RESUMEN
Prolonged exposure to opioid agonists can induce adaptive changes resulting in tolerance and dependence. Here, rats were rendered tolerant by subcutaneous injections of increasing doses of morphine from 10 to 60 mg/kg for 3, 5, or 10 consecutive days. Binding parameters of the mu-opioid receptor in subcellular fractions were measured with [(3)H]DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin). Although the density of surface mu-sites did not change after the 5-day morphine treatment, up-regulation of synaptic plasma membrane binding was detected after the 10-day drug administration. In contrast, the number of mu-binding sites in a light vesicle or microsomal fraction (MI) was elevated by 68 and 30% after 5 and 10 days of morphine exposure, respectively. The up-regulated MI mu-sites displayed enhanced coupling to G proteins compared with those detected in saline-treated controls. Pertussis toxin catalyzed ADP ribosylation, and Western blotting with specific antisera was used to quantitate chronic morphine-induced changes in levels of various G protein alpha-subunits. Morphine treatment of 5 days and longer induced significant increases in levels of Galpha(o), Galpha(i1), and Galpha(i2) in MI fractions that are part of an adaptation process. Up-regulation of intracellular mu-sites may be the result of post-translational changes and in part de novo synthesis. The results provide the first evidence that distinct regulation of intracellular mu-opioid receptor G protein coupling and G protein levels may accompany the development of morphine tolerance.
Asunto(s)
Analgésicos Opioides/farmacología , Encéfalo/metabolismo , Proteínas de Unión al GTP/metabolismo , Dependencia de Morfina/metabolismo , Dependencia de Morfina/fisiopatología , Morfina/farmacología , Receptores Opioides mu/fisiología , Animales , Encéfalo/efectos de los fármacos , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacocinética , Proteínas de Unión al GTP/efectos de los fármacos , Especificidad de Órganos , Dolor/fisiopatología , Dolor/prevención & control , Ratas , Receptores Opioides mu/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Phosphorylation of the MAPK isoform ERK by G protein-coupled receptors involves multiple signaling pathways. One of these pathways entails growth factor receptor transactivation followed by ERK activation. This study demonstrates that a similar signaling pathway is used by the mu-opioid receptor (MOR) expressed in HEK293 cells and involves calmodulin (CaM). Stimulation of MOR resulted in both epidermal growth factor receptor (EGFR) and ERK phosphorylation. Data obtained with inhibitors of EGFR Tyr kinase and membrane metalloproteases support an intermediate role of EGFR activation, involving release of endogenous membrane-bound epidermal growth factor. Previous studies had demonstrated a role for CaM in opioid signaling based on direct CaM binding to MOR. To test whether CaM contributes to EGFR transactivation and ERK phosphorylation by MOR, we compared wild-type MOR with mutant K273A MOR, which binds CaM poorly, but couples normally to G proteins. Stimulation of K273A MOR with [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin (10-100 nm) resulted in significantly reduced ERK phosphorylation. Furthermore, wild-type MOR stimulated EGFR Tyr phosphorylation 3-fold more than K273A MOR, indicating that direct CaM-MOR interaction plays a key role in the transactivation process. Inhibitors of CaM and protein kinase C also attenuated [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin-induced EGFR transactivation in wild-type (but not mutant) MOR-expressing cells. This novel pathway of EGFR transactivation may be shared by other G protein-coupled receptors shown to interact with CaM.
Asunto(s)
Calmodulina/metabolismo , Receptores ErbB/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores Opioides mu/metabolismo , Activación Transcripcional , Animales , Línea Celular , Colforsina/farmacología , Reactivos de Enlaces Cruzados/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Mutación , Fenantrolinas/farmacología , Fosforilación , Pruebas de Precipitina , Inhibidores de Proteasas/farmacología , Unión Proteica , Isoformas de Proteínas , Proteína Quinasa C/metabolismo , Ratas , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
Chronic treatment with micro or kappa opioid agonists (>/=2 h) inhibits EGF-induced ERK activation in opioid receptor overexpressing COS-7 cells. Although acute mu and kappa opioids activate ERK via a pertussis toxin-sensitive G protein, pertussis toxin insensitivity of the chronic mu (but not kappa) action was observed. Here, we tested several pertussis toxin-insensitive G proteins as candidates to transduce acute and/or chronic opioid modulation of ERK. Overexpressed Galpha(z) (but not Galpha(12)) transduced acute mu (but not kappa) ERK activation in pertussis toxin-treated COS-7 cells. Chronic mu (but not kappa) inhibited EGF stimulation of ERK in pertussis toxin-treated cells overexpressing Galpha(z) or Galpha(12). Transfection of Galpha(13) or Galpha(q) blocked inhibition under the same conditions. Overexpressed interfering and non-interfering Galpha(z) mutants differentially affected mu inhibition of ERK consistent with G(z) transduction. In this and prior studies, Galpha(z) and Galpha(12) immunoreactivity were detected in untransfected COS-7 cells, suggesting that these G proteins may be endogenous mediators of chronic mu inhibitory actions on ERK.
Asunto(s)
Bencenoacetamidas , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides kappa/metabolismo , Transducción Genética , Analgésicos/farmacología , Analgésicos Opioides/farmacología , Animales , Células COS , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Activación Enzimática , Ácidos Grasos/metabolismo , Proteínas de Unión al GTP/genética , Immunoblotting , Mutagénesis Sitio-Dirigida , Toxina del Pertussis , Pirrolidinas/farmacología , Receptores Opioides kappa/agonistas , Receptores Opioides mu/metabolismo , Factores de Tiempo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
As reports on G protein-coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous kappa-opioid receptors in C6 glioma cells, an astrocytic model system. We find that the kappa-opioid receptor-selective agonist U69,593 stimulates phospholipase C activity, extracellular signal-regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593-stimulated extracellular signal-regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin-sensitive G proteins, L-type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen-activated protein or extracellular signal-regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant-negative or sequestering mutants, we provide evidence that extracellular signal-regulated kinase 1/2 phosphorylation is Ras-dependent and transduced by Gbetagamma subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous kappa-opioid receptor in C6 glioma cells.
Asunto(s)
Bencenoacetamidas , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Proteínas de Unión al GTP Heterotriméricas , Mitosis/fisiología , Receptores Opioides kappa/fisiología , Transducción de Señal/fisiología , Animales , ADN/biosíntesis , Quinasa 2 de Adhesión Focal , Proteínas de Unión al GTP/fisiología , Glioma/patología , Glioma/fisiopatología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosfatidilinositoles/antagonistas & inhibidores , Fosfatidilinositoles/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/metabolismo , Pirrolidinas/farmacología , Ratas , Receptores Opioides kappa/agonistas , Células Tumorales Cultivadas , Proteínas ras/fisiologíaRESUMEN
In previous studies we found that mu-opioids, acting via mu-opioid receptors, inhibit endothelin-stimulated C6 glioma cell growth. In the preceding article we show that the kappa-selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with mu-opioid agonists for 1 h results in the inhibition of kappa-opioid mitogenic signaling. The mu-selective agonist endomorphin-1 attenuates kappa-opioid-stimulated DNA synthesis, phosphoinositide turnover, and extracellular signal-regulated kinase phosphorylation. To investigate the role of receptor endocytosis in signaling, we have examined the effects of dynamin-1 and its GTPase-defective, dominant suppressor mutant (K44A) on opioid modulation of extracellular signal-regulated kinase phosphorylation in C6 cells. Overexpression of dynamin K44A in C6 cells does not affect kappa-opioid phosphorylation of extracellular signal-regulated kinase. However, it does block the inhibitory action on kappa-opioid signaling mediated by the kappa-opioid receptor. Our results are consistent with a growing body of evidence of the opposing actions of mu- and kappa-opioids and provide new insight into the role of opioid receptor trafficking in signaling.
Asunto(s)
Bencenoacetamidas , GTP Fosfohidrolasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores Opioides kappa/fisiología , Receptores Opioides mu/agonistas , Animales , ADN/antagonistas & inhibidores , ADN/biosíntesis , Dinamina I , Dinaminas , Endotelinas/farmacología , Glioma/metabolismo , Glioma/patología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Morfina/farmacología , Oligopéptidos/farmacología , Fosfatidilinositoles/antagonistas & inhibidores , Fosfatidilinositoles/metabolismo , Fosforilación/efectos de los fármacos , Pirrolidinas/metabolismo , Pirrolidinas/farmacología , Ratas , Receptores Opioides kappa/agonistas , Receptores Opioides mu/fisiología , Células Tumorales CultivadasRESUMEN
Previously, we implicated the opioid receptor (OR), Gbetagamma subunits, and Ras in the opioid activation of extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family involved in mitogenic signaling. We now report that OR endocytosis also plays a role in the opioid stimulation of ERK activity. COS-7 and HEK-293 cells were cotransfected with the cDNA of delta-, mu;-, or kappa-OR, dynamin wild-type (DWT), or the dominant suppressor mutant dynamin K44A, which blocks receptor endocytosis. The activation of ERK by opioid agonists in the presence of DWT was detected. In contrast, parallel ectopic coexpression of the K44A mutant with OR, followed by agonist treatment, resulted in a time-dependent attenuation of ERK activation. Immunofluorescence confocal microscopy of delta-OR and DWT-cotransfected COS-7 cells revealed that agonist exposure for 10 min resulted in an ablation of cell surface delta-OR immunoreactivity (IR) and an intensification of cytoplasmic (presumably endosomal) staining as seen in the absence of overexpressed DWT. After 1 hr of delta-agonist exposure the cells displayed substantial internalization of delta-OR IR. If the cells were cotransfected with delta-OR and dynamin mutant K44A, OR IR was retained on the cell surface even after 1 hr of delta-agonist treatment. Parallel immunofluorescence confocal microscopy, using an anti-ERK antibody, showed that agonist-induced time-dependent ERK IR trafficking into perinuclear and nuclear loci was impaired in the internalization-defective cells. Thus, both biochemical and immunofluorescence confocal microscopic evidence supports the hypothesis that the opioid activation of ERK requires receptor internalization in transfected mammalian cells.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/efectos de los fármacos , Receptores Opioides/agonistas , Transducción de Señal/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Células COS , Dinaminas , Encefalina D-Penicilamina (2,5) , Leucina Encefalina-2-Alanina/farmacología , Encefalinas/farmacología , GTP Fosfohidrolasas/farmacología , Inmunohistoquímica , Microscopía Confocal , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Estimulación QuímicaRESUMEN
Previous in vivo studies revealed that buprenorphine can down-regulate mu and up-regulate delta2 and kappa1 opioid receptors in adult and neonatal rat brain. To assess gestational effects of buprenorphine on offspring, pregnant rats were also administered this drug and opioid receptor binding parameters (Kd and Bmax values) were measured by homologous binding assays of postnatal day 1 (P1) brain membranes. Buprenorphine concentrations of 2.5 mg/kg injected into dams elicited an up-regulation of kappa1 opioid receptors as detected with the kappa1-selective agonist 3H-U69593. Parallel studies with the mu-selective agonist [D-ala2, mephe4,gly-ol5] enkephalin revealed a buprenorphine-induced down-regulation in receptor density at 0.3, 0.6 or 2.5 mg/kg drug treatment. A greater down-regulation of mu receptors for P1 males than for their female counterparts was observed. Buprenorphine did not cause a reduction in binding affinity in these experiments. Changes in opioid receptor adaptation induced by buprenorphine were further supported by data from cross-linking of 125I-beta-endorphin to brain membrane preparations. RT-PCR analysis of opioid receptor expression was also estimated in P1 brains. However, significant changes in neither mu nor kappa receptor message were detected in P1 brains as a result of prenatal buprenorphine treatment under the conditions of these experiments. Since buprenorphine is being evaluated in clinical trials for the treatment of heroin abuse, the in utero actions of the drug have ramifications for its use in the treatment of maternal drug abuse.
Asunto(s)
Adaptación Fisiológica/fisiología , Encéfalo/metabolismo , Buprenorfina/farmacología , Antagonistas de Narcóticos/farmacología , Efectos Tardíos de la Exposición Prenatal , Receptores Opioides/fisiología , Animales , Animales Recién Nacidos/metabolismo , Encéfalo/efectos de los fármacos , Reactivos de Enlaces Cruzados/farmacología , Regulación hacia Abajo/fisiología , Electroforesis en Gel de Poliacrilamida , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Femenino , Embarazo , Ratas , Receptores Opioides/efectos de los fármacos , Receptores Opioides/metabolismo , Receptores Opioides mu/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , betaendorfina/efectos de los fármacos , betaendorfina/metabolismoRESUMEN
The astrocytoma cell line rat C6 glioma has been used as a model system to study the mechanism of various opioid actions. Nevertheless, the type of opioid receptor(s) involved has not been established. Here we demonstrate the presence of high-affinity U69,593, endomorphin-1, morphine, and beta-endorphin binding in desipramine (DMI)-treated C6 cell membranes by performing homologous and heterologous binding assays with [3H]U69,593, [3H]morphine, or 125I-beta-endorphin. Naive C6 cell membranes displayed U69,593 but neither endomorphin-1, morphine, nor beta-endorphin binding. Cross-linking of 125I-beta-endorphin to C6 membranes gave labeled bands characteristic of opioid receptors. Moreover, RT-PCR analysis of opioid receptor expression in control and DMI-treated C6 cells indicate that both kappa- and mu-opioid receptors are expressed. There does not appear to be a significant difference in the level of mu nor kappa receptor expression in naive versus C6 cells treated with DMI over a 20-h period. Collectively, the data indicate that kappa- and mu-opioid receptors are present in C6 glioma cells.
Asunto(s)
Bencenoacetamidas , Glioma/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Analgésicos/metabolismo , Analgésicos Opioides/metabolismo , Animales , Unión Competitiva , Desipramina/farmacología , Glioma/patología , Morfina/metabolismo , Reacción en Cadena de la Polimerasa , Pirrolidinas/metabolismo , Ratas , Transcripción Genética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , betaendorfina/metabolismoRESUMEN
Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with mu-, delta-, or kappa-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding mu- ([D-Ala2,Me-Phe4,Gly-ol5]enkephalin)-, delta- ([D-Pen2,D-Pen5]enkephalin)-, or kappa- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a betagamma scavenger, CD8- beta-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8- beta-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and betagamma subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.
Asunto(s)
Bencenoacetamidas , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Receptores Opioides delta/fisiología , Receptores Opioides kappa/fisiología , Receptores Opioides mu/fisiología , Proteínas ras/biosíntesis , Animales , Células COS , Chlorocebus aethiops , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina D-Penicilamina (2,5) , Encefalinas/farmacología , Factor de Crecimiento Epidérmico/farmacología , Cinética , Sustancias Macromoleculares , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Toxina del Pertussis , Pirrolidinas/farmacología , Receptores Opioides delta/agonistas , Receptores Opioides delta/biosíntesis , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/biosíntesis , Receptores Opioides mu/agonistas , Receptores Opioides mu/biosíntesis , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
BACKGROUND: Opioid agonists can inhibit cell proliferation in various neural tumor cell lines, including rat gliomas. Because opioid antimitogenic effects are mediated by opioid receptors, it was of interest to the authors to determine opioid receptor levels in human brain tumors. METHODS: Specimens obtained at craniotomy from 30 patients with glioma and nonneoplastic brain disorders were evaluated for their kappa-opioid receptor binding. Kd and Bmax values were estimated from homologous competition binding curves with the kappa1-selective radioligand [3H]U69,593. RESULTS: Receptor binding density was greatest in nonneoplastic brain tissue, less in Grade 2 and 3 astrocytoma, and least in glioblastoma multiforme. CONCLUSIONS: These results suggest that opioid receptor-based stratification of grade may have clinical utility in distinguishing glioblastoma multiforme from lower grade astrocytomas, and thereby may facilitate diagnosis and treatment.
Asunto(s)
Astrocitoma/metabolismo , Astrocitoma/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Receptores Opioides kappa/metabolismo , Encefalopatías/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patología , HumanosRESUMEN
In our initial purification of dihydrobenzophenanthridine oxidase from Sanguinaria canadensis plant cell cultures, we reported that our most purified preparations contained a major band at 77 kDa and minor lower Mr bands. Here we present evidence on highly purified dihydrobenzophenanthridine oxidase from elicited S. canadensis cultures to indicate that this enzyme is the 77-kDa protein and that lower Mr bands include an isozyme(s) of the polyphenol oxidase family that copurifies with it. An antibody raised against the 77-kDa protein and an anti-polyphenol oxidase antibody that recognizes a 70-kDa band were used to monitor chromatographic fractions by immunoblot analysis of the oxidases. Oxidase-containing eluates from DEAE-Sephadex, CM, and HiTrap blue were compared to corresponding flow-through fractions. Bands at 77 and 88 kDa were detected with anti-dihydrobenzophenanthridine oxidase antibody in eluates displaying high dihydrobenzophenanthridine oxidase activity. Polyphenol oxidase specific activity and immunoreactivity partitioned both in flow-through and eluate fractions of the CM and HiTrap columns. Estimation of the dihydrobenzophenanthridine oxidase and polyphenol oxidase specific activities for each step showed increasing enrichment of alkaloidal enzyme accompanied by variable dihydrobenzophenanthridine oxidase/polyphenol oxidase activity ratios. Taken together these observations indicate that the dihydrobenzophenanthridine and polyphenol oxidases have Mr values of 77 and 70 kDa, respectively, and the two enzymes are different entities.
Asunto(s)
Oxidorreductasas/aislamiento & purificación , Plantas Medicinales/enzimología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Catecol Oxidasa/aislamiento & purificación , Expresión Génica , Immunoblotting/métodos , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Oxidorreductasas/inmunología , Análisis de SecuenciaRESUMEN
Opioid-receptor adaptation may lead to changes in transcriptional regulation by sequence-specific DNA-binding proteins. Gel-shift assays of nuclear extracts from NG108-15 cells revealed that an increase of AP-1 DNA-binding activity ensues under conditions previously established to induce down- or up-regulation of delta-opioid receptors.
Asunto(s)
Leucina Encefalina-2-Alanina/farmacología , Naltrexona/farmacología , Oligodesoxirribonucleótidos/metabolismo , Receptores Opioides delta/biosíntesis , Factor de Transcripción AP-1/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Secuencia de Consenso , Regulación hacia Abajo/efectos de los fármacos , Glioma , Células Híbridas , Ratones , Neuroblastoma , Oligodesoxirribonucleótidos/química , Ratas , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Dihydrobenzophenanthridine (DHBP) oxidase catalyses the last step in the biogenesis of the benzo[c]phenanthridine alkaloid sanguinarine. Addition of autoclaved fungal preparations or putative plant defence signalling intermediates (jasmonic acid (JA), methyl jasmonate (MeJ), acetylsalicylic acid (ASA)) to Sanguinaria canadensis cell suspension cultures elicited an increase in the activity of DHBP oxidase. MeJ and ASA were better inducers of oxidase activity than were the fungal elicitor and JA. Enzyme-specific activity could be induced in a dose- and time-dependent manner up to 4- to 14-fold, respectively, when cells were treated with MeJ or with ASA. A change in total enzyme activity in cultured cells was observed only at the highest concentration of MeJ and not at any level of ASA tested. The results suggest that MeJ and ASA may play a role in the S. canadensis defence against pathogens by eliciting the enzymes involved in the synthesis of the phytoalexin benzophenanthridine alkaloids.
Asunto(s)
Oxidorreductasas/biosíntesis , Plantas/enzimología , Células Cultivadas , Inducción Enzimática , Oxidorreductasas/metabolismo , Células VegetalesRESUMEN
The delta opioid binding sites in subcellular fractions from NG108-15 cells were characterized with respect to their relative molecular size and levels under conditions of receptor adaptation. 125I-beta-Endorphin was cross-linked to preparations enriched in plasma membranes (P20), nuclear membranes or nuclear matrices. Five cross-linked bands appear in all subcellular fractions. The largest molecular size reaction product in nuclear matrix preparations (approximately 72 kDa) differed from that in the other two fractions-(approximately 83 kDa). Immunoblot analyses with an antibody to the delta opioid receptor gave a P20 band pattern similar to that for the corresponding cross-linked products. To determine which cross-linked products in P20 are glycoproteins, labeled membranes were solubilized and purified by wheat germ agglutinin chromatography. The absence of a approximately 36 kDa band after purification suggests that this product is not a glycoprotein. The remaining four bands were present in N-acetyl-D-glucosamine eluates, although their % distribution changes in favor of the largest molecular size band (approximately 83 kDa). Immunoblotting of the eluate gave a single diffuse band at approximately 73 kDa, suggesting the native glycoprotein has a molecular size in the 70-80 kDa range. Etorphine-induced desensitization of cell surface receptors increased the amount of some cross-linked products associated with nuclear membranes. The same treatment did not affect the relative density of the four larger molecular size bands in P20, but increased the density of the approximately 26 kDa product two fold. Etorphine-induced down-regulation evoked an elevation of cross-linked products in nuclear matrix preparations, while all band densities of P20 were diminished. These results suggest that nuclear matrix associated opioid binding sites represent internalized, truncated forms of the glycosylated delta opioid receptor found in P20.
Asunto(s)
Receptores Opioides delta/metabolismo , betaendorfina/metabolismo , Línea Celular , Membrana Celular/metabolismo , Cromatografía , Reactivos de Enlaces Cruzados/farmacología , Densitometría , Regulación hacia Abajo , Etorfina/farmacología , Immunoblotting , Membranas Intracelulares , Matriz Nuclear/metabolismo , Tamaño de la Partícula , Receptores Opioides delta/agonistas , Fracciones SubcelularesRESUMEN
Previous in vivo studies revealed that the mixed agonist-antagonist buprenorphine can down-regulate mu and up-regulate delta 2 and kappa 1 opioid receptors in rat brain. In this report brain regional differences in opioid receptor adaptation were addressed. Rats received i.p. injections with buprenorphine (0.5-2.5 mg/kg) and were killed 20 h later. Membranes from 7 brain regions were analyzed for mu (3H-[D-Ala2,N-mephe4,Gly-ol5] enkephalin), kappa 1 (3H-U-69593), delta 1 (3H-[D-Pen2, D-Pen5] enkephalin) and delta 2 (3H-deltorphin II) receptor binding parameters. Buprenorphine induced down-regulation of mu receptors in frontal cortex, occipital cortex, thalamus, hippocampus, striatum and brain stem. Kd values for 3H-[D-Ala2,N-mephe4,Gly-ol5] enkephalin were unchanged from controls. Up-regulation of kappa 1 receptors was observed in frontal, parietal, occipital cortexes and striatum. Binding to delta 2 sites was elevated in frontal and parietal cortexes. Buprenorphine did not alter delta 1 binding in any of the regions examined. Changes in opioid receptor adaptation induced by buprenorphine were further supported by data from cross-linking of 125I-beta-endorphin to cortical membrane preparations. A reduction in a 60- to 65-kDa band was detected in frontal and occipital cortices in which binding assays revealed down-regulation of mu receptors. In parietal cortex neither the 60- to 65-kDa product nor Bmax changes were observed. These results indicate that buprenorphine is a useful tool to study brain opioid receptor adaptation in vivo and the information accrued may be relevant to the mode of action of this drug in the treatment of heroin and cocaine abuse.
Asunto(s)
Encéfalo/efectos de los fármacos , Buprenorfina/farmacología , Receptores Opioides/efectos de los fármacos , Adaptación Fisiológica , Analgésicos/farmacología , Animales , Encefalina D-Penicilamina (2,5) , Encefalinas/farmacología , Cinética , Masculino , Oligopéptidos/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-DawleyRESUMEN
Previously, opioid peptide analogues, beta-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via kappa- and mu-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 microM, all dynorphins tested and beta-endorphin inhibited [3H]thymidine incorporation into DNA by 20-38% in 7-day rat brain cell aggregates. The putative epsilon-antagonist beta-endorphin (1-27) did not prevent the effect of beta-endorphin, suggesting that the epsilon-receptor is not involved in opioid inhibition of DNA synthesis. The kappa-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a kappa-opioid receptor. In dose-dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete kappa-receptor subtype. The IC50 value of 0.1 nM estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the kappa-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33-86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express kappa-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.
Asunto(s)
Encéfalo/embriología , ADN/biosíntesis , Dinorfinas/fisiología , Feto/metabolismo , Animales , Agregación Celular , Relación Dosis-Respuesta a Droga , Dinorfinas/farmacología , Feto/citología , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Timidina/antagonistas & inhibidores , Timidina/metabolismoRESUMEN
Opioid binding sites were found in nuclear matrix preparations from NG108-15 neurohybrid cells. Binding parameters of delta-specific radioligands indicated that high-affinity binding sites discovered in purified nuclei were present in nuclear membranes and nuclear matrix fractions. Agonists bind with low affinity, if at all, to nuclear matrix preparations. Neither sensitivity of agonist binding to the GTP analog 5-guanylylimidodiphosphate nor adenylyl cyclase activity were detected in this fraction, suggesting the presence of guanine nucleotide binding regulatory protein/effector uncoupled sites. Opioid inhibition of basal and forskolin-stimulated adenylyl cyclase activity was found in nuclear membrane preparations. Cycloheximide treatment of cells inhibited opioid binding to nuclear membrane fractions to a greater extent than that associated with membranes sedimenting at 20,000 x g (P20) or nuclear matrix. Colchicine, a microtubule disrupter and inhibitor of receptor internalization, caused up-regulation of nuclear membrane and P20 opioid receptors and a loss in nuclear matrix associated sites. Taxol, a microtubule stabilizing agent, prevented the effect of colchicine. Etorphine-elicited down-regulation increased nuclear matrix associated binding while diminishing that in nuclear membranes and P20 fractions. Agonist-induced desensitization completely abolished nuclear matrix binding. In vitro preincubation of nuclear matrix preparations with protein kinase A catalytic subunit mimicked the desensitization effect. Forskolin treatment of cells potentiated nuclear matrix and P20 binding. These data suggest that nuclear membrane opioid receptors represent newly synthesized molecules en route to the cell surface, whereas nuclear matrix contains internalized delta sites.
Asunto(s)
Narcóticos/metabolismo , Membrana Nuclear/metabolismo , Matriz Nuclear/metabolismo , Receptores Opioides delta/metabolismo , Analgésicos/metabolismo , Línea Celular , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Etorfina/farmacología , Microscopía Electrónica , Antagonistas de Narcóticos/metabolismo , Matriz Nuclear/ultraestructura , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inhibidores , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Opioid agonists inhibit DNA synthesis in C6 rat glioma cells that express opioid receptors, induced by desipramine (DMI). This inhibition was not observed in cells that were not treated with DMI, and thus did not express opioid-binding sites. Endothelin, a known mitogen, increased thymidine incorporation dose dependently (up to 1.7-fold) in DMI-treated C6 cells. This increase was reversed by an anti-idiotypic antibody to opioid receptors, Ab2AOR, which has opioid agonist properties. The opioid antagonist naltrexone blocked the inhibition caused by Ab2AOR. Endothelin also stimulated phosphoinositide (PI) turnover and this effect was inhibited by morphine (50%) or by Ab2AOR (72%) in DMI-treated but not in DMI-untreated C6 cells. These actions of morphine and Ab2AOR were reversed by naltrexone. The inhibition of PI turnover and of thymidine incorporation by Ab2AOR or morphine was insensitive to pertussis toxin (PTX). Since PI turnover is known to induce Ca2+ mobilization, it was of interest to examine the effects of the applied opioids on intracellular Ca2+ concentrations. Endothelin increased the concentration of cytosolic free Ca2+ in the cells while Ab2AOR, morphine, and beta-endorphin reversed the endothelin-induced Ca2+ mobilization in DMI-treated but not in DMI-untreated C6 cells. The effect of these agonists was also blocked by naltrexone. The results indicate that glial cells can be a target of an opioid receptor-mediated antimitogenic action and that an abatement in PI turnover and Ca2+ mobilization may be associated with this mechanism.
Asunto(s)
Calcio/metabolismo , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/efectos de los fármacos , Endotelinas/farmacología , Glioma/metabolismo , Morfina/farmacología , Fosfatidilinositoles/metabolismo , Animales , Anticuerpos Antiidiotipos/farmacología , Desipramina/farmacología , Relación Dosis-Respuesta a Droga , Endotelinas/antagonistas & inhibidores , Naltrexona/farmacología , Ratas , Receptores Opioides/efectos de los fármacos , Timidina/metabolismo , Células Tumorales Cultivadas , betaendorfina/farmacologíaRESUMEN
Gestational actions of the mixed agonist-antagonist buprenorphine on mu- and kappa 1-opioid binding in neonatal and maternal rat brain were investigated. Upon exposure of pregnant rats to 0.5 mg/kg buprenorphine for 7 days prior to birth, postnatal day-one (P1) and P7 offspring brain mu-binding parameters (Kd and Bmax) were assessed with 3H-labeled [D-Ala2,Mephe4,Gly-ol5] enkephalin (DAMAGE). DAMAGE binding was attenuated by 64% in P1 membranes, whereas P7 preparations showed no changes. The same buprenorphine regimen resulted in diminished DAMGE Bmax values in mothers' brains, 2 but not 7 days after cessation of drug administration. Receptor density changes were not accompanied by alteration of mu-binding affinities. Although the postnatal developmental profile of kappa 1 opioid receptors in rat brain measured with [3H]U69593 revealed the presence of an ample number of sites for detection, their binding parameters in P1, P7 pups and mothers were unaffected by 0.5 mg/kg buprenorphine. In summary, buprenorphine administration to pregnant rats transiently down-regulates mu opioid receptors in neonatal and maternal brain.
Asunto(s)
Animales Recién Nacidos/metabolismo , Bencenoacetamidas , Química Encefálica/fisiología , Buprenorfina/farmacología , Regulación hacia Abajo/fisiología , Receptores Opioides mu/metabolismo , Analgésicos/farmacología , Animales , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Femenino , Membranas/efectos de los fármacos , Membranas/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal , Pirrolidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Opioides kappa/metabolismoRESUMEN
Opioid antagonists such as naltrexone, naloxone, and ICI174864 induce a transient downregulation of delta opioid receptors prior to upregulation in NG108-15 cells. Here we show that naltrexone can also elicit a transient downregulation of delta 2 opioid receptors preceding upregulation in brain. A 1 h treatment of rats with naltrexone (IP, 10 mg/kg) resulted in lowered 3H-[D-Ser2,L-Leu5]enkephalyl-Thr Bmax values in hindbrain, but not in striatum, hippocampus, or cortex. The decrease in hindbrain delta 2 receptor density was not accompanied by changes in Kd values, indicating that downregulation rather than receptor blockade occurred. Longer naltrexone treatment (48 h), caused twofold upregulation of delta opioid binding in all four regions. These data suggest that the process of upregulation of delta opioid receptors by antagonists in vivo can entail an initial, transient downregulation.