Opiates such as morphine are the choice analgesic in the treatment of chronic pain. However their long-term use is limited because of the development of tolerance and dependence. Due to its importance in therapy, different strategies have been considered for making opiates such as morphine more effective, while curbing its liability to be abused. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular, ␦ opioid receptor ligands have been useful in enhancing morphine's potency. The underlying molecular basis for these observations is not understood. We propose the modulation of receptor function by physical association between and ␦ opioid receptors as a potential mechanism. In support of this hypothesis, we show that -␦ interacting complexes exist in live cells and native membranes and that the occupancy of ␦ receptors (by antagonists) is sufficient to enhance opioid receptor binding and signaling activity. Furthermore, ␦ receptor antagonists enhance morphine-mediated intrathecal analgesia. Thus, heterodimeric associations between -␦ opioid receptors can be used as a model for the development of novel combination therapies for the treatment of chronic pain and other pathologies. Opioid receptors belong to the rhodopsin family of G proteincoupled receptors (GPCRs). Like many GPCRs, these receptors were thought to function as single units. This notion has been revised in recent years by a number of studies showing that GPCRs associate with each other to form dimers and͞or oligomers (1-3). Of particular significance are the studies with rhodopsin, a prototypical member of the GPCR family, where infrared-laser atomic-force microscopy of native mouse disk membranes showed the receptors to be arranged in crystalline arrays of dimeric units (4, 5). Also, data from x-ray crystallographic studies with rhodopsin (6, 7) and the N terminus of metabotropic glutamate receptors (8), support the notion that dimerization is an integral feature of these receptors and could play a key role in modulating their function.The three types of opioid receptors (, ␦, and ) have been shown to associate with each other in a homotypic or heterotypic fashion when expressed in heterologous cells (9-11). Furthermore, heterotypic interactions appear to alter the ligand-binding and signaling properties of these receptors (12). However, until now, it was not clear whether these interactions occurred in live cells and in endogenous tissues and whether they were physiologically relevant. In this study, we addressed these questions by using multiple approaches. We used the bioluminescence resonance energy transfer (BRET) assay to show that and ␦ receptors interact in living cells. In addition, we show that signaling by clinically relevant drugs, such as morphine, fentanyl, and methadone can be enhanced by ␦ receptor ligands. This potentiation of receptor signaling by the ␦ receptor antagonist is seen in membranes from WT mice and not in membranes from ␦ receptor lacking mice (␦ k͞o). Finally, w...

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The μ and δ types of opioid receptors form heteromers that exhibit pharmacological and functional properties distinct from those of homomeric receptors. To characterize these complexes in the brain, we generated antibodies that selectively recognize the μ-δ heteromer and blocked its in vitro signaling. With these antibodies, we showed that chronic, but not acute, morphine treatment caused an increase in the abundance of μ-δ heteromers in key areas of the central nervous system that are implicated in pain processing. Because of its distinct signaling properties, the μ-δ heteromer could be a therapeutic target in the treatment of chronic pain and addiction.

To date, the endogenous ligands described for cannabinoid receptors have been derived from membrane lipids. To identify a peptide ligand for CB 1 cannabinoid receptors, we used the recently described conformation-state sensitive antibodies and screened a panel of endogenous peptides from rodent brain or adipose tissue. This led to the identification of hemopressin (PVNFKFLSH) as a peptide ligand that selectively binds CB 1 cannabinoid receptors. We find that hemopressin is a CB 1 receptor-selective antagonist, because it is able to efficiently block signaling by CB 1 receptors but not by other members of family A G protein-coupled receptors (including the closely related CB2 receptors). Hemopressin also behaves as an inverse agonist of CB 1 receptors, because it is able to block the constitutive activity of these receptors to the same extent as its well characterized antagonist, rimonabant. Finally, we examine the activity of hemopressin in vivo using different models of pain and find that it exhibits antinociceptive effects when administered by either intrathecal, intraplantar, or oral routes, underscoring hemopressin's therapeutic potential. These results represent a demonstration of a peptide ligand for CB 1 cannabinoid receptors that also exhibits analgesic properties. These findings are likely to have a profound impact on the development of novel therapeutics targeting CB 1 receptors.

Adolescence is a critical phase of active brain development often characterized by the initiation of marijuana (Cannabis sativa) use. Limited information is known regarding the endogenous cannabinoid system of the adolescent brain as well as related neurotransmitters that appear sensitive to cannabis exposure. We recently observed that adult rats pre-exposed to Δ-9-tetrahydrocannabinol (THC) during adolescence self-administered higher amounts of heroin and had selective impairments of the enkephalin opioid system within the nucleus accumbens (NAc) implicated in reward-related behavior. To explore the ontogeny of the cannabinoid and opioid neuronal systems in association with adolescence THC exposure, rats were examined at different adolescent stages during an intermittent THC paradigm (1.5 mg/kg i.p. every third day) from postnatal days (PNDs) 28-49. Rat brains were examined 24 hours after injection at PND 29 (early adolescence), PND 38 (mid adolescence) and PND 50 (late adolescence) and analyzed for endocannabinoids (anandamide and 2-arachidonoylglycerol), Met-enkephalin, cannabinoid CB 1 receptors and µ opioid receptors (µOR) in the NAc, caudate-putamen and prefrontal cortex (PFC). Of the markers studied, the endocannabinoid levels had the most robust alterations throughout adolescence and were specific to the PFC and NAc. Normal correlations between anandamide and 2-arachidonoylglycerol concentrations in the NAc (positive) and PFC (negative) were reversed by THC. Other significant THC-induced effects were confined to the NAc -increased anandamide, decreased Met-enkephalin and decreased µORs. These findings emphasize the dynamic nature of the mesocorticolimbic endocannabinoid system during adolescence and the selective mesocorticolimbic disturbance as a consequence of adolescent cannabis exposure.