RESUMEN
Cholangiopathies encompass a heterogeneous group of disorders affecting biliary epithelial cells (i.e. cholangiocytes). Early diagnosis, prognosis and treatment still remain clinically challenging for most of these diseases and are critical for adequate patient care. In the past decade, extensive research has emphasized microRNAs (miRs) as potential non-invasive biomarkers and tools to accurately identify, predict and treat cholangiopathies. MiRs can be released extracellularly conjugated with lipoproteins or encapsulated in extracellular vesicles (EVs). Research on EVs is also gaining attention since they are present in multiple biological fluids and may represent a relevant source of novel non-invasive biomarkers and be vehicles for new therapeutic approaches. This review highlights the most promising candidate miRs and EV-related biomarkers in cholangiopathies, as well as their relevant roles in biliary pathophysiology. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen. RESEARCH STRATEGY: PubMed search (April 2017) was done with the following terms: "microRNA", "miRNA", "miR", "extracellular vesicles", "EV", "exosomes", "primary biliary cholangitis", "primary biliary cholangitis", "PBC", "primary sclerosing cholangitis", "PSC", "cholangiocarcinoma", "CCA", "biliary atresia", "BA", "polycystic liver diseases", "PLD", "cholangiopathies", "cholestatic liver disease". Most significant articles in full-text English were selected. The reference lists of selected papers were also considered.
Asunto(s)
Enfermedades de los Conductos Biliares/diagnóstico , Conductos Biliares/patología , Células Epiteliales/patología , Vesículas Extracelulares/metabolismo , MicroARNs/metabolismo , Animales , Enfermedades de los Conductos Biliares/etiología , Enfermedades de los Conductos Biliares/patología , Conductos Biliares/citología , Conductos Biliares/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Humanos , MicroARNs/análisisRESUMEN
Polycystic liver diseases (PLDs) include a heterogeneous group of congenital disorders inherited as dominant or recessive genetic traits; they are manifested alone or in association with polycystic kidney disease. Ductal plate malformation during embryogenesis and the loss of heterozygosity linked to second-hit mutations may promote the dilatation and/or development of a large number (> 20) of biliary cysts, which are the main cause of morbidity in these patients. Surgical procedures aimed to eliminate symptomatic cysts show short-term beneficial effects, but are not able to block the disease progression. Therefore, liver transplantation is the only curative option. Intense studies on the molecular mechanisms involved in the pathogenesis of PLDs have resulted in different clinical trials, some of them with promising outcomes. Here the authors summarize the key aspects of PLD etiology, pathogenesis, and therapy, highlighting the most recent advances and future research directions.
Asunto(s)
Quistes , Hepatopatías , Quistes/genética , Quistes/patología , Quistes/terapia , Progresión de la Enfermedad , Humanos , Hepatopatías/genética , Hepatopatías/patología , Hepatopatías/terapia , Trasplante de Hígado , Mutación , FenotipoRESUMEN
Treatment of chronic pain has created a "silent epidemic," a term that describes the serious public health problem of the abuse of opioid painkillers and other prescription drugs. Conventional pharmacotherapy is limited by the loss of effectiveness in the long-term and by potentially lethal side effects. Efforts need to be focused on the development of nonpharmacological approaches. As significant progress is made in the viral vector technology, gene therapy involving recombinant viruses as vehicles may become a viable alternative for treatment of severe pain. Virus-based gene therapy has several advantages: (1) the transfer of a therapeutic gene to produce/release bioactive therapeutic molecules in a specific location in the nervous system thus minimizing the risks of off-target side effects, and (2) sustained long-term production of the therapeutic agent. This review compiles recently developed strategies for gene therapy targeting specific mechanisms of specific chronic pain conditions. A few successful studies on animal models of chronic pain have been translated to human clinical trials.
Asunto(s)
Dolor Crónico/genética , Dolor Crónico/terapia , Terapia Genética , Analgésicos Opioides/uso terapéutico , Animales , Dolor Crónico/complicaciones , Dolor Crónico/fisiopatología , Humanos , Inflamación/complicaciones , Inflamación/patología , Neuralgia/complicaciones , Neuralgia/genética , Neuralgia/fisiopatología , Neuralgia/terapia , NocicepciónRESUMEN
AIMS: To investigate plasmid-mediated fosfomycin resistance related to fosA3 in Escherichia coli isolates collected from different animals in Hong Kong, China, 2008-2010. METHODS AND RESULTS: In total, 2106 faecal specimens from 210 cattle, 214 pigs, 460 chickens, 398 stray cats, 368 stray dogs and 456 wild rodents were cultured. The faecal colonization rates of fosfomycin-resistant E. coli were as follows: 11.2% in pigs, 8.6% in cattle, 7.3% in chickens, 2.4% in dogs, 0.8% in cats and 1.5% in rodents. The cultures yielded 1693 isolates of which 831 were extended-spectrum ß-lactamases (ESBL) producers. Fosfomycin-resistant isolates were more likely than fosfomycin-susceptible isolates to be producers of ESBL and to have resistance to chloramphenicol, ciprofloxacin, cotrimoxazole, gentamicin and tetracycline. Of the 101 fosfomycin-resistant isolates, 97 (96.0%) isolates were fosA3 positive and 94 (93.1%) were bla(CTX) (-M) positive. PCR mapping showed that the fosA3-containing regions were flanked by IS26, both upstream and downstream in 81 (83.5%) isolates, and by an upstream bla(CTX-M-14) -containing transposon-like structure (ΔISEcp1-bla(CTX-M-14) -ΔIS903 or ISEcp1-IS10 -bla(CTX-M-14) -ΔIS903) and a downstream IS26 in 14 (14.4%) isolates. For the remaining two isolates, fosA3 was flanked by a downstream IS26 but the upstream part cannot be defined. In a random subset of 18 isolates, fosA3 was carried on transferable plasmids with sizes of 50-200 kb and the following replicons: F2:A-B- (n = 3), F16:A1:B- (n = 2), F24:A-B- (n = 1), N (n = 1), B/O (n = 1) and untypeable (n = 3). SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the emergence of fosA3-mediated fosfomycin resistance among multidrug-resistant E. coli isolates from various animals. IS26 transposon-like structures might be the main vehicles for dissemination of fosA3.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Escherichia coli/veterinaria , Escherichia coli/efectos de los fármacos , Fosfomicina/farmacología , Animales , Gatos , Bovinos , Pollos , Elementos Transponibles de ADN , ADN Bacteriano/genética , Perros , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Heces/microbiología , Hong Kong , Ganado , Pruebas de Sensibilidad Microbiana , Plásmidos , Reacción en Cadena de la Polimerasa , Roedores , Porcinos , beta-Lactamasas/genéticaRESUMEN
Direct injection of double-stranded adeno-associated virus type 2 (dsAAV2) with a mu-opioid receptor (MOR) mutant [S4.45(196)A], and a reporter protein (enhanced green fluorescent protein) into the spinal cord (S2/S3) dorsal horn region of ICR mice resulted in antinociceptive responses to systemic injection of opioid antagonist naloxone without altering the acute agonist morphine responses and no measurable tolerance or dependence development during subchronic naloxone treatment. To develop further such mutant MORs into a therapeutic agent in pain management, a less invasive method for virus delivery is needed. Thus, in current studies, the dsAAV2 was locally injected into the subarachnoid space of the spinal cord by intrathecal administration. Instead of using the MORS196A mutant, we constructed the dsAAV2 vector with the MORS196ACSTA mutant, a receptor mutant in which naloxone has been shown to exhibit full agonistic properties in vitro. After 2 weeks of virus injection, naloxone (10 mg/kg s.c.) elicited antinociceptive effect (determined by tail-flick test) without tolerance (10 mg/kg s.c., b.i.d. for 6 days) and significant withdrawal symptoms. On the other hand, subchronic treatment with morphine (10 mg/kg s.c., b.i.d.) for 6 days induced significant tolerance (4.8-fold) and withdrawal symptoms. Furthermore, we found that morphine, but not naloxone, induced the rewarding effects (determined by conditioned place preference test). These data suggest that local expression of MORS196ACSTA in spinal cord and systemic administration of naloxone has the potential to be developed into a new strategy in the management of pain without addiction liability.
Asunto(s)
Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Manejo del Dolor , Dimensión del Dolor/efectos de los fármacos , Receptores Opioides mu/efectos de los fármacos , Receptores Opioides mu/genética , Animales , Condicionamiento Operante/efectos de los fármacos , Tolerancia a Medicamentos , Técnicas de Transferencia de Gen , Terapia Genética , Inyecciones Espinales , Masculino , Ratones , Ratones Endogámicos ICR , Mutación , Trastornos Relacionados con Opioides/psicología , Recompensa , Espacio SubaracnoideoRESUMEN
We previously reported that mutations in the mu-opioid receptor (MOR), S196L or S196A, rendered MOR responsive to the opioid antagonist naloxone without altering the agonist phenotype. Subsequently, a mouse strain carrying the S196A mutation exhibited in vivo naloxone antinociceptive activity without the development of tolerance. In this study we investigated the possibility of combining the in vivo site-directed delivery of MORS196A and systemic naloxone administration as a paradigm for pain management. Double-stranded adenoassociated virus type 2 (dsAAV2) was used to deliver MORS196A-EGFP by injecting the virus into the spinal cord (S2/S3) dorsal horn region of ICR mice. MORS196A-EGFP fluorescence colocalized with some calcitonin gene-related peptide and neuron-specific protein immunoreactivity in the superficial layers of the dorsal horn 1 week after injection and lasted for at least 6 months. In mice injected with the mutant receptor, morphine induced similar antinociceptive responses and tolerance development or precipitated withdrawal symptoms and reward effects, similar to those in the control mice (saline injected into the spinal cord). Conversely, in the dsAAV2-injected mice, naloxone produced antinociceptive effects at the spinal level but not at the supraspinal level, whereas naloxone had no measurable effect on the control mice. Furthermore, the chronic administration of naloxone to mice injected with dsAAV2-MORS196A-EGFP did not induce tolerance, dependence, or reward responses. Thus, our current approach to activate a mutant receptor, but not the endogenous receptor, with an opioid antagonist represents an alternative to the use of traditional opioid agonists for pain management.
Asunto(s)
Dependovirus/genética , Genes Reporteros/genética , Dimensión del Dolor , Receptores Opioides mu/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Animales , Dependovirus/clasificación , Tolerancia a Medicamentos , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Masculino , Ratones , Morfina/uso terapéutico , Mutación/genética , Naloxona/farmacología , Dolor/tratamiento farmacológico , Dolor/genética , Dolor/metabolismo , Receptores Opioides mu/genética , Serina/genética , Serina/metabolismo , Factores de TiempoRESUMEN
Glucose transporter type 1 deficiency syndrome (Glut1DS) is the result of autosomal-dominant loss-of-function mutation of the glucose transporter type 1 gene (GLUT1) leading to brain energy failure and epileptic encephalopathy. In this study, the protein products of the Glut1DS-associated GLUT1 missense mutations, S66F, R126C, and T295M, were characterized using the Glut1-green fluorescent protein (GFP) fusion expressed in CHO cells. Glut1-GFP expression was confirmed by Western blot and confocal microscopy. The applicability of this Glut1-GFP expression model in reporting Glut1 functional deficits was validated by re-confirming the glucose transport defects of the previously reported pathogenic mutations R126H, R126L, and R333W. While S66F, R126C, and T295M mutants were expressed and targeted to the cell membrane, these Glut1 mutants have significantly diminished membrane association and glucose transport activity (p<0.05) relative to the wild-type Glut1 protein. Consistent with the reduced Glut1 membrane association, glucose transport kinetics studies showed that S66F, R126C, and T295M mutants have significantly reduced (p<0.05) Vmax but not Km. Thus, Glut1 single amino acid substitute mutants S66F, R126C, and T295M impair glucose transport function and constitute Glut1-deficiency states in vitro. These results support the pathogenicity of Glut1 S66F, R126C, and T295M in vivo.
Asunto(s)
Sustitución de Aminoácidos , Errores Innatos del Metabolismo de los Carbohidratos/genética , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Mutación , Animales , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Glucosa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , HumanosRESUMEN
Long-term opioid agonist treatment results in adenylyl cyclase superactivation. A recent "RAVE" theory implicates a direct correlation between the ability of agonist to induce receptor internalization and the magnitude of adenylyl cyclase superactivation. We decided to test such a theory by examining the adenylyl cyclase superactivation after long-term activation of mu-opioid receptor (MOR) in an EcR293 cell model. We examined the magnitudes of adenylyl cyclase superactivation in the presence of naloxone after long-term treatment with morphine, etorphine, and methadone, three agonists reported to have differential activities in promoting MOR internalization. It can be shown that the magnitudes of adenylyl cyclase superactivation after treating with these three agonists, although different, were dependent on MOR density. Blunting MOR internalization with the dominant-negative mutant of dynamin, K44E, did not alter the magnitude of either morphine- or etorphine-induced adenylyl cyclase superactivation. In the presence of diprenorphine, the magnitude of adenylyl cyclase superactivation after etorphine treatment was identical to that observed with morphine. It could be demonstrated further that adenylyl cyclase superactivation is dependent on the cell surface-located MOR. Sucrose gradient fractionation demonstrated the colocalization of MOR and adenylyl cyclase V/VI with caveolin-1, a marker for lipid rafts. After long-term agonist treatment, the majority of MOR remained at the lipid rafts. Methyl-beta-cyclodextrin (MbetaCD) completely blunted the adenylyl cyclase superactivation and agonist-induced receptor internalization. These MbetaCD actions were reversed by incubating the cells with cholesterol. Thus, the adenylyl cyclase superactivation is not dependent on agonist-induced receptor internalization. Rather, the location of MOR at lipid rafts is an absolute requirement for the observed adenylyl cyclase superactivation.
Asunto(s)
Adenilil Ciclasas/metabolismo , Metabolismo de los Lípidos , Receptores Opioides mu/agonistas , Animales , AMP Cíclico/metabolismo , Endocitosis , Activación Enzimática , Ratas , Receptores Opioides mu/metabolismoRESUMEN
The phosphatidylinositol 3-kinase-Akt/protein kinase B (PKB) survival signaling is very important for cancer cell survival and growth. Constitutively active phosphatidylinositol 3-kinase-Akt/PKB signaling in small cell lung cancer (SCLC) is a major factor for the survival of SCLC cells. Inhibitors of this signaling pathway would be potential antitumor agents, particularly for SCLC. Here we report that naltrindole, which has been used as a classic delta opioid antagonist, inhibited growth and induced apoptosis in the three characteristic SCLC cell lines, NCI-H69, NCI-H345, and NCI-H510. Naltrindole treatment reduced constitutive phosphorylation of Akt/PKB on serine 473 and threonine 308 in cells. We found that the levels of constitutive phosphorylation of Akt/PKB on serine 473 correlate with the sensitivity of the three cell lines to naltrindole treatment. Furthermore, naltrindole treatment not only reduced the phosphorylation of the Akt/PKB upstream kinase phosphoinositide-dependent kinase-1, but also its downstream effectors glycogen synthase kinase-3beta and the Forkhead transcription factors AFX and FKHR. DNA array analysis of 205 apoptosis-related genes indicated that some Akt/PKB-dependent genes were either up- or down-regulated by naltrindole. Flow cytometric and microscopic analyses clearly showed that naltrindole induced apoptosis in SCLC cells. RNA interference experiments confirmed that naltrindole-induced cell death was associated with the Akt/PKB survival pathway. Together, these results show that naltrindole is a new inhibitor of the Akt/PKB signaling pathway, suggesting that naltrindole could be a potential lead for the development of a new type of inhibitors that target the constitutively active Akt/PKB signaling-dependent SCLC cells.
Asunto(s)
Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/enzimología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Naltrexona/análogos & derivados , Naltrexona/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Sitios de Unión , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Drug addiction has great social and economical implications. In order to resolve this problem, the molecular and cellular basis for drug addiction must be elucidated. For the past three decades, our research has focused on elucidating the molecular mechanisms behind morphine tolerance and dependence. Although there are many working hypotheses, it is our premise that cellular modulation of the receptor signaling, either via transcriptional or post-translational control of the receptor, is the basis for morphine tolerance and dependence. Thus, in the current review, we will summarize our recent work on the transcriptional and post-translational control of the opioid receptor, with special emphasis on the mu-opioid receptor, which is demonstrated to mediate the in vivo functions of morphine.
Asunto(s)
Narcóticos/farmacología , Trastornos Relacionados con Opioides/genética , Trastornos Relacionados con Opioides/fisiopatología , Receptores Opioides/biosíntesis , Receptores Opioides/metabolismo , Receptores Opioides/fisiología , Transcripción Genética/genética , Animales , Tolerancia a Medicamentos , Humanos , Ratones , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Opioides/efectos de los fármacos , Receptores Opioides/genética , Transducción de Señal/efectos de los fármacosRESUMEN
The mechanism for the differential regulation of the mu-opioid receptor by agonists is investigated by identifying the receptor domains used to define the relative efficacies of three mu-opioid receptor-selective agonists: [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), morphine, and [N-MePhe3,D-Pro4]-morphiceptin (PL017) to inhibit forskolin-stimulated intracellular cAMP production in human embryonic kidney 293 cells. This was accomplished by systematically deleting four to five amino acids clusters within the third intracellular loop of rat mu-opioid receptor, Arg258 to Arg280, followed by Ala substitution and scanning studies of the 276RRITR280 sequence, the putative G protein-coupling motif. Deletion of the four to five amino acid clusters resulted in differential effects on the affinities of the agonists and antagonists, and also on the potencies and coupling efficiencies of the three opioid agonists. Ala scanning studies of the 276RRITR280 sequence revealed also the differences between [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), morphine, and PL017. Substitution of Arg276 or Ile278 with Ala reduced the potency of DAMGO but not that of morphine PL017. Meanwhile, mutation of Thr279 to Ala increased the potencies of morphine and PL017 but not that of DAMGO. The I278A mutation decreased the DAMGO coupling efficiency but increased the PL017 coupling efficiency. The R280A mutation resulted in the increase in PL017 potency and coupling efficiency without altering those of DAMGO and morphine. Thus, these mutation studies suggested that the activation of mu-opioid receptor and interaction between the critical domains such as RRITR within third intracellular loop and the G proteins are agonist-selective.
Asunto(s)
Analgésicos Opioides/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Proteínas de la Membrana/metabolismo , Receptores Opioides mu/metabolismo , Adenilil Ciclasas/metabolismo , Alanina/metabolismo , Células Cultivadas , Encefalinas/farmacología , Eliminación de Gen , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Morfina/farmacología , Antagonistas de Narcóticos/farmacología , Toxina del Pertussis/farmacología , Estructura Terciaria de Proteína , Receptores Opioides mu/química , Receptores Opioides mu/efectos de los fármacos , Receptores Opioides mu/genéticaRESUMEN
Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named 'reporter affinity labels' because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.
Asunto(s)
Marcadores de Afinidad , Receptores Opioides/efectos de los fármacos , Sitios de Unión , HumanosAsunto(s)
Marcadores de Afinidad/síntesis química , Colorantes Fluorescentes/síntesis química , Naltrexona/síntesis química , Receptores Opioides delta/agonistas , Marcadores de Afinidad/metabolismo , Marcadores de Afinidad/farmacología , Analgésicos/síntesis química , Analgésicos/metabolismo , Analgésicos/farmacología , Animales , Células CHO , Cricetinae , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/farmacología , Cobayas , Íleon/efectos de los fármacos , Íleon/fisiología , Técnicas In Vitro , Ligandos , Masculino , Ratones , Modelos Moleculares , Naltrexona/análogos & derivados , Naltrexona/metabolismo , Naltrexona/farmacología , Receptores Opioides delta/metabolismo , Conducto Deferente/efectos de los fármacos , Conducto Deferente/fisiologíaRESUMEN
Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between mu-opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. Among those 12 Ser/Thr residues, Ser(363), Thr(370), and Ser(375) have been identified as phosphorylation sites. In the absence of the agonist, a basal phosphorylation of Ser(363) and Thr(370) was observed, whereas [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO)-induced receptor phosphorylation occurs at Thr(370) and Ser(375) residues. Furthermore, the role of these phosphorylation sites in regulating the internalization of MOR was investigated. The mutation of Ser(375) to Ala reduced the rate and extent of receptor internalization, whereas mutation of Ser(363) and Thr(370) to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though mu-receptors and delta-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.
Asunto(s)
Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Serina , Treonina , Alanina , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Diprenorfina/farmacocinética , Endocitosis , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfatos/metabolismo , Fosforilación , Fosfoserina , Fosfotreonina , Ratas , Receptores Opioides mu/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Opioid receptors often couple to multiple effectors within the same cell. To examine potential mechanisms that contribute to the specificity by which delta-receptors couple to distinct intracellular effectors, we stably transfected rat pituitary GH(3) cells with cDNAs encoding for delta-opioid receptors. In cells transfected with a relatively low delta-receptor density of 0.55 pmol/mg of protein (GH(3)DOR), activation of delta-receptors produced inhibition of adenylyl cyclase activity but was unable to alter L-type Ca(2+) current. In contrast, activation of delta-receptors in a clone that contained a higher density of delta-receptors (2.45 pmol/mg of protein) and was also coexpressed with mu-opioid receptors (GH(3)MORDOR), resulted in not only the expected inhibition of adenylyl cyclase activity but also produced inhibition of L-type Ca(2+) current. The purpose of the present study was to determine whether these observations resulted from differences in delta-opioid receptor density between clones or interaction between delta- and mu-opioid receptors to allow the activation of different G proteins and signaling to Ca(2+) channels. Using the delta-opioid receptor alkylating agent SUPERFIT, reduction of available delta-opioid receptors in GH(3)MORDOR cells to a density similar to that of delta-opioid receptors in the GH(3)DOR clone resulted in abolishment of coupling to Ca(2+) channels, but not to adenylyl cyclase. Furthermore, although significantly greater amounts of all G proteins were activated by delta-opioid receptors in GH(3)MORDOR cells, delta-opioid receptor activation in GH(3)DOR cells resulted in coupling to the identical pattern of G proteins seen in GH(3)MORDOR cells. These findings suggest that different threshold densities of delta-opioid receptors are required to activate critical amounts of G proteins needed to produce coupling to specific effectors and that delta-opioid receptors couple more efficiently to adenylyl cyclase than to L-type Ca(2+) channels.
Asunto(s)
Adenilil Ciclasas/metabolismo , Canales de Calcio Tipo L/metabolismo , Fentanilo/análogos & derivados , Naltrexona/análogos & derivados , Hipófisis/metabolismo , Receptores Opioides delta/metabolismo , Toxina de Adenilato Ciclasa , Inhibidores de Adenilato Ciclasa , Alquilantes/farmacología , Analgésicos Opioides/farmacología , Animales , Bario/metabolismo , Bario/fisiología , Encefalina D-Penicilamina (2,5)/farmacología , Fentanilo/farmacología , Proteínas de Unión al GTP Heterotriméricas/biosíntesis , Proteínas de Unión al GTP Heterotriméricas/fisiología , Canales Iónicos/fisiología , Naltrexona/farmacología , Hipófisis/citología , Hipófisis/efectos de los fármacos , Ratas , Receptores Opioides delta/genética , Receptores Opioides delta/fisiología , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Receptores Opioides mu/fisiología , Transfección , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.
Asunto(s)
Receptores Opioides delta/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Línea Celular , Encefalina D-Penicilamina (2,5)/farmacología , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Receptores Opioides delta/genética , Serina/metabolismo , Relación Estructura-Actividad , Treonina/metabolismoRESUMEN
Previously, we reported that the time course for the rapid phosphorylation rate of mu-opioid receptor expressed in human embryonic kidney (HEK)293 cells did not correlate with the slow receptor desensitization rate induced by [D-Ala(2),N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO). However, others have suggested that receptor phosphorylation is the trigger for mu-opioid receptor desensitization. In this study, we demonstrated the relatively slow rate of receptor desensitization could be attributed partially to the recycling of internalized receptor as determined by fluorescence-activated cell-sorting analysis. However, the blockade of the endocytic and Golgi transport events in HEK293 cells with monensin and brefeldin A did not increase the initial rate of receptor desensitization. But the desensitization rate was increased by reduction of the mu-opioid receptor level with beta-furnaltrexamine (betaFNA). The reduction of the receptor level with 1 microM betaFNA significantly increased the rate of etorphine-induced receptor desensitization. By blocking the ability of receptor to internalize with 0.4 M sucrose, a significant degree of receptor being rapidly desensitized was observed in HEK293 cells pretreated with betaFNA. Hence, mu-opioid receptor is being resensitized during chronic agonist treatment. The significance of resensitization of the internalized receptor in affecting receptor desensitization was demonstrated further with human neuroblastoma SHSY5Y cells that expressed a low level of mu-opioid receptor. Although DAMGO could not induce a rapid desensitization in these cells, in the presence of monensin and brefeldin A, DAMGO desensitized the mu-opioid receptor's ability to regulate adenylyl cyclase with a t(1/2) = 9.9 +/- 2.1 min and a maximal desensitized level at 70 +/- 4.7%. Furthermore, blockade of receptor internalization with 0.4 M sucrose enhanced the DAMGO-induced receptor desensitization, and the inclusion of monensin prevented the resensitization of the mu-opioid receptor after chronic agonist treatment in SHSY5Y cells. Thus, the ability of the mu-opioid receptor to resensitize and to recycle, and the relative efficiency of the receptor to regulate adenylyl cyclase activity, contributed to the observed slow rate of mu-opioid receptor desensitization in HEK293 cells.
Asunto(s)
Receptores Opioides mu/metabolismo , Brefeldino A/farmacología , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Ionóforos/farmacología , Monensina/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Receptores Opioides mu/agonistas , Transducción de Señal/efectos de los fármacosRESUMEN
Similar to other G protein-coupled receptors, rapid phosphorylation of the delta-opioid receptor in the presence of agonist has been reported. Hence, agonist-induced desensitization of the delta-opioid receptor has been suggested to be via the receptor phosphorylation, arrestin-mediated pathway. However, due to the highly efficient coupling between the delta-opioid receptor and the adenylyl cyclase, the direct correlation between the rates of receptor phosphorylation and receptor desensitization as measured by the adenylyl cyclase activity could not be established. In the current studies, using an ecdysone-inducible expression system to control the delta-opioid receptor levels in HEK293 cells, we could demonstrate that the rate of deltorphin II-induced receptor desensitization is dependent on the receptor level. Only at receptor concentrations =90 fmol/mg of protein were rapid desensitizations (t(12) <10 min) observed. Apparently, deltorphin II-induced receptor desensitization involves cellular events in addition to receptor phosphorylation. Mutation of Ser(363) in the carboxyl tail of the delta-opioid receptor to Ala completely abolished the deltorphin II-induced receptor phosphorylation but not the desensitization response. Although the magnitude of desensitization was attenuated, the rate of deltorphin II-induced receptor desensitization remained the same in the S363A mutant as compared with wild type. Also, the S363A mutant could internalize in the presence of deltorphin II. Only when the agonist-induced clathrin-coated pit-mediated receptor internalization was blocked by 0.4 m sucrose that the deltorphin II-induced receptor desensitization was abolished in the S363A mutant. Similarly, 0.4 m sucrose could partially block the agonist-induced rapid desensitization in HEK293 cells expressing the wild type delta-opioid receptor. Taken together, these data supported the hypothesis that rapid desensitization of the delta-opioid receptor involves both the phosphorylation and the internalization of the receptor.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Endocitosis/efectos de los fármacos , Oligopéptidos/farmacología , Receptores Opioides delta/metabolismo , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Sustitución de Aminoácidos , Western Blotting , Línea Celular , Vesículas Cubiertas por Clatrina/efectos de los fármacos , Vesículas Cubiertas por Clatrina/metabolismo , Ecdisterona/análogos & derivados , Ecdisterona/farmacología , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Oligopéptidos/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Receptores Opioides delta/agonistas , Receptores Opioides delta/genética , Serina/genética , Serina/metabolismo , Sacarosa/farmacologíaAsunto(s)
Naltrexona/análogos & derivados , Antagonistas de Narcóticos/síntesis química , Receptores Opioides/metabolismo , o-Ftalaldehído/análogos & derivados , o-Ftalaldehído/síntesis química , Marcadores de Afinidad , Animales , Células CHO , Cricetinae , Ligandos , Ratones , Naltrexona/síntesis química , Naltrexona/química , Naltrexona/metabolismo , Naltrexona/farmacología , Antagonistas de Narcóticos/química , Antagonistas de Narcóticos/metabolismo , Antagonistas de Narcóticos/farmacología , Dimensión del Dolor , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , o-Ftalaldehído/química , o-Ftalaldehído/metabolismoRESUMEN
Cloning of multiple opioid receptors has presented opportunities to investigate the mechanisms of multiple opioid receptor signaling and the regulation of these signals. The subsequent identification of receptor gene structures has also provided opportunities to study the regulation of receptor gene expression and to manipulate the concentration of the gene products in vivo. Thus, in the current review, we examine recent advances in the delineation basis for the multiple opioid receptor signaling, and their regulation at multiple levels. We discuss the use of receptor knockout animals to investigate the function and the pharmacology of these multiple opioid receptors. The reasons and basis for the multiple opioid receptor are addressed.