RESUMEN
This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Regulación hacia Abajo , Complejos Multienzimáticos/metabolismo , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Línea Celular , Humanos , Cinética , Fosforilación , Complejo de la Endopetidasa Proteasomal , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Ubiquitinas/metabolismoRESUMEN
Several non-peptidic opioids have been synthesized recently as part of a program to develop selective delta receptor agonists. In this study, the affinities of a set of compounds for cloned delta and mu opioid receptors expressed in HEK 293 cell lines were determined by competition analysis of [3H]bremazocine binding to membrane preparations. All compounds studied exhibited high affinity and selectivity, with apparent dissociation constants in the range of 0.6-1.7 nM for the delta opioid receptor and 240-1165 nM for the mu opioid receptor. We next sought to determine which domain of the delta receptor was critical for mediating the highly selective binding by analysis of ligand affinities for mu/delta receptor chimeras. Receptor binding profiles suggested that a critical site of receptor/ligand interaction was located between transmembrane domain 5 (TM5) and TM7 of the delta receptor. Substitution of tryptophan 284, located at the extracellular surface of TM6, with lysine, which is found at the equivalent position in the mu opioid receptor, led to a spectrum of effects on affinities, depending on the ligand tested. Affinities of SB 219825 and SB 222941 were particularly sensitive to the substitution, displaying a 50-fold and 70-fold decrease in affinity, respectively. Activities of the delta receptor-selective agonists were tested in two functional assays. Brief exposure of HEK 293 cells expressing delta opioid receptors with selective ligands induced phosphorylation of MAP kinase, although the non-peptidic ligands were less efficacious than the enkephalin derivative DADL (Tyr-D-Ala-Gly-Phe-D-Leu). Similarly, chronic exposure of HEK 293 cells expressing delta opioid receptors with selective, non-peptidic ligands, with the exception of SB 206848, caused receptor down-regulation, however, the SB compounds were less efficacious than DADL.
Asunto(s)
Receptores Opioides delta , Secuencia de Aminoácidos , Analgésicos/metabolismo , Analgésicos/farmacología , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacología , Benzomorfanos/metabolismo , Benzomorfanos/farmacología , Unión Competitiva , Células Cultivadas , Clonación Molecular , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Leucina Encefalina-2-Alanina/farmacología , Proteínas de Unión al GTP/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Indoles/química , Indoles/farmacología , Isoquinolinas/química , Isoquinolinas/farmacología , Riñón/citología , Ligandos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Morfina/metabolismo , Morfina/farmacología , Mutagénesis Sitio-Dirigida , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacología , Ensayo de Unión Radioligante , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides delta/genética , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/genética , TritioRESUMEN
Exposure of A2780 human ovarian tumor cells to a low concentration of melphalan in vitro for 7 days resulted in the development of melphalan resistance. This resistance was not a stable characteristic of the cells since it was lost after 2 weeks in culture in the absence of drug. The melphalan-resistant cells exhibited significant cross-resistance to cisplatin but only minor cross-resistance to doxorubicin. The resistant cells had elevated levels of glutathione-S-transferase activity and mRNA. Exposure of the cells to the ethacrynic acid resulted in a decrease in enzyme activity as well as a reversal of their drug-resistant phenotype, indicating that the enzyme is involved in the resistance. When ethacrynic acid was present during the 7-day exposure of the cells to melphalan, the development of drug resistance was prevented. This system may serve as a useful preliminary step in screening for agents which can prevent the development of chemotherapy-induced drug resistance in human cancer.