RESUMO
We identified, via high-throughput screening using a FLIPR® calcium assay, compound 1, which incorporated a dihydroquinolinyl-2-oxoethylsulfanyl-(1H,5H)-pyrimidinedione core and activated the µ-opioid receptor (MOR) in the presence of naloxone or naltrexone. A structure-activity relationship study of the analogs of 1 led to the design of compound 21, which activated MOR in the presence of naloxone with an EC50 of 3.3 ± 0.2 µM. MOR activation by the compound 21-antagonist pair was antagonist-dependent. Compound 21 did not affect the potency of the orthosteric agonist, morphine, toward MOR, indicating that it affected the function of MOR antagonists rather than that of the agonists. Computer modeling of the compound 21-MOR-naloxone complex revealed major interactions between compound 21 and MOR, including hydrogen bonding with Ser196, π-π stacking with Tyr149, and sulfur-aromatic interaction with Trp192. This study may pave the way for developing agents capable of safe and effective MOR modulation.
Assuntos
Naloxona , Naltrexona , Analgésicos Opioides , Imidazóis , Naloxona/farmacologia , Naltrexona/farmacologia , Receptores Opioides , Sulfonamidas , TiofenosRESUMO
Although the roles of opioid receptors in neurogenesis have been implicated in previous studies, the mechanism by which κ-opioid receptor (OPRK1) regulates adult neurogenesis remains elusive. We now demonstrate that two agonists of OPRK1, U50,488H and dynorphin A, inhibit adult neurogenesis by hindering neuronal differentiation of mouse hippocampal neural stem cells (NSCs), both in vitro and in vivo. This effect was blocked by nor-binaltorphimine (nor-BNI), a specific antagonist of OPRK1. By examining neurogenesis-related genes, we found that OPRK1 agonists were able to downregulate the expression of Pax6, Neurog2, and NeuroD1 in mouse hippocampal NSCs, in a way that Pax6 regulates the transcription of Neurog2 and Neurod1 by directly interacting with their promoters. Moreover, this effect of OPRK1 was accomplished by inducing expression of miR-7a, a miRNA that specifically targeted Pax6 by direct interaction with its 3'-UTR sequence, and thereby decreased the levels of Pax6, Neurog2, and NeuroD1, thus resulted in hindrance of neuronal differentiation of NSCs. Thus, by modulating Pax6/Neurog2/NeuroD1 activities via upregulation of miR-7a expression, OPRK1 agonists hinder the neuronal differentiation of NSCs and hence inhibit adult neurogenesis in mouse hippocampus.
Assuntos
MicroRNAs/genética , Células-Tronco Neurais/citologia , Neurogênese/genética , Fator de Transcrição PAX6/genética , Receptores Opioides kappa/genética , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Dinorfinas/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Humanos , Camundongos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Proteínas do Tecido Nervoso/genética , Neurogênese/efeitos dos fármacos , Receptores Opioides kappa/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
Opioids, like morphine and naloxone, regulate the proliferation and neuronal differentiation of neural stem cells (NSCs) in a receptor-independent and ten-eleven translocation methylcytosine dioxygenase (TET1)-dependent manner in vitro. Whether naloxone regulates hippocampal NSCs and contextual learning in vivo in a similar manner was determined. Naloxone infusion increased the Ki67 and Doublecortin positive cells in subgranular zone of wild type mice, which suggested the increased proliferation and differentiation of hippocampal NSCs in vivo and was consistent with the in vitro functions of naloxone. In addition, naloxone infusion also facilitated the contextual learning and memory of wild type mice. To determine the contribution of µ-opioid receptor (OPRM1) and TET1 to these functions of naloxone, several types of knockout mice were used. Since Tet1-/- mice have high deficiency in contextual learning and memory, Tet1+/- mice were used instead. The abilities of naloxone to regulate NSCs and to facilitate contextual learning were significantly impaired in Tet1+/- mice. In addition, these abilities of naloxone were not affected in Oprm1-/- mice. Therefore, naloxone facilitates contextual learning and memory in a receptor-independent and Tet1-dependent manner, which provides new understanding on the receptor-independent functions of opioids.
Assuntos
Proteínas de Ligação a DNA/deficiência , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Proteínas Proto-Oncogênicas/deficiência , Receptores Opioides mu/deficiência , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Camundongos Knockout , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Opioides mu/genéticaRESUMO
It is estimated that nearly a third of people who abuse drugs started with prescription opioid medicines. Approximately, 11.5 million Americans used prescription drugs recreationally in 2016, and in 2018, 46,802 Americans died as the result of an opioid overdose, including prescription opioids, heroin, and illicitly manufactured fentanyl (National Institutes on Drug Abuse (2020) Opioid Overdose Crisis. https://www.drugabuse.gov/drugs-abuse/opioids/opioid-overdose-crisis . Accessed 06 June 2020). Yet physicians will continue to prescribe oral opioids for moderate-to-severe pain in the absence of alternative therapeutics, underscoring the importance in understanding how drug choice can influence detrimental outcomes. One of the opioid prescription medications that led to this crisis is oxycodone, where misuse of this drug has been rampant. Being one of the most highly prescribed opioid medications for treating moderate-to-severe pain as reflected in the skyrocketed increase in retail sales of 866% between 1997 and 2007, oxycodone was initially suggested to be less addictive than morphine. The false-claimed non-addictive formulation of oxycodone, OxyContin, further contributed to the opioid crisis. Abuse was often carried out by crushing the pills for immediate burst release, typically by nasal insufflation, or by liquefying the pills for intravenous injection. Here, we review oxycodone pharmacology and abuse liability as well as present the hypothesis that oxycodone may exhibit a unique pharmacology that contributes to its high likability and abuse susceptibility. We will discuss various mechanisms that likely contribute to the high abuse rate of oxycodone including clinical drug likability, pharmacokinetics, pharmacodynamics, differences in its actions within mesolimbic reward circuity compared to other opioids, and the possibility of differential molecular and cellular receptor interactions that contribute to its selective effects. We will also discuss marketing strategies and drug difference that likely contributes to the oxycodone opioid use disorders and addiction.
Assuntos
Analgésicos Opioides/efeitos adversos , Comportamento Aditivo/epidemiologia , Epidemia de Opioides , Transtornos Relacionados ao Uso de Opioides/epidemiologia , Oxicodona/efeitos adversos , Recompensa , Analgésicos Opioides/administração & dosagem , Animais , Comportamento Aditivo/psicologia , Humanos , Transtornos Relacionados ao Uso de Opioides/psicologia , Oxicodona/administração & dosagem , Dor/tratamento farmacológico , Dor/epidemiologia , Dor/psicologiaRESUMO
The abilities of opioids to activate downstream signaling pathways normally depend on the binding between opioids and their receptors. However, opioids may also function in a receptor-independent manner, especially in neural stem cells (NSCs) in which the expression of opioid receptors and endogenous opioid agonists is low. When two opioids, morphine and naloxone, were used during the early stage of NSC differentiation, increased neurogenesis was observed. However, naloxone methiodide, a membrane impenetrable analog of naloxone, did not affect the NSC differentiation. The abilities of morphine and naloxone to facilitate neurogenesis were also observed in opioid receptor-knockout NSCs. Therefore, morphine and naloxone promote neurogenesis in a receptor-independent manner at least during the early stage. In addition, the receptor-independent functions of opioids were not observed in methylcytosine dioxygenase ten-eleven translocation 1 (Tet1) knockout NSCs. When the expression of opioid receptors increased and the expression of Tet1 decreased during the late stage of NSC differentiation, morphine, but not naloxone, inhibited neurogenesis via traditional receptor-dependent and miR181a-Prox1-Notch-related pathway. In summary, the current results demonstrated the time-dependent effects of opioids during the differentiation of NSCs and provided additional insight on the complex functions of opioids.
Assuntos
Diferenciação Celular , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Naloxona/farmacologia , Células-Tronco Neurais/citologia , Neurogênese , Receptores Opioides mu/fisiologia , Animais , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Antagonistas de Entorpecentes/farmacologia , Entorpecentes/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismoRESUMO
Normally, opioids function in a receptor-dependent manner. They bind to opioid receptors, activate or inhibit receptor activation, and subsequently modulate downstream signal transduction. However, the complex functions of opioids and the low expression of opioid receptors and their endogenous peptide agonists in neural stem cells (NSCs) suggest that some opioids may also modulate NSCs via a receptor-independent pathway. In the current study, two opioids, morphine and naloxone, are demonstrated to facilitate NSC proliferation via a receptor-independent and ten-eleven translocation methylcytosine dioxygenase 1 (TET1)-dependent pathway. Morphine and naloxone penetrate cell membrane, bind to TET1 protein via three key residues (1,880-1,882), and subsequently result in facilitated proliferation of NSCs. In addition, the two opioids also inhibit the DNA demethylation ability of TET1. In summary, the current results connect opioids and DNA demethylation directly at least in NSCs and extend our understanding on both opioids and NSCs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Morfina/farmacologia , Naloxona/farmacologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Opioides/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Desmetilação do DNA/efeitos dos fármacos , Camundongos Endogâmicos ICR , Células-Tronco Neurais/efeitos dos fármacosRESUMO
Oxycodone, a widely prescribed and very potent oral opioid analgesic agent, is highly addictive and has many side effects, including troublesome constipation. Our studies in mice indicated that pretreatment of naltrindole did not significantly affect the analgesic efficacy of oxycodone but attenuated the tolerance and withdrawal induced by chronic oxycodone administration. Naltrindole also attenuated the oxycodone-induced rewarding and re-instatement behaviors, as shown by the conditioned place preference test. Further, oxycodone-induced decrease in intestinal transit (i.e., constipation) was reduced by naltrindole. However, naltrindole did not block the respiratory depression produced by oxycodone. Taken together, these data suggest that naltrindole can attenuate some major side effects while retaining the analgesic efficacy of oxycodone in mice. Naltrindole and oxycodone may have the potential to be a potent analgesic combination with much lower levels of oxycodone's side effects of addictive liability and constipation.
Assuntos
Constipação Intestinal/tratamento farmacológico , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/farmacologia , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Oxicodona/efeitos adversos , Receptores Opioides delta/antagonistas & inibidores , Analgésicos/efeitos adversos , Animais , Constipação Intestinal/induzido quimicamente , Tolerância a Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naltrexona/farmacologia , Naltrexona/uso terapêutico , Antagonistas de Entorpecentes/uso terapêutico , Transtornos Relacionados ao Uso de Opioides/complicações , Transtornos Relacionados ao Uso de Opioides/etiologia , Insuficiência Respiratória/complicaçõesRESUMO
Morphine is widely used for the treatment of severe pain. This analgesic effect is mediated principally by the activation of µ-opioid receptors (MOR). However, prolonged activation of MOR also results in tolerance, dependence, addiction, constipation, nausea, sedation, and respiratory depression. To address this problem, we sought alternative ways to activate MOR - either by use of novel ligands, or via a novel activation mechanism. To this end, a series of compounds were screened using a sensitive CHO-K1/MOR/Gα15 cell-based FLIPR® calcium high-throughput screening (HTS) assay, and the bithiazole compound 5a was identified as being able activate MOR in combination with naloxone. Structural modifications of 5a resulted in the discovery of lead compound 5j, which could effectively activate MOR in combination with the MOR antagonist naloxone or naltrexone. In vivo, naloxone in combination with 100â¯mg/kg of compound 5j elicited antinociception in a mouse tail-flick model with an ED50 of 17.5⯱â¯4â¯mg/kg. These results strongly suggest that the mechanism by which the 5j/naloxone combination activates MOR is worthy of further study, as its discovery has the potential to yield an entirely novel class of analgesics.
Assuntos
Analgésicos/farmacologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/uso terapêutico , Receptores Opioides mu/agonistas , Tiazóis/farmacologia , Aminas , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Quimioterapia Combinada , Muridae , Antagonistas de Entorpecentes/farmacologia , Relação Estrutura-AtividadeRESUMO
M1 muscarinic acetylcholine receptors are highly expressed in key areas that control cognition, such as the cortex and hippocampus, representing one potential therapeutic target for cognitive dysfunctions of Alzheimer's disease and schizophrenia. We have reported that M1 receptors facilitate cognition by promoting membrane insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AMPA receptor subunit 1 (GluA1) through phosphorylation at Ser845. However, the signaling pathway is still unclear. Here we showed that adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and PKA inhibitor KT5720 inhibited enhancement of phosphorylation of Ser845 and membrane insertion of GluA1 induced by M1 receptor activation. Furthermore, PI3K inhibitor LY294002 and protein kinase B (Akt) inhibitor IV blocked the effects of M1 receptors as well. Remarkably, the increase of the activity of PI3K-Akt signaling induced by M1 receptor activation could be abolished by cAMP-PKA inhibitors. Moreover, inhibiting the mammalian target of rapamycin (mTOR) complex 1, an important downstream effector of PI3K-Akt, by short-term application of rapamycin attenuated the effects of M1 receptors on GluA1. Furthermore, such effect was unrelated to possible protein synthesis promoted by mTOR. Taken together, these data demonstrate that M1 receptor activation induces membrane insertion of GluA1 via a signaling linking cAMP-PKA and PI3K-Akt-mTOR pathways but is irrelevant to protein synthesis.-Zhao, L.-X., Ge, Y.-H., Li, J.-B., Xiong, C.-H., Law, P.-Y., Xu, J.-R., Qiu, Y., Chen, H.-Z. M1 muscarinic receptors regulate the phosphorylation of AMPA receptor subunit GluA1 via a signaling pathway linking cAMP-PKA and PI3K-Akt.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Muscarínico M1/metabolismo , Receptores de AMPA/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Morphine is a unique opioid analgesic that activates the mu-opioid receptor (MOR) without efficiently promoting its endocytosis that may underlie side effects. Our objective was to discover a novel enhancer of ligand-induced MOR endocytosis and determine its effects on analgesia, tolerance and dependence. We used high-throughput screening to identify convallatoxin as an enhancer of ligand-induced MOR endocytosis with high potency and efficacy. Treatment of cells with convallatoxin enhanced morphine-induced MOR endocytosis through an adaptor protein 2 (AP2)/clathrin-dependent mechanism, attenuated morphine-induced phosphorylation of MOR, and diminished desensitization of membrane hyperpolarization. Furthermore, co-treatment with chronic convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance and dependence in morphine-tolerant mice. These findings suggest convallatoxin are potentially therapeutic for morphine side effects and open a new avenue to study MOR trafficking.
Assuntos
Analgésicos/farmacologia , Morfina/farmacologia , Receptores Opioides mu/genética , Estrofantinas/farmacologia , Analgesia/métodos , Analgésicos/química , Animais , Modelos Animais de Doenças , Endocitose/efeitos dos fármacos , Humanos , Ligantes , Camundongos , Receptores Opioides mu/efeitos dos fármacosRESUMO
Mortality due to opioid use has grown to the point where, for the first time in history, opioid-related deaths exceed those caused by car accidents in many states in the United States. Changes in the prescribing of opioids for pain and the illicit use of fentanyl (and derivatives) have contributed to the current epidemic. Less known is the impact of opioids on hippocampal neurogenesis, the functional manipulation of which may improve the deleterious effects of opioid use. We provide new insights into how the dysregulation of neurogenesis by opioids can modify learning and affect, mood and emotions, processes that have been well accepted to motivate addictive behaviours.
Assuntos
Afeto/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Encéfalo/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Receptores Opioides/metabolismoRESUMO
We have previously reported that the miR-181a/Prox1/Notch1 pathway mediates the effect of morphine on modulating lineage-specific differentiation of adult neural stem/progenitor cells (NSPCs) via a PKCε-dependent pathway, whereas fentanyl shows no such effect. However, the role of the PKCε/Prox1 pathway in mediating drug-associated contextual memory remains unknown. The current study investigated the effect of PKCε/Prox1 on morphine-induced inhibition of adult neurogenesis and drug-associated contextual memory in mice, while the effect of fentanyl was tested simultaneously. By using BrdU labeling, we were able to examine the lineages of differentiated NSPCs in adult DG. PKCε knockout blocked morphine's effects on inducing in vivo astrocyte-preferential differentiation of NSPCs, but did not alter NSPC lineages upon fentanyl treatment. Inhibited adult neurogenesis further resulted in prolonged extinction and enhanced reinstatement of morphine-induced CPP, as well as prolonged extinction of space reference memory indicated by the Morris water maze paradigm. However, after fentanyl administration, no significant changes were found between wild-type and PKCε knockout mice, during either CPP or water maze tasks. When the lentivirus encoding Nestin-promoter-controlled Prox1 cDNA was injected into hippocampi of wildtype and PKCε knockout adult mice to modulate PKCε/Prox1 activity, similar effects were discovered in adult mice injected with lentivirus encoding Prox1, and more dramatic effects were found in PKCε knockout mice with concurrent Prox1 overexpression. In conclusion, morphine mediates lineage-specific NSPC differentiation, inhibits adult neurogenesis and regulates contextual memory retention via the PKCε/Prox1 pathway, which are implicated in the eventual context-associated relapse.
Assuntos
Extinção Psicológica/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Memória/efeitos dos fármacos , Morfina/farmacologia , Neurogênese/efeitos dos fármacos , Proteína Quinase C-épsilon/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Condicionamento Psicológico/efeitos dos fármacos , Giro Denteado/metabolismo , Fentanila/farmacologia , Vetores Genéticos/administração & dosagem , Proteínas de Homeodomínio/genética , Lentivirus/genética , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Knockout , Microinjeções , Morfina/antagonistas & inibidores , Células-Tronco Neurais/citologia , Proteína Quinase C-épsilon/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor/genéticaRESUMO
M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.
Assuntos
Cognição/fisiologia , Subunidades Proteicas/metabolismo , Receptor Muscarínico M1/metabolismo , Receptores de AMPA/metabolismo , Animais , Pareamento Cromossômico/fisiologia , Hipocampo/metabolismo , Aprendizagem/fisiologia , Masculino , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/metabolismo , Fosforilação/fisiologia , Receptores Muscarínicos/metabolismoRESUMO
The treatment of opioid addiction is challenging because addicts are highly prone to relapse when the memory of the former drug experience is triggered by emotional or environmental cues. An emerging and promising concept in addiction biology is that by manipulating adult hippocampal neurogenesis, a phenomenon involved in learning and memory, drug reward-like behaviors and relapse can be attenuated. We tested a new synthetic compound, KHS101, in an animal model of drug-associated contextual memory. KHS101 has been reported to increase the expression of neurogenic differentiation 1 (NeuroD1), a transcription factor involved in adult neurogenesis, and to specifically induce neuronal differentiation both in vitro and in vivo. Our results indicated that the subcutaneous injection of 3 mg/kg KHS101 for 7 days before conditioned place preference (CPP) training prolonged CPP extinction, while the same treatment after training accelerated extinction. This effect paralleled that observed following temporally controlled, tetracycline-induced NeuroD1 overexpression. Furthermore, the effect of KHS101 may occur via its induction of NeuroD1 expression as demonstrated by the abolition of the KHS101-mediated modulation of morphine-induced CPP extinction after the stereotaxic injection of lentiviral NeuroD1 small interfering RNA into the dentate gyrus (DG) of the hippocampus. These results suggest that the KHS101-mediated modulation of neurogenesis at a critical stage of the conditioning or the extinction of an opioid-associated experience may disrupt the memory trace of the existing opioid-associated experience to facilitate the extinction of drug-associated contextual memory. This implies that KHS101 has therapeutic potential for the treatment of opioid addiction.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Memória/efeitos dos fármacos , Morfina/administração & dosagem , Entorpecentes/administração & dosagem , Neurogênese/efeitos dos fármacos , Tiazóis/administração & dosagem , Animais , Condicionamento Clássico/efeitos dos fármacos , Extinção Psicológica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos Endogâmicos C57BLRESUMO
Opiate withdrawal/negative reinforcement has been implicated as one of the mechanisms for the progression from impulsive to compulsive drug use. Increase in the intracellular cAMP level and protein kinase A (PKA) activities within the neurocircuitry of addiction has been a leading hypothesis for opiate addiction. This increase requires the phosphorylation of µ-opioid receptor (MOR) at Tyr336 by Src after prolonged opiate treatment in vitro Here, we report that the Src-mediated MOR phosphorylation at Tyr336 is a prerequisite for opiate withdrawal in mice. We observed the recruitment of Src in the vicinity of MOR and an increase in phosphorylated Tyr336 (pY336) levels during naloxone-precipitated withdrawal. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses attenuated pY336 levels, and several somatic withdrawal signs. This was also observed in Fyn-/- mice. The stereotaxic injection of wild-type MOR, but not mutant (Y336F) MOR, lentiviruses into the locus coeruleus of MOR-/- mice restored somatic withdrawal jumping. Regulating pY336 levels during withdrawal might be a future target for drug development to prevent opiate addictive behaviors.
Assuntos
Receptores Opioides mu/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Benzodioxóis/farmacologia , Peso Corporal/efeitos dos fármacos , Células HEK293 , Humanos , Locomoção/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fyn/deficiência , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Quinazolinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Opioides mu/deficiência , Receptores Opioides mu/genética , Tirosina/química , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
The Mu opioid receptor (MOR) mediates various functions of opioid-induced analgesia, euphoria and respiratory depression, and is a major target of opioid analgesics. Understanding of MOR gene expression among species is important for understanding its analgesic function in humans. In the current study, the polypyrimidine/polypurine (PPy/u) region, a key element of MOR gene expression, was compared in humans and mice. The mouse PPy/u element is highly homologous to its human element (84%), and the mouse MOR (mMOR) reporter drives luciferase activity 35-fold more effectively than the human MOR (hMOR) reporter. The structural study of reporter plasmids using S1 nuclease indicates that the mouse PPy/u element has a particular conformational structure, namely a single-stranded DNA (ssDNA) region that promotes strong promoter activity. DNA electrophoretic mobility shift assays demonstrated that the recombinant α-complex protein 1 (α-CP1) is capable of binding to a single-stranded mouse PPy/u sequence. Furthermore, plasmid-expressing α-CP1 activated the expression of a luciferase reporter when cotransfected with a single-stranded (p336/306) construct. In addition, the α-CP1 gene induced the mMOR gene in mouse neuronal cells and did not induce the human neuronal MOR gene. The current study demonstrates that α-CP1 functions as a transcriptional activator in the mMOR gene, but does not function in the hMOR gene due to species-specific structural differences. The differences in human and mouse MOR gene expression are based on α-CP1 and the ssDNA structure of the MOR promoter. The MOR gene is species-specifically regulated, as the PPy/u element adopts a unique species-specific conformation and α-CP1 recruitment.
RESUMO
The development of tolerance to morphine, one of the most potent analgesics, in the management of chronic pain is a significant clinical problem and its mechanisms are poorly understood. Morphine exerts its pharmacological effects via the µ-opioid receptor (MOR). Tolerance is highly connected to G-protein-coupled receptors (GPCR) phosphorylation and desensitization increase. Because morphine desensitization previously has been shown to be MOR phosphorylation- and ß-arrestin2-independent (in contrast to agonists such as fentanyl), we examined the contribution of phosphorylation of the entire C-terminus to the development of antinociceptive tolerance to the partial (morphine) and full (fentanyl) MOR agonists in vivo. In MOR knockout (MORKO) mice, we delivered via lentivirus the genes encoding the wild-type MOR (WTMOR) or a phosphorylation-deficient MOR (Cterm(-S/T)MOR) in which all of the serine and threonine residues were mutated to alanine into the ventrolateral periaqueductal grey matter (vlPAG) or lumbar spinal cord (SC), structures that are involved in nociception. We compared the analgesic ED50 in WTMOR- and Cterm(-S/T)MOR-expressing MORKO mice before and after morphine or fentanyl tolerance was induced. Morphine acute antinociception was partially restored in WTMOR- or Cterm(-S/T)MOR-transferred MORKO mice. Fentanyl acute antinociception was observed only in MORKO mice with the transgenes expressed in the SC. Morphine antinociceptive tolerance was not affected by expressing Cterm(-S/T)MOR in the vlPAG or SC of MORKO mice. Fentanyl-induced tolerance in MORKO mice expressing WTMOR or Cterm(-S/T)MOR, is greater than morphine-induced tolerance. Thus, MOR C-terminus phosphorylation does not appear to be critical for morphine tolerance in vivo.
Assuntos
Analgésicos Opioides/farmacologia , Morfina/farmacologia , Receptores Opioides mu/metabolismo , Medula Espinal/efeitos dos fármacos , Animais , Tolerância a Medicamentos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Receptores Opioides mu/genética , Medula Espinal/metabolismoRESUMO
BACKGROUND: The authors investigated the pharmacology and signaling pathways of the opioid receptors modulated by compound 1, 1-(2,4-dibromophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one. METHODS: In vitro studies of compound 1 were assessed by using a radioligand-binding assay (n = 3), a cyclic adenosine monophosphate assay (n = 3), a ß-arrestin assay (n = 3), an internalization assay (n = 3), and an immunohistochemistry (n = 8). In vivo studies of compound 1 were characterized using a tail-flick test (n = 5 to 6), tail-clip test (n = 7), von Frey hair test (n = 5), and charcoal meal test (n = 5). RESULTS: Compound 1 elicited robust effects in µ-opioid (mean ± SD; binding affinity: 15 ± 2 nM; cyclic adenosine monophosphate assay: 24 ± 6 nM), δ-opioid (82 ± 7 nM; 1.9 ± 0.1 µM), and κ-opioid (76 ± 9 nM; 1.4 ± 0.5 µM) receptor-expressing cells. Compound 1 acts as a full agonist of ß-arrestin-2 recruitment in µ-opioid (1.1 ± 0.3 µM) and δ-opioid (9.7 ± 1.9 µM) receptor-expressing cells. Compound 1 caused less gastrointestinal dysfunction (charcoal meal test: morphine: 82 ± 5%; compound 1: 42 ± 5%) as well as better antinociception in mechanical pain hypersensitivity (tail-clip test: morphine: 10 ± 3 s; compound 1: 19 ± 1 s) and in cancer-induced pain (von Frey hair test: morphine: 0.1 ± 0.1 g; compound 1: 0.3 ± 0.1 g) than morphine at equi-antinociceptive doses. CONCLUSIONS: Compound 1 produced antinociception with less gastrointestinal dysfunction than morphine.
Assuntos
Gastroenteropatias/induzido quimicamente , Indazóis/farmacologia , Morfina , Receptores Opioides/agonistas , Analgésicos Opioides/farmacologia , Animais , Modelos Animais de Doenças , Gastroenteropatias/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Since the discovery of µ-opioid receptor (MOR) gene two decades ago, various regulatory factors have been shown to interact with the MOR promoter and modulate transcript levels. However, the majority of early transcriptional studies on MOR gene have not addressed how intracellular signaling pathways mediate extracellular modulators. In this study, we demonstrate that MOR epigenetic regulation requires multiple coordinated signals converging at the MOR promoter, involving mitogen-activated protein kinase (MAPK) activation and mitogen- and stress-activated protein kinase 1 (MSK1)-ranges of intracellular signaling pathways similar to those activated by opioid agonists. Inhibiting p38 MAPK or extracellular signal-regulated kinase (ERK) 1/2 MAPK (upstream activators of MSK1) reduced MOR expression levels; accordingly, the functional role of MSK1, but not MSK2, was demonstrated using genetic approaches. However, for maximal MSK1 effect, an open chromatin configuration was required, because in vitro CpG methylation of the MOR promoter abolished MSK1 activity. Finally, endogenous MSK1 levels concomitantly increased to regulate MOR gene expression during neuronal differentiation of P19 cells, suggesting a conserved role of this kinase in the epigenic activation of MOR in neurons. Taken together, our findings indicate that the expression of MOR gene requires the activity of intracellular signaling pathways that have been implicated in the behavioral outcomes of opioid drugs, which suggests that an autoregulatory mechanism may function in opioid systems.
Assuntos
Epigênese Genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Receptores Opioides mu/genética , Acetilação/efeitos dos fármacos , Animais , Diferenciação Celular/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Camundongos , Modelos Biológicos , Neurônios/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Gene regulation at the post-transcriptional level is frequently based on cis- and trans-acting factors on target mRNAs. We found a C-rich element (CRE) in mu-opioid receptor (MOR) 3'-untranslated region (UTR) to which poly (rC) binding protein 1 (PCBP1) binds, resulting in MOR mRNA stabilization. RNA immunoprecipitation and RNA EMSA revealed the formation of PCBP1-RNA complexes at the element. Knockdown of PCBP1 decreased MOR mRNA half-life and protein expression. Stimulation by forskolin increased cytoplasmic localization of PCBP1 and PCBP1/MOR 3'-UTR interactions via increased serine phosphorylation that was blocked by protein kinase A (PKA) or (phosphatidyl inositol-3) PI3-kinase inhibitors. The forskolin treatment also enhanced serine- and tyrosine-phosphorylation of AU-rich element binding protein (AUF1), concurrent with its increased binding to the CRE, and led to an increased interaction of poly A binding protein (PABP) with the CRE and poly(A) sites. AUF1 phosphorylation also led to an increased interaction with PCBP1. These findings suggest that a single co-regulator, PCBP1, plays a crucial role in stabilizing MOR mRNA, and is induced by PKA signaling by conforming to AUF1 and PABP.