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.

Intrathecal administration of the neuropeptide neurotensin (NT) was shown previously to exert antinociceptive effects in a variety of acute spinal pain paradigms including hotplate, tail-flick, and writhing tests. In the present study, we sought to determine whether some of these antinociceptive effects might be elicited via stimulation of low-affinity NTS2 receptors. We first established, using immunoblotting and immunohistochemical techniques, that NTS2 receptors were extensively associated with putative spinal nociceptive pathways, both at the level of the dorsal root ganglia and of the superficial layers of the dorsal horn of the spinal cord. We then examined the effects of intrathecal administration of NT or selective NTS2 agonists on acute thermal pain. Both NT and NTS2 agonists, levocabastine and Boc-Arg-Arg-Pro-Tyr⌿(CH 2 NH)Ile-Leu-OH (JMV-431), induced dose-dependent antinociceptive responses in the tail-flick test. The effects of levocabastine and of JMV-431 were unaffected by coadministration of the NTS1-specific antagonist 2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxy-phenyl)pyrazol-3-yl)carboxylamino]tricyclo)3.3.1.1. 3.7)-decan-2-carboxylic acid (SR48692), confirming that they were NTS2 mediated. In contrast, the antinociceptive effects of NT were partly abolished by coadministration of SR48692, indicating that NTS1 and NTS2 receptors were both involved. These results suggest that NTS2 receptors play a role in the regulation of spinal nociceptive inputs and that selective NTS2 agonists may offer new avenues for the treatment of acute pain.

Abstract-We previously showed that prolonged morphine treatment and chronic inflammation both enhanced delta opioid receptor (␦OR) cell surface density in lumbar spinal cord neurons. Here, we sought to determine whether administration of morphine to rats with chronic inflammation would further increase the bio-availability of ␦OR, and thereby the analgesic properties of the ␦OR agonist deltorphin, over that produced by inflammation alone. We found that chronic inflammation produced by injection of complete Agonists acting through the mu opioid receptor ( OR), such as morphine and its derivatives, induce potent analgesic effects (Bodnar and Klein, 2004). However, they also give rise to undesirable side-effects such as nausea, constipation, and respiratory depression (Colpaert, 1996;Kreek, 1996). In addition, chronic stimulation of OR induces tolerance and physical dependence (Cowan et al., 1988). By contrast, drugs acting on ␦OR produce more limited analgesia, but also give rise to considerably less undesirable side-effects and induce virtually no tolerance (Porreca et al., 1984;May et al., 1989; Sheldon et al., 1990;Szeto et al., 1999;Gallantine and Meert, 2005). For these reasons, ␦OR agonists have been proposed as possible alternatives to OR agonists for the treatment of chronic pain, including neuropathic (Mika et al., 2001;Petrillo et al., 2003;Morinville et al., 2004a) and chronic inflammatory pain (Desmeules et al., 1993;Stewart and Hammond, 1994;Fraser et al., 2000;Hurley and Hammond, 2000;Qiu et al., 2000;Cahill et al., 2003;Petrillo et al., 2003).One of the reasons for the relatively poor analgesic efficiency of ␦OR agonists may be that only a small proportion of ␦OR is actually present on neuronal plasma membranes under baseline conditions (Cheng et al., 1995(Cheng et al., , 1997Elde et al., 1995;Zhang et al., 1998;Cahill et al., 2001a). However, under conditions of chronic inflammation, such as produced in rodents by injection of complete Freund's adjuvant (CFA) in the hind paw, we observed a massive recruitment of ␦OR from intracellular stores to the plasma membrane in neurons of the dorsal horn of the spinal cord Morinville et al., 2004b). This increase in the pharmacological availability of ␦OR was postulated to account for the enhanced antihyperalgesic efficacy of the centrally administered ␦OR agonists reported in conditions of chronic inflammation (Hylden et al., 1991;Stewart and Hammond, 1994;Fraser et al., 1 Present address: Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada, J1H 5N4. *Corresponding author. Tel: ϩ1-514-398-1913; fax: ϩ1-514-398-5871. E-mail address: alain.beaudet@mcgill.ca (A. Beaudet). Abbreviations: CFA, complete Freund's adjuvant; DRG, dorsal root ganglion; Fluo-DLT, -Bodipy 576/589 deltorphin-I 5-aminopentylamide; i.t., intrathecal/intrathecally; MPAHE, maximum possible antihyperalgesic effect; MPE, maximum possible antinociceptive effect; MS, morphine sulfate; OR, mu opioid receptor; PB, phosphate buffer; RI...

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.