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...
Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.
To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.
Repeated forced-swim stress (FSS) produced analgesia, immobility and potentiation of cocaine-conditioned place preference (CPP) in wild-type C57Bl/6 mice, but not in littermates lacking the kappa opioid receptor (KOR) gene. These results were surprising because kappa agonists are known to produce conditioned place aversion and to suppress cocaine-CPP when coadministered with cocaine. The possibility that disruption of the kappa system blocked the stress response by adversely affecting the hypothalamic-pituitary axis was examined by measuring plasma corticosterone levels. However, disruption of the dynorphin/kappa system by gene deletion or receptor antagonism did not reduce the FSS-induced elevation of plasma corticosterone levels. A second explanation for the difference is that kappa receptor activation caused by FSS occurred prior to cocaine conditioning rather than contemporaneously. To test this hypothesis, we measured the effects of the kappa agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488) administered to mice at various intervals preceding cocaine conditioning. The results showed that the interaction between the kappa system and cocaine reinforcement depended on the timing of the drug pairing. Mice given U50,488 60 min prior to cocaine showed a robust, nor-BNI-sensitive potentiation of cocaine-CPP, whereas administration 15 min before cocaine significantly suppressed cocaine-CPP. In the absence of cocaine, U50,488 given 60 min prior to saline conditioning produced no place preference, whereas administration 15 min before saline conditioning produced significant place aversion. The results of this study suggest that kappa receptor activation induced by FSS prior to the cocaine-conditioning session may be both necessary and sufficient for potentiation of the reinforcing actions of cocaine.
Pain remains a pervasive problem throughout medicine, transcending all specialty boundaries. Despite the extraordinary insights into pain and its mechanisms over the past few decades, few advances have been made with analgesics. Most pain remains treated by opiates, which have significant side effects that limit their utility. We now describe a potent opiate analgesic lacking the traditional side effects associated with classical opiates, including respiratory depression, significant constipation, physical dependence, and, perhaps most important, reinforcing behavior, demonstrating that it is possible to dissociate side effects from analgesia. Evidence indicates that this agent acts through a truncated, six-transmembrane variant of the G protein-coupled mu opioid receptor MOR-1. Although truncated splice variants have been reported for a number of G protein-coupled receptors, their functional relevance has been unclear. Our evidence now suggests that truncated variants can be physiologically important through heterodimerization, even when inactive alone, and can comprise new therapeutic targets, as illustrated by our unique opioid analgesics with a vastly improved pharmacological profile.opiate receptor | rewarding behavior | kappa 3 receptor T he utility of opioids in the management of pain is not disputed, but they come at a price. Along with their ability to relieve pain comes a variety of opioid receptor-mediated side effects, including respiratory depression, constipation, physical dependence, and reward behavior felt by many to contribute to their addictive potential. Most of the clinical opioids act through mu receptors, which mediate both analgesia and these side effects. Pharmacological studies have long suggested subtypes of mu receptors (1) and the possibility of dissociating analgesia from respiratory depression (2, 3), physical dependence (4), and the inhibition of gastrointestinal transit (5, 6). However, attempts to develop opiate analgesics that avoid these side effects have not been very fruitful. The isolation of a series of splice variants of the cloned mu opioid receptor from mice ( Fig. 1), rats, and humans with similar splicing patterns (7, 8) reveals a complexity far exceeding the pharmacological classification of mu receptor subtypes (1). However, this complexity has yet to be exploited in generating new classes of opioid analgesics. We now report an unexpected and unusual target for potent opioid analgesic drugs that lack respiratory depression, physical dependence, reward behavior, and significant constipation. ResultsRecently, we synthesized iodobenzoylnaltrexamide (IBNtxA), a naltrexone derivative (Fig. 2) (9). In vivo, it is a very potent analgesic (ED 50 = 0.48 ± 0.05 mg/kg s.c.) (Fig. 3A and Fig. S1), ∼10-fold more potent than morphine (4.6 ± 0.97 mg/kg s.c.) (10), with a mechanism of action quite distinct from traditional opiates. It was active s.c. as well as orally (Fig. S1), with a peak effect after oral administration that was delayed relative to parenteral administration. In ...
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