RESUMO
The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which, like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a converging regulator of autophagy and GPCR turnover and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking, and metabolism.
Assuntos
Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Obesidade/metabolismo , Alinhamento de SequênciaRESUMO
The harmful side effects of opioid drugs such as respiratory depression, tolerance, dependence, and abuse potential have limited the therapeutic utility of opioids for their entire clinical history. However, no previous attempt to develop effective pain drugs that substantially ameliorate these effects has succeeded, and the current opioid epidemic affirms that they are a greater hindrance to the field of pain management than ever. Recent attempts at new opioid development have sought to reduce these side effects by minimizing engagement of the regulatory protein arrestin-3 at the mu-opioid receptor, but there is significant controversy around this approach. Here, we discuss the ongoing effort to develop safer opioids and its relevant historical context. We propose a new model that reconciles results previously assumed to be in direct conflict to explain how different signaling profiles at the mu-opioid receptor contribute to opioid tolerance and dependence. Our goal is for this framework to inform the search for a new generation of lower liability opioid analgesics.
Assuntos
Analgésicos Opioides , Transdução de Sinais , Humanos , Analgésicos Opioides/efeitos adversos , Tolerância a MedicamentosRESUMO
Potentiation of glutamate responses is a critical synaptic response to cocaine exposure in ventral tegmental area (VTA) neurons. However, the mechanism by which cocaine exposure promotes potentiation of NMDA receptors (NMDARs) and subsequently AMPA receptors (AMPARs) is not fully understood. In this study we demonstrate that repeated cocaine treatment causes loss of D2 dopamine receptor functional responses via interaction with lysosome-targeting G-protein-associated sorting protein1 (GASP1). We also show that the absence of D2 downregulation in GASP1-KO mice prevents cocaine-induced potentiation of NMDAR currents, elevation of the AMPA/NMDA ratio, and redistribution of NMDAR and AMPAR subunits to the membrane. As a pharmacological parallel, coadministration of the high-affinity D2 agonist, aripiprazole, reduces not only functional downregulation of D2s in response to cocaine but also potentiation of NMDAR and AMPAR responses in wild-type mice. Together these data suggest that functional loss of D2 receptors is a critical mechanism mediating cocaine-induced glutamate plasticity in VTA neurons.
Assuntos
Cocaína/farmacologia , Receptores de AMPA/fisiologia , Receptores de Dopamina D2/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/fisiologia , Animais , Antipsicóticos/farmacologia , Aripiprazol , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Ácido Glutâmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Técnicas de Cultura de Órgãos , Piperazinas/farmacologia , Quinolonas/farmacologia , Receptores de Dopamina D2/agonistas , Potenciais Sinápticos/efeitos dos fármacos , Potenciais Sinápticos/fisiologiaRESUMO
It is well established that dopamine neurons of the ventral tegmental area (VTA) play a critical role in reward and aversion as well as pathologies including drug dependence and addiction. The distinct effects of acute and chronic opioid exposure have been previously characterized at VTA synapses. Recent work suggests that distinct VTA projections that target the medial and lateral shell of the nucleus accumbens (NAc), may play opposing roles in modulating behavior. It is possible that these two anatomically and functionally distinct pathways also have disparate roles in opioid reward, tolerance, and withdrawal in the brain. In this study we monitored dopamine release in the medial or lateral shell of the NAc of male mice during a week-long morphine treatment paradigm. We measured dopamine release in response to an intravenous morphine injection both acutely and following a week of repeated morphine. We also measured dopamine in response to a naloxone injection both prior to and following repeated morphine treatment. Morphine induced a transient increase in dopamine in the medial NAc shell that was much larger than the slower rise observed in the lateral shell. Surprisingly, chronic morphine treatment induced a sensitization of the medial dopamine response to morphine that opposed a diminished response observed in the saline-treated control group. This study expands on our current understanding of the medial NAc shell as hub of opioid-induced dopamine fluctuation. It also highlights the need for future opioid studies to appreciate the heterogeneity of dopamine neurons. Significance Statement: The social and economic consequences of the opioid epidemic are tragic and far-reaching. Yet, opioids are indisputably necessary in clinical settings where they remain the most useful treatment for severe pain. To combat this crisis, we must improve our understanding of opioid function in the brain, particularly the neural mechanisms that underlie opioid dependence and addictive behaviors. This study uses fiber photometry to examine dopamine changes that occur in response to repeated morphine, and morphine withdrawal, at multiple stages of a longitudinal opioid-dependence paradigm. We reveal key differences in how dopamine levels respond to opioid administration in distinct sub-regions of the ventral striatum and lay a foundation for future opioid research that appreciates our contemporary understanding of the dopamine system.
RESUMO
The therapeutic benefits of opioids are compromised by the development of analgesic tolerance, which necessitates higher dosing for pain management thereby increasing the liability for dependence and addiction. Rodent models indicate opposing roles of the gut microbiota in tolerance: morphine-induced gut dysbiosis exacerbates tolerance, whereas probiotics ameliorate tolerance. Not all individuals develop tolerance which could be influenced by differences in microbiota, and yet no study has capitalized upon this natural variation to identify specific features linked to tolerance. We leveraged this natural variation in a murine model of voluntary oral morphine self-administration to elucidate the mechanisms by which microbiota influences tolerance. Although all mice shared similar and predictive morphine-driven microbiota changes that largely masked informative associations with variability in tolerance, our high-resolution temporal analyses revealed a divergence in the progression of dysbiosis that best explained differences in the development in tolerance. Mice that did not develop tolerance also maintained a higher abundance of taxa capable of producing the short-chain fatty acid (SCFA) butyrate, known to bolster intestinal barriers, suppress inflammation, and promote neuronal homeostasis. Furthermore, dietary butyrate supplementation significantly reduced the development of tolerance. These findings could inform immediate therapies to extend the analgesic efficacy of opioids.
RESUMO
Opioid drugs are potent analgesics that mimic the endogenous opioid peptides, endorphins and enkephalins, by activating the µ-opioid receptor. Opioid use is limited by side effects, including significant risk of opioid use disorder. Improvement of the effect/side effect profile of opioid medications is a key pursuit of opioid research, yet there is no consensus on how to achieve this goal. One hypothesis is that the degree of arrestin-3 recruitment to the µ-opioid receptor impacts therapeutic utility. However, it is not clear whether increased or decreased interaction of the µ-opioid receptor with arrestin-3 would reduce compulsive drug-seeking. To examine this question, we utilized three genotypes of mice with varying abilities to recruit arrestin-3 to the µ-opioid receptor in response to morphine in a novel longitudinal operant self-administration model. We demonstrate that arrestin-3 knockout and wild type mice have highly variable drug-seeking behavior with few genotype differences. In contrast, in mice where the µ-opioid receptor strongly recruits arrestin-3, drug-seeking behavior is much less varied. We created a quantitative method to define compulsivity in drug-seeking and found that mice lacking arrestin-3 were more likely to meet the criteria for compulsivity whereas mice with enhanced arrestin-3 recruitment did not develop a compulsive phenotype. Our data suggest that opioids that engage both G protein and arrestin-3, recapitulating the endogenous signaling pattern, will reduce abuse liability.
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Neuropeptides are ubiquitous in the nervous system. Research into neuropeptides has been limited by a lack of experimental tools that allow for the precise dissection of their complex and diverse dynamics in a circuit-specific manner. Opioid peptides modulate pain, reward and aversion and as such have high clinical relevance. To illuminate the spatiotemporal dynamics of endogenous opioid signaling in the brain, we developed a class of genetically encoded fluorescence sensors based on kappa, delta and mu opioid receptors: κLight, δLight and µLight, respectively. We characterized the pharmacological profiles of these sensors in mammalian cells and in dissociated neurons. We used κLight to identify electrical stimulation parameters that trigger endogenous opioid release and the spatiotemporal scale of dynorphin volume transmission in brain slices. Using in vivo fiber photometry in mice, we demonstrated the utility of these sensors in detecting optogenetically driven opioid release and observed differential opioid release dynamics in response to fearful and rewarding conditions.
RESUMO
Dopamine receptors are important for diverse biological functions and are important pharmacological targets in human medicine. Signal transduction from the dopamine receptors is controlled at many levels, including by the process of receptor trafficking. Little is known regarding the endocytic and postendocytic trafficking properties of the D5 dopamine receptor. Here, we show that endocytosis of the D5 receptor can be achieved both homologously, through direct receptor activation by agonist, and also heterologously, due to independent activation of protein kinase C (PKC). In contrast, the D1 receptor is endocytosed only in response to agonist but not PKC activation. We have identified the residue in the third intracellular loop of the D5 receptor that is both necessary for PKC-mediated endocytosis of the D5 receptor and sufficient to induce PKC-mediated endocytosis when introduced to the D1 receptor. In addition, we show that endocytosis of D5 through both pathways is dependent on clathrin and dynamin but that only agonist-induced endocytosis engages ß-arrestin 2. Together, these data show that the D5 receptor shows a trafficking profile distinct from that of any of the other dopamine receptors.
Assuntos
Endocitose/fisiologia , Isoformas de Proteínas/metabolismo , Transporte Proteico/fisiologia , Receptores de Dopamina D5/metabolismo , Transdução de Sinais/fisiologia , Arrestinas/metabolismo , Clatrina/metabolismo , Dopamina/metabolismo , Dinaminas/metabolismo , Endocitose/efeitos dos fármacos , Ativação Enzimática , Células HEK293 , Humanos , Proteína Quinase C/metabolismo , Interferência de RNA , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/genética , Acetato de Tetradecanoilforbol/farmacologia , beta-Arrestina 2 , beta-ArrestinasRESUMO
Drugs targeting G-protein-coupled receptors (GPCRs) make up more than 25% of all prescribed medicines. The ability of GPCRs to form heteromers with unique signaling properties suggests an entirely new and unexplored pool of drug targets. However, current in vitro assays are ill equipped to detect heteromer-selective compounds. We have successfully adapted an approach, using fusion proteins of GPCRs and chimeric G proteins, to create an in vitro screening assay (in human embryonic kidney cells) in which only activated heteromers are detectable. Here we show that this assay can demonstrate heteromer-selective G-protein bias as well as measure transinhibition. Using this assay, we reveal that the δ-opioid receptor agonist ADL5859, which is currently in clinical trials, has a 10-fold higher potency against δ-opioid receptor homomers than δ/µ-opioid receptor heteromers (pEC(50) = 6.7 ± 0.1 versus 5.8 ± 0.2). The assay enables the screening of large compound libraries to identify heteromer-selective compounds that could then be used in vivo to determine the functional role of heteromers and develop potential therapeutic agents.
Assuntos
Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Benzamidas/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio , Clonagem Molecular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Encefalina Leucina/farmacologia , Ensaio de Imunoadsorção Enzimática , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Piperazinas/farmacologia , Reação em Cadeia da Polimerase , Receptores Acoplados a Proteínas G/genética , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides delta/genética , Proteínas Recombinantes de FusãoRESUMO
BACKGROUND: Second-generation antipsychotics (SGAs) are frontline treatments for serious mental illness. Often, individual patients benefit only from some SGAs and not others. The mechanisms underlying this unpredictability in treatment efficacy remain unclear. All SGAs bind the dopamine D3 receptor (D3R) and are traditionally considered antagonists for dopamine receptor signaling. METHODS: Here, we used a combination of two-photon calcium imaging, in vitro signaling assays, and mouse behavior to assess signaling by SGAs at D3R. RESULTS: We report that some clinically important SGAs function as arrestin-3 agonists at D3R, resulting in modulation of calcium channels localized to the site of action potential initiation in prefrontal cortex pyramidal neurons. We further show that chronic treatment with an arrestin-3 agonist SGA, but not an antagonist SGA, abolishes D3R function through postendocytic receptor degradation by GASP1 (G protein-coupled receptor-associated sorting protein-1). CONCLUSIONS: These results implicate D3R-arrestin-3 signaling as a source of SGA variability, highlighting the importance of including arrestin-3 signaling in characterizations of drug action. Furthermore, they suggest that postendocytic receptor trafficking that occurs during chronic SGA treatment may contribute to treatment efficacy.
Assuntos
Antipsicóticos , Dopamina , Camundongos , Animais , beta-Arrestina 2/metabolismo , Antipsicóticos/farmacologia , Receptores de Dopamina D3/metabolismo , Agonistas de Dopamina/farmacologia , Tolerância a Medicamentos , Receptores de Dopamina D1/metabolismoRESUMO
Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.
Assuntos
Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais , Quimiocinas/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citomegalovirus/genética , Citomegalovirus/metabolismo , Endocitose , Humanos , Fosfatos de Inositol/metabolismo , Ligantes , NF-kappa B/metabolismo , Proteínas/metabolismo , Receptores de Quimiocinas/genética , Receptores Acoplados a Proteínas G/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
The D(3) dopamine receptor is endocytosed through a heterologous mechanism mediated by phorbol esters. Here, we show that following this endocytosis the D(3) dopamine receptors fail to recycle and are instead targeted for degradation through an interaction with the G protein-coupled receptor (GPCR)-associated sorting protein-1 (GASP-1). Furthermore, we identified a specific binding motif in the C terminus common to the D(3) and D(2) that confers GASP-1 binding. shRNA knockdown of GASP-1 delayed post-endocytic degradation of both the D(2) and D(3) dopamine receptors. In addition, mutation of the D(2) and D(3) receptor C termini to resemble the D(4), which does not interact with GASP-1, not only inhibited GASP-1 binding but slowed degradation after endocytosis. Conversely, mutation of the C terminus of the D(4) to resemble that of the D(2) and D(3) facilitated GASP-1 binding and promoted post-endocytic degradation of the mutant D(4) receptor. Thus, we have identified a motif that is both necessary and sufficient to promote GASP-1 binding and receptor degradation. In addition, these data demonstrated that GASP-1 can mediate post-endocytic degradation of dopamine receptors that have been endocytosed not only as a consequence of dopamine activation but also as a consequence of activation by phorbol esters.
Assuntos
Endocitose/fisiologia , Transporte Proteico/fisiologia , Proteínas/metabolismo , Receptores de Dopamina D3/metabolismo , Sequência de Aminoácidos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Endocitose/efeitos dos fármacos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Mutagênese , Ésteres de Forbol/farmacologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas/genética , RNA Interferente Pequeno , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/química , Receptores de Dopamina D3/genética , Receptores de Dopamina D4/química , Receptores de Dopamina D4/genética , Receptores de Dopamina D4/metabolismoRESUMO
The bioconjugation of organometallic complexes with peptides has proven to be a novel approach for drug discovery. We report the facile and chemoselective reaction of tyrosine-containing G-protein-coupled receptor (GPCR) peptides with [Cp*Rh(H(2)O)(3)](OTf)(2), in water, at room temperature, and at pH 5-6. We have focused on three important GPCR peptides; namely, [Tyr(1)]-leu-enkephalin, [Tyr(4)]-neurotensin(8-13), and [Tyr(3)]-octreotide, each of which has a different position for the tyrosine residue, together with competing functionalities. Importantly, all other functional groups present, i.e., amino, carboxyl, disulfide, phenyl, and indole, were not prominent sites of reactivity by the Cp*Rh tris aqua complex. Furthermore, the influence of the Cp*Rh moiety on the structure of [Tyr(3)]-octreotide was characterized by 2D NMR, resulting in the first representative structure of an organometallic-peptide complex. The biological consequences of these Cp*Rh-peptide complexes, with respect to GPCR binding and growth inhibition of MCF7 and HT29 cancer cells, will be presented for [(η(6)-Cp*Rh-Tyr(1))-leu-enkephalin](OTf)(2) and [(η(6)-Cp*Rh-Tyr(3))-octreotide](OTf)(2).
Assuntos
Modelos Moleculares , Compostos Organometálicos/química , Peptídeos/química , Receptores Acoplados a Proteínas G/química , Ródio/química , Tirosina/química , Ligação Competitiva , Neoplasias da Mama/tratamento farmacológico , Feminino , Células HT29 , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Compostos Organometálicos/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Chronic treatment of pain with opiate drugs can lead to analgesic tolerance and drug dependence. Although all opiate drugs can promote tolerance and dependence in practice, the severity of those unwanted side effects differs depending on the drug used. Although each opiate drug has its own unique set of pharmacological profiles, methadone is the only clinically used opioid drug that produces substantial receptor endocytosis at analgesic doses. Here, we examined whether moderate doses of methadone carry any benefits over chronic use of equianalgesic morphine, the prototypical opioid. Our data show that chronic administration of methadone produces significantly less analgesic tolerance than morphine. Furthermore, we found significantly reduced precipitated withdrawal symptoms after chronic methadone treatment than after chronic morphine treatment. Finally, using a novel animal model with a degrading µ-opioid receptor we showed that, although endocytosis seems to protect against tolerance development, endocytosis followed by receptor degradation produces a rapid onset of analgesic tolerance to methadone. Together, these data indicated that opioid drugs that promote receptor endocytosis and recycling, such as methadone, may be a better choice for chronic pain treatment than morphine and its derivatives that do not.
Assuntos
Metadona/farmacologia , Metadona/uso terapêutico , Morfina/farmacologia , Morfina/uso terapêutico , Dor/prevenção & controle , Analgésicos Opioides , Animais , Área Sob a Curva , Relação Dose-Resposta a Droga , Tolerância a Medicamentos/fisiologia , Endocitose/fisiologia , Técnicas de Introdução de Genes , Metadona/administração & dosagem , Metadona/economia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfina/administração & dosagem , Morfina/economia , Mutação/fisiologia , Naloxona/farmacologia , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Síndrome de Abstinência a Substâncias/diagnóstico , Transtornos Relacionados ao Uso de Substâncias/diagnósticoRESUMO
Opioids and their receptors have an important role in analgesia and alcohol and substance use disorders (ASUD). We have identified several naturally occurring amino acid changing variants of the human mu-opioid receptor (MOR), and assessed the functional consequences of these previously undescribed variants in stably expressing cell lines. Several of these variants had altered trafficking and signaling properties. We found that an L85I variant showed significant internalization in response to morphine, in contrast to the WT MOR, which did not internalize in response to morphine. Also, when L85I and WT receptor were coexpressed, WT MOR internalized with the L85I MOR, suggesting that, in the heterozygous condition, the L85I phenotype would be dominant. This finding is potentially important, because receptor internalization has been associated with development of tolerance to opiate analgesics. In contrast, an R181C variant abolished both signaling and internalization in response to saturating doses of the hydrolysis-resistant enkephalin [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO). Coexpression of the R181C and WT receptor led to independent trafficking of the 2 receptors. S42T and C192F variants showed a rightward shift in potency of both morphine and DAMGO, whereas the S147C variant displayed a subtle leftward shift in morphine potency. These data suggest that these and other such variants may have clinical relevance to opioid responsiveness to both endogenous ligands and exogenous drugs, and could influence a broad range of phenotypes, including ASUD, pain responses, and the development of tolerance to morphine.
Assuntos
Mutação , Receptores Opioides mu/fisiologia , Substituição de Aminoácidos , Analgésicos Opioides/farmacologia , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Variação Genética , Humanos , Imuno-Histoquímica , Microscopia Confocal , Morfina/farmacologia , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , TrítioRESUMO
GABA transmission in the ventral tegmental area (VTA) is critical for fine tuning the activity of dopamine neurons in response to opioids. However, the precise mechanism by which GABA input shapes opioid reward is poorly understood. We observed a reduction of conditioned place preference for low doses of the opioid [d-Ala2, N-MePhe4, Gly5-ol]-enkephalin (DAMGO) and a switch in the functional effects of µ-opioid receptor modulation of GABA postsynaptic currents in the mouse VTA 1 d after chronic morphine treatment. Specifically, whereas in naive mice DAMGO inhibits GABA postsynaptic currents, GABAergic currents are potentiated by DAMGO after chronic morphine treatment. Importantly, pretreatment with the cAMP signaling inhibitor (R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium both restored DAMGO reward and reversed the DAMGO-mediated potentiation, thereby reestablishing the inhibitory effects of opioids on GABA currents. Thus, a paradoxical bidirectionality in µ-receptor-mediated control of GABA transmission following chronic morphine treatment is a critical mechanism that determines the expression of opioid reward in the VTA.
Assuntos
Analgésicos Opioides/farmacologia , Morfina/farmacologia , Recompensa , Área Tegmentar Ventral/efeitos dos fármacos , Ácido gama-Aminobutírico/fisiologia , Analgésicos Opioides/administração & dosagem , Animais , Condicionamento Operante/efeitos dos fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/farmacologia , AMP Cíclico/fisiologia , Eletrofisiologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/administração & dosagem , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfina/administração & dosagem , Receptores Opioides mu/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Tionucleotídeos/farmacologiaRESUMO
Chronic morphine drives adaptations in synaptic transmission thought to underlie opiate dependence. Here we examine the role of micro-opioid receptor (MOR) trafficking in one of these adaptations, specifically, changes in GABA transmission in the ventral tegmental area (VTA). To address this question, we used a knock-in mouse, RMOR (for recycling MOR), in which genetic change in the MOR promotes morphine-induced receptor desensitization and endocytosis in GABA interneurons of the VTA. In wild-type mice (postnatal days 23-28) chronic morphine (10 mg/kg, s.c., twice daily for 5 d), induced a cAMP-dependent increase in the probability of GABA release onto VTA dopamine neurons. The increased GABA release frequency correlated with physical dependence on morphine measured by counting somatic signs of morphine withdrawal, such as, tremors, jumps, rears, wet-dog shakes, and grooming behavior precipitated by subcutaneous administration of naloxone (NLX) (2 mg/kg). This adaptation in GABA release was prevented in RMOR mice given the same morphine treatment, implicating MOR trafficking in this morphine-induced change in plasticity. Importantly, treatment with the cAMP activity inhibitor rp-cAMPS [(R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium] (50 ng/0.5 microl), directly to the VTA, attenuated somatic withdrawal signs to systemic morphine produced by intra-VTA NLX (500 ng/0.5 microl), directly tying enhanced cAMP-driven GABA release to naloxone-precipitated morphine withdrawal in the VTA.
Assuntos
Dependência de Morfina/metabolismo , Receptores Opioides mu/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/fisiologia , Animais , Quimera , AMP Cíclico/análogos & derivados , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Modelos Animais de Doenças , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Técnicas de Introdução de Genes , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfina/farmacologia , Dependência de Morfina/genética , Dependência de Morfina/fisiopatologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Entorpecentes/farmacologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Síndrome de Abstinência a Substâncias/genética , Transmissão Sináptica/fisiologia , Tionucleotídeos/farmacologia , Área Tegmentar Ventral/metabolismoRESUMO
Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence.
Assuntos
Tolerância a Medicamentos , Endocitose/efeitos dos fármacos , Dependência de Morfina/metabolismo , Morfina/farmacologia , Receptores Opioides mu/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Síndrome de Abstinência a Substâncias/metabolismoRESUMO
Heteromerization of opioid receptors has been shown to alter opioid receptor pharmacology. However, how receptor heteromerization affects the processes of endocytosis and postendocytic sorting has not been closely examined. This question is of particular relevance for heteromers of the µ-opioid receptor (MOR) and δ-opioid receptor (DOR), because the MOR is recycled primarily after endocytosis and the DOR is degraded in the lysosome. Here, we examined the endocytic and postendocytic fate of MORs, DORs, and DOR/MOR heteromers in human embryonic kidney 293 cells stably expressing each receptor alone or coexpressing both receptors. We found that the clinically relevant MOR agonist methadone promotes endocytosis of MOR but also the DOR/MOR heteromer. Furthermore, we show that DOR/MOR heteromers that are endocytosed in response to methadone are targeted for degradation, whereas MORs in the same cell are significantly more stable. It is noteworthy that we found that the DOR-selective antagonist naltriben mesylate could block both methadone- and [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin-induced endocytosis of the DOR/MOR heteromers but did not block signaling from this heteromer. Together, our results suggest that the MOR adopts novel trafficking properties in the context of the DOR/MOR heteromer. In addition, they suggest that the heteromer shows "biased antagonism," whereby DOR antagonist can inhibit trafficking but not signaling of the DOR/MOR heteromer.
Assuntos
Endocitose/efeitos dos fármacos , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides delta/metabolismo , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismo , Animais , Cálcio/metabolismo , Células HEK293 , Humanos , Ligantes , Metadona/metabolismo , Metadona/farmacologia , Camundongos , Naltrexona/análogos & derivados , Naltrexona/metabolismo , Naltrexona/farmacologia , Transporte Proteico , Receptores Opioides delta/agonistas , Receptores Opioides delta/genética , Receptores Opioides mu/agonistas , Receptores Opioides mu/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The role of µ-opioid receptor (MOR) down-regulation in opioid tolerance remains controversial. In this study, we used a novel knock-in mouse to examine how changing the extent of MOR down-regulation alters the development of morphine tolerance. These mice express a mutant MOR, degrading MOR (DMOR), that differs from the wild-type (WT) MOR in two ways: 1) unlike the recycling WT MOR, the mutant DMOR is targeted for degradation after its internalization, thus facilitating down-regulation; and 2) unlike the WT MOR, DMOR is efficiently internalized in response to morphine activation. We found that both WT MOR and DMOR mice develop tolerance to morphine, but DMOR mice exhibit a more rapid onset of tolerance and show receptor down-regulation. WT MOR mice develop morphine tolerance more slowly but even once profoundly tolerant show no receptor down-regulation. Furthermore, WT mice show significantly more morphine dependence than DMOR mice after long-term treatment as indicated by withdrawal. Taken together these data indicate that tolerance mediated by receptor down-regulation manifests differently both at the behavioral and biochemical level than does the actual morphine tolerance that occurs in WT mice and that loss of receptor function is not a major contributor to morphine tolerance in WT MOR mice.