1 gene expression in granule cells plays a role in regulated neurogenesis after global ischemia. The finding that programmed neuronal death after global ischemia was not associated with SDF-1 upregulation or recruitment of CXCR4-expressing cells is in sharp contrast to SDF-1/CXCR4-mediated infiltration of infarct tissue after focal ischemia. Hence, the different modes of neuronal death after focal and global ischemia are associated with distinct SDF-1 and PACAP gene regulation patterns and distinct reorganization mechanisms.
Asunto(s)
Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Regulación de la Expresión Génica , Isquemia/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , ARN Mensajero/metabolismo , Animales , Autorradiografía , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Modelos Animales de Enfermedad , Isquemia/clasificación , Isquemia/patología , Masculino , Neuronas/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G(i/o) proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Delta9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the zeta-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.
Asunto(s)
Cannabinoides/farmacología , Activación de Linfocitos/efectos de los fármacos , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/metabolismo , Ácidos Araquidónicos/antagonistas & inhibidores , Ácidos Araquidónicos/farmacología , Proteína Tirosina Quinasa CSK , Cannabinoides/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico , Dronabinol/antagonistas & inhibidores , Dronabinol/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Indoles/farmacología , Interleucina-2/inmunología , Interleucina-2/metabolismo , Células Jurkat , Activación de Linfocitos/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Morfolinas/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Proteínas Tirosina Quinasas , Psicotrópicos/análisis , Psicotrópicos/antagonistas & inhibidores , Psicotrópicos/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/inmunología , Receptor Cannabinoide CB2/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Familia-src QuinasasRESUMEN
Endocytosis of the mu-opioid receptor (MOPr) has been shown to play a protective role against the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated and ADP-ribosylation factor (ARF)-dependent activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis. In this study, we investigated which particular ARF protein is involved in opioid-mediated PLD2 activation and what are the mechanisms of ARF function in MOPr trafficking and signaling. By coexpressing the MOPr and dominant negative or constitutively active ARF mutants in human embryonic kidney (HEK) 293 cells and primary cultured cortical neurons as well as by using siRNA technology, we identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. We also found that expression of an effector domain mutant of ARF6, which is incapable of activating PLD, blocked agonist-induced endocytosis suggesting that ARF6 function in MOPr trafficking is PLD2-mediated. Analogously, opioid-mediated activation of PLD2 is blocked in the presence of dominant negative ARF6 mutants. Finally, we also showed that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. Together, these results provide evidence that ARF6 protein regulates MOPr trafficking and signaling via PLD2 activation and hence affects the development of opioid receptor desensitization and tolerance.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Fosfolipasa D/metabolismo , Transporte de Proteínas , Receptores Opioides mu/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Animales , Línea Celular , Células Cultivadas , Endocitosis/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Morfina/farmacología , Mutación , Narcóticos/farmacología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/agonistas , Transducción de SeñalRESUMEN
Transient prenatal vitamin D deficiency is considered a neurodevelopmental animal model in schizophrenia research. Vitamin D deficiency in female rats causes morphological, cellular and molecular changes in the brain and alters behaviour and nerve growth factors expression in their offspring. Prenatal depleted animals showed a significant impairment of latent inhibition, a feature often associated with schizophrenia and of hole board habituation. Interestingly, memory consolidation of brightness discrimination was improved. Possible functional effects of altered brain development that results from prenatal vitamin D deficiency were characterized by investigation of potentiation phenomena in the hippocampus in freely moving rats. Transient prenatal vitamin D deficiency induced an enhancement of long-term potentiation (LTP) using either weak tetanic or strong tetanic stimulation, whereas the response to test stimuli was not changed. The classic neuroleptic drug haloperidol (Hal) and the atypical neuroleptic risperidone (Ris) in doses, which normalized behavioural disturbances in prenatal vitamin D-deficient animals without any side effects on the normal behaviour decreased the enhanced LTP in the experimental group to control level. Interestingly, the effect of the substances was different in experimental and control rats. The LTP was enhanced in control animals by the low doses of the drugs effective in our behavioural experiments. It can be suggested, that changes in brain development induced by prenatal vitamin D deficiency lead to specific functional alterations in hippocampal synaptic plasticity. LTP is considered a cellular correlate of learning and memory. The better retention performance in brightness discrimination seems in accordance with enhanced potentiation level.
Asunto(s)
Giro Dentado/fisiopatología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Deficiencia de Vitamina D/fisiopatología , Animales , Giro Dentado/efectos de los fármacos , Femenino , Haloperidol/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Embarazo , Ratas , Ratas Sprague-Dawley , Risperidona/farmacología , Factores de TiempoRESUMEN
We have recently shown that the activation of the rat mu-opioid receptor (MOPr, also termed MOR1) by the mu-agonist [D-Ala(2), Me Phe(4), Glyol(5)]enkephalin (DAMGO) leads to an increase in phospholipase D2 (PLD2) activity and an induction of receptor endocytosis, whereas the agonist morphine which does not induce opioid receptor endocytosis fails to activate PLD2. We report here that MOPr-mediated activation of PLD2 stimulates production of reactive oxygen molecules via NADH/NADPH oxidase. Oxidative stress was measured with the fluorescent probe dichlorodihydrofluorescein diacetate and the role of PLD2 was assessed by the PLD inhibitor D-erythro-sphingosine (sphinganine) and by PLD2-small interfering RNA transfection. To determine whether NADH/NADPH oxidase contributes to opioid-induced production of reactive oxygen species, mu-agonist-stimulated cells were pre-treated with the flavoprotein inhibitor, diphenylene iodonium, or the specific NADPH oxidase inhibitor, apocynin. Our results demonstrate that receptor-internalizing agonists (like DAMGO, beta-endorphin, methadone, piritramide, fentanyl, sufentanil, and etonitazene) strongly induce NADH/NADPH-mediated ROS synthesis via PLD-dependent signaling pathways, whereas agonists that do not induce MOPr endocytosis and PLD2 activation (like morphine, buprenorphine, hydromorphone, and oxycodone) failed to activate ROS synthesis in transfected human embryonic kidney 293 cells. These findings indicate that the agonist-selective PLD2 activation plays a key role in the regulation of NADH/NADPH-mediated ROS formation by opioids.
Asunto(s)
Analgésicos Opioides/farmacología , Estrés Oxidativo/efectos de los fármacos , Fosfolipasa D/efectos de los fármacos , Especies Reactivas de Oxígeno/agonistas , Receptores Opioides mu/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , NAD/efectos de los fármacos , NAD/metabolismo , NADP/efectos de los fármacos , NADP/metabolismo , Estrés Oxidativo/fisiología , Fosfolipasa D/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Receptores Opioides mu/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Opioids are widely used for the treatment of severe pain. However, it is also known that opioids, in particular morphine, cause immunosuppression. Therefore, their use may complicate treatment of persons with an already impaired immune system, e.g., patients suffering from cancer or AIDS. We investigated the mechanisms of opioid-induced immunosuppression in primary human T lymphocytes and the human T cell line Jurkat. We demonstrated that morphine and the endogenous opioid beta-endorphin inhibited the transcription of IL-2 in activated human T lymphocytes as well as the activation of the transcription factors AP-1, NFAT, and NF-kappaB, which transactivate IL-2. In addition, the TCR-induced calcium flux and MAPK activation were inhibited by the opioids, as well as proximal signaling events, such as the phosphorylation of the linker for activation of T cells and Zap70. A more detailed characterization of the mechanism revealed that incubation of T cells with the opioids caused a marked increase in cAMP. This in turn activated protein kinase A, which augmented the kinase activity of C-terminal Src kinase bound to phosphoprotein associated with glycosphingolipid-enrich microdomains, resulting in a further enhancement of the tonic inhibition of the leukocyte-specific protein tyrosine kinase Lck, thereby blocking the initiation of TCR signaling. These effects were mediated by mu opioid receptors. Our findings contribute to the understanding of immunosuppressive side effects of morphine. Since beta-endorphin is expressed and secreted by immune effector cells, including T cells, and up-regulated in these cells by various stimuli, our data also suggest an inhibitory role for beta-endorphin in the physiological regulation of T cell activation.