RESUMO
Morphine induces profound analgesic tolerance in vivo despite inducing little internalization of the mu opioid receptor (muOR). Previously proposed explanations suggest that this lack of internalization could either lead to prolonged signaling and associated compensatory changes in downstream signaling systems, or that the receptor is unable to recycle and resensitize and so loses efficacy, either mechanism resulting in tolerance. We therefore examined, in cultured neurons, the relationship between muOR internalization and desensitization in response to two agonists, D-Ala2, N-MePhe4, Gly5-ol-enkephalin (DAMGO) and morphine. In addition, we studied the chimeric mu/delta opioid receptor (mu/ partial differentialOR) which could affect internalization and desensitization in neurons. Dorsal root ganglia neurons from muOR knockout mice were transduced with an adenovirus expressing either receptor and their respective internalization, desensitization and trafficking profiles determined. Both receptors desensitized equally, measured by Ca2+ current inhibition, during the first 5 min of agonist exposure to DAMGO or morphine treatment, although the mu/partial differentialOR desensitized more extensively. Such rapid desensitization was unrelated to internalization as DAMGO, but not morphine, internalized both receptors after 20 min. In response to DAMGO the mu/partial differentialOR internalized more rapidly than the muOR and was trafficked through Rab4-positive endosomes and lysosomal-associated membrane protein-1-labeled lysosomes whereas the muOR was trafficked through Rab4 and Rab11-positive endosomes. Chronic desensitization of the Ca2+ current response, after 24 h of morphine or DAMGO incubation, was seen in the DAMGO, but not morphine-treated, muOR-expressing cells. Such persistence of signaling after chronic morphine treatment suggests that compensation of downstream signaling systems, rather than loss of efficacy due to poor receptor recycling, is a more likely mechanism of morphine tolerance in vivo. In contrast to the muOR, the mu/partial differentialOR showed equivalent desensitization whether morphine or DAMGO treated, but internalized further with DAMGO than morphine. Such ligand-independent desensitization could be a result of the observed higher rate of synthesis and degradation of this chimeric receptor.
Assuntos
Gânglios Espinais/citologia , Neurônios/fisiologia , Receptores Opioides mu/fisiologia , Analgésicos Opioides/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Baclofeno/farmacologia , Células Cultivadas , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Encefalinas/farmacologia , Citometria de Fluxo/métodos , Agonistas GABAérgicos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Knockout , Morfina/farmacologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Transporte Proteico/fisiologia , Receptores Opioides mu/deficiência , Fatores de Tempo , Transfecção/métodosRESUMO
There is growing evidence that tonic activity of the opioid system may be important in the modulation of affective state. Naloxone produces a conditioned place aversion in rodents, an effect that is centrally mediated. Previous pharmacological data using antagonists with preferential actions at mu-, delta-, and kappa-opioid receptors indicate the importance of the mu-opioid receptor in mediating this effect. We sought to test the mu-opioid receptor selectivity of naloxone aversion using mu-opioid receptor knock-out mice. mu-Opioid receptor knock-out and wild-type mice were tested for naloxone (10 mg/kg, s.c.) aversion using a place conditioning paradigm. As a positive control for associative learning, knock-out mice were tested for conditioned place aversion to a kappa agonist, U50,488H (2 mg/kg, s.c.). Naloxone produced a significant place aversion in wild-type mice, but failed to have any effect in mu-opioid receptor knock-out mice. On the other hand, both knock-out and wild-type mice treated with U50,488H spent significantly less time in the drug-paired chamber compared to their respective vehicle controls. We conclude that the mu-opioid receptor is crucial for the acquisition of naloxone-induced conditioned place aversion. Furthermore, in a separate experiment using C57BL/6 mice, the delta-selective antagonist naltrindole (10 or 30 mg/kg, s.c.) failed to produce conditioned place aversion.Taken together, these data further support the notion that naloxone produces aversion by antagonizing tonic opioid activity at the mu-opioid receptor.
Assuntos
Aprendizagem da Esquiva/fisiologia , Encéfalo/metabolismo , Condicionamento Psicológico/fisiologia , Naloxona/farmacologia , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/farmacologia , Peptídeos Opioides/metabolismo , Receptores Opioides mu/deficiência , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Analgésicos não Narcóticos/farmacologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Habituação Psicofisiológica/efeitos dos fármacos , Habituação Psicofisiológica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Naltrexona/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Peptídeos Opioides/antagonistas & inibidores , Fenótipo , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/efeitos dos fármacos , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/genética , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Although mu opioid receptors desensitize in various cell lines in vitro, the relationship of this change in signaling efficacy to the development of tolerance in vivo remains uncertain. It is clear that a system is needed in which functional mu opioid receptor expression is obtained in appropriate neurons so that desensitization can be measured, manipulated, and mutated receptors expressed in this environment. We have developed a recombinant system in which expression of a flag-tagged mu opioid receptor is returned to dorsal root ganglia neurons from mu opioid receptor knockout mice in vitro. Flow cytometry analysis showed that adenoviral-mediated expression of the amino-terminal flag-tagged mu opioid receptor in neurons resulted in approximately 1.3x10(6) receptors/cell. Many mu opioid receptor cell lines express a similar density of receptors but this is approximately 7x greater than the number of endogenous receptors expressed by matched wild-type neurons. Inhibition of the high voltage-activated calcium currents in dorsal root ganglia neurons by the mu agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), was not different between the endogenous and flag-tagged receptor at several concentrations of DAMGO used. Both receptors desensitized equally over the first 6 h of DAMGO pre-incubation, but after 24 h the response of the endogenous receptor to DAMGO had desensitized further than the flag- tagged receptor (71+/-3 vs 29+/-7% respectively; P<0.002), indicating less desensitization in neurons expressing a higher density of receptor. Using flow cytometry to quantify the percentage of receptors remaining on the neuronal cell surface, the flag-tagged receptor internalized by 17+/-1% after 20 min and 55+/-2% after 24 h of DAMGO. These data indicate that this return of function model in neurons recapitulates many of the characteristics of endogenous mu opioid receptor function previously identified in non-neuronal cell lines.
Assuntos
Citomegalovirus/metabolismo , DNA Viral/biossíntese , Gânglios Espinais/metabolismo , Neurônios/metabolismo , Receptores Opioides mu/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Citomegalovirus/genética , DNA Viral/genética , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Gânglios Espinais/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Receptores Opioides mu/deficiência , Receptores Opioides mu/genéticaRESUMO
The capacity to reestablish locomotor function after complete spinal cord transection in the adult mammal is now well documented. Further studies have shown different neurotransmitters to be involved in the initiation and maintenance of these locomotor patterns. However, there has been no in vivo evidence of the changes in glutamate or any other neurotransmitter in the extracellular space of the dorsal horn during an alternating motor pattern such as hindlimb stepping. This study describes an in vivo microdialysis technique to measure extracellular glutamate in the dorsal horn of the spinal cord in the fully awake intact rat. A concentric microdialysis probe was placed in the dorsal horn at L5, and 18 h later dialysate samples were collected at 20-min intervals before, during, and after 20 min of hindlimb stepping. During stepping, extracellular glutamate rose 150% above resting levels and returned to resting levels 40 min later. This increase may have occurred either as a result of primary afferent depolarization or modulation by the descending and ascending supraspinal pathways. In another series of experiments extracellular glutamate was, therefore, measured in the dorsal horn of the chronic spinally transected rat during 20 min of hindlimb stepping. Although the spinal group did not take as many steps as the intact group, those taking more than 40 steps showed a significant rise in extracellular glutamate, and the number of steps taken by the individual spinal rats correlated positively with the individual values of extracellular glutamate (r2 = 0.63). These results are consistent with glutamate being an important neurotransmitter in the spinal cord in normal locomotion.
Assuntos
Espaço Extracelular/metabolismo , Membro Posterior/fisiologia , Locomoção/fisiologia , Medula Espinal/metabolismo , Animais , Ácido Aspártico/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Ácido Glutâmico/metabolismo , Histidina/metabolismo , Microdiálise , Ratos , Ratos Sprague-Dawley , Espectrometria de Fluorescência , Medula Espinal/químicaRESUMO
A previous phase III clinical trial failed to show significant therapeutic benefit of repeated subcutaneous nerve growth factor (NGF) administration in the treatment of diabetic neuropathy. Animal studies have since shown that site-specific viral-mediated expression of NGF in the lumbar dorsal root ganglia prevents peripheral nerve dysfunction associated with chemically induced neuropathy. Using a Herpes simplex virus expression vector, we have investigated the effect of localized NGF expression in a genetic mouse model of progressive diabetic neuropathy, the +/+ Leprdb mouse. We found that site-specific delivery of NGF initially delayed the appearance of hypoalgesia, assessed by the Hargreaves test, by 1 month and effectively attenuated this deficit for 2 months over the approximately 10 months normal life-span of these animals. Once the disease progressed into its more severe stages, NGF, although still capable of altering the electrophysiological profile of the sensory A- and C-fibers and influencing the expression of p75 and substance P in the dorsal root ganglia, could no longer maintain normal nociception. These data suggest that maximal therapeutic benefit in future NGF-based gene therapy trials will be gained from early applications of such viral-mediated neurotrophin delivery.