RESUMO
We recently demonstrated that prolonged treatment with morphine increases the antinociceptive potency of the delta-opioid receptor (deltaOR) agonist deltorphin and promotes cell surface targeting of deltaORs in neurons of the dorsal horn of the rat spinal cord (Cahill et al., 2001b). In the present study we examined whether these effects were mediated selectively via muOR. Using the same intermittent treatment regimen as for morphine, we found that methadone and etorphine, but not fentanyl, enhanced [D-Ala2]-deltorphin-mediated antinociception. However, continuous delivery of fentanyl for 48 hr resulted in augmented deltaOR-mediated antinociception when compared with saline-infused animals. Time course studies confirmed that a 48 hr treatment with morphine was necessary for the establishment of enhanced deltaOR-mediated antinociception. The observed increases in deltaOR agonist potency and deltaOR plasma membrane density were reversed fully 48 hr after discontinuation of morphine injections. Wild-type C57BL/6 mice pretreated with morphine for 48 hr similarly displayed enhanced deltaOR-mediated antinociception in a tonic pain paradigm. Accordingly, the percentage of plasma membrane-associated deltaOR in the dorsal horn of the spinal cord, as assessed by immunogold electron microscopy, increased from 6.6% in naive to 12.4% in morphine-treated mice. In contrast, morphine treatment of muOR gene knock-out (KO) mice did not produce any change in deltaOR plasma membrane density. These results demonstrate that selective activation of muOR is critical for morphine-induced targeting of deltaOR to neuronal membranes, but not for basal targeting of this receptor to the cell surface.
Assuntos
Receptores Opioides delta/metabolismo , Receptores Opioides mu/deficiência , Animais , Área Sob a Curva , Comportamento Animal/efeitos dos fármacos , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Etorfina/farmacologia , Feminino , Fentanila/administração & dosagem , Fentanila/farmacologia , Masculino , Metadona/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Entorpecentes/farmacologia , Medição da Dor/efeitos dos fármacos , Células do Corno Posterior/citologia , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/agonistas , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
Amphiphysins are SH3 domain-containing proteins thought to function in clathrin-mediated endocytosis. To investigate the potential role of amphiphysin II in cellular trafficking of G protein-coupled somatostatin (SRIF) receptors, we generated an AtT-20 cell line stably overexpressing amphiphysin IIb, a splice variant that does not bind clathrin. Endocytosis of (125)I-[d-Trp(8)]SRIF was not affected by amphiphysin IIb overexpression. However, the maximal binding capacity (B(max)) of the ligand on intact cells was significantly lower in amphiphysin IIb overexpressing than in non-transfected cells. This difference was no longer apparent when the experiments were performed on crude cell homogenates, suggesting that amphiphysin IIb overexpression interferes with SRIF receptor targeting to the cell surface and not with receptor synthesis. Accordingly, immunofluorescence experiments demonstrated that, in amphiphysin overexpressing cells, sst(2A) and sst(5) receptors were segregated in a juxtanuclear compartment identified as the trans-Golgi network. Amphiphysin IIb overexpression had no effect on corticotrophin-releasing factor 41-stimulated adrenocorticotropic hormone secretion, suggesting that it is not involved in the regulated secretory pathway. Taken together, these results suggest that amphiphysin II is not necessary for SRIF receptor endocytosis but is critical for its constitutive targeting to the plasma membrane. Therefore, amphiphysin IIb may be an important component of the constitutive secretory pathway.