CHO细胞表达人源μ型阿片受体及其活性分析

μ型阿片受体在阿片类药物镇痛与成瘾中发挥重要作用 .从人脑组织总RNA通过一次反转录和两次PCR法扩增获得 μ型阿片受体的cDNA ,将其克隆至pcDNA3 1 (+)中 ,转染CHO细胞后 ,筛选单克隆细胞株并制备膜受体 ,检测重组细胞株表达的 μ型阿片受体与特异性配体的结合能力 .通过饱和性结合和竞争性结合试验证实 ,重组细胞株表达的 μ型阿片受体与天然的 μ型阿片受体具有基本一致的生物学特性 ,为进一步研究阿片受体与配体相互作用的分子机制打下了基础     

Opioid Receptor Trafficking and Signaling: What Happens After Opioid Receptor Activation?

Prolonged opioid treatment leads to a comprehensive cellular adaptation mediated by opioid receptors, a basis to understand the development of opioid tolerance and dependence. However, the molecular mechanisms underlying opioid-induced cellular adaptation remain obscure. Recent advances in opioid receptor trafficking and signaling in cells have extensively increased our insight into the network of intracellular signal integration. This review focuses on those important intracellular biochemical processes that play critical roles in the development of opioid tolerance and dependence after opioid receptor activation, and tries to explain what happens after opioid receptor activation, and how the cellular adaptation develops from cell membrane to nucleus. Decades of research have delineated a network on opioid receptor trafficking and signaling, but the challenge remains to explain opioid tolerance and dependence from a single cellular signal network.     

Binding mode characterization of 6α- and 6β-N-heterocyclic substituted naltrexamine derivatives via docking in opioid receptor crystal structures and site-directed mutagenesis studies: Application of the ‘message–address’ concept in development of mu opioid receptor selective antagonists

Highly selective opioid receptor antagonists are essential pharmacological probes in opioid receptor structural characterization and opioid agonist functional studies. Currently, there is no highly selective, nonpeptidyl and reversible mu opioid receptor antagonist available. Among a series of naltrexamine derivatives that have been designed and synthesized, two compounds, NAP and NAQ, were previously identified as novel leads for this purpose based on their in vitro and in vivo pharmacological profiles. Both compounds displayed high binding affinity and selectivity to the mu opioid receptor. To further study the interaction of these two ligands with the three opioid receptors, the recently released opioid receptor crystal structures were employed in docking studies to further test our original hypothesis that the ligands recognize a unique ‘address’ domain in the mu opioid receptor involving Trp318 that facilitates their selectivity. These modeling results were supported by site-directed mutagenesis studies on the mu opioid receptor, where the mutants Y210A and W318A confirmed the role of the latter in binding. Such work not only enriched the ‘message–address’ concept, also facilitated our next generation ligand design and development.     

Oligomerization of opioid receptors

Opioid receptors belong to the family of G-protein-coupled receptors characterized by their seven transmembrane domains. The activation of these receptors by agonists such as morphine and endogenous opioid peptides leads to the activation of inhibitory G-proteins followed by a decrease in the levels of intracellular cAMP. Opioid receptor activation is also associated with the opening of K(+) channels and the inhibition of Ca(2+) channels. A number of investigations, prior to the development of opioid receptor cDNAs, suggested that opioid receptor types interacted with each other. Early pharmacological studies provided evidence for the probable interaction between opioid receptors. More recent studies using receptor selective antagonists, antisense oligonucleotides, or animals lacking opioid receptors further suggested that interactions between opioid receptor types could modulate their activity. We examined opioid receptor interactions using biochemical, biophysical, and pharmacological techniques. We used differential epitope tagging and selective immunoisolation of receptor complexes to demonstrate homotypic and heterotypic interactions between opioid receptor types. We also used the proximity-based bioluminescence resonance energy transfer assay to explore opioid receptor-receptor interactions in living cells. In this article we describe the biochemical and biophysical methods involved in the detection of receptor dimers. We also address some of the concerns and suggest precautions to be taken in studies examining receptor-receptor interactions.     

Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization

Determining which domains and amino acid residues of the mu opioid receptor are phosphorylated is critical for understanding the mechanism of mu opioid receptor phosphorylation. The role of the C-terminus of the receptor was investigated by examining the C-terminally truncated or point-mutated mu opioid receptors in receptor phosphorylation and desensitization. Both wild-type and mutated receptors were stably expressed in Chinese hamster ovary (CHO) cells. The receptor expression was confirmed by receptor radioligand binding and immunoblottting. After exposure to 5 microM of DAMGO, phosphorylation of the C-terminally truncated receptor and the mutant receptor T394A was reduced to 40 and 10% of that of the wild-type receptor, respectively. Mutation effects on agonist-induced desensitization were studied using adenylyl cyclase inhibition assays. The C-terminally truncated receptor and mutant receptor T394A both showed complete loss of DAMGO-induced desensitization, while the mutant T/S-7A receptor only lost part of its ability to desensitize. Taken together, these results suggest that the C-terminus of the mu opioid receptor participates in receptor phosphorylation and desensitization with threonine 394, a crucial residue for both features. DAMGO-induced mu opioid receptor phosphorylation and desensitization are associated and appear to involve both the mu opioid receptor C-terminus and other domains of the receptor.     

14-O-Heterocyclic-substituted naltrexone derivatives as non-peptide mu opioid receptor selective antagonists: Design,synthesis, and biological studies

Mu opioid receptor antagonists have clinical utility and are important research tools. To develop non-peptide and highly selective mu opioid receptor antagonist, a series of 14-O-heterocyclic-substituted naltrexone derivatives were designed, synthesized, and evaluated. These compounds showed subnanomolar-to-nanomolar binding affinity for the mu opioid receptor. Among them, compound 1 exhibited the highest selectivity for the mu opioid receptor over the delta and kappa receptors. These results implicated an alternative ‘address’ domain in the extracellular loops of the mu opioid receptor.     

Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.     

The design and synthesis of a novel quinolizidine template for potent opioid and opioid receptor-like (ORL1, NOP) receptor ligands

A new class of high affinity opioid and opioid receptor-like receptor (ORL1 receptor, NOP receptor) ligands has been designed by conformational restriction of piperidine-based NOP receptor ligands, resulting in a novel quinolizidine scaffold. Different modifications of the pendant functional groups on the scaffold provide differential activities at the opioid and NOP receptors. While the conformational rigidity will provide an improved understanding of the NOP and opioid receptor binding pockets, these compounds also provide a new template for the design of novel opiate and NOP ligands.     

Differential down-regulation of delta opioid binding sites during physical dependence on methionine enkephalin in the rat

Post-synaptic receptor modulation is thought to be one important mechanism involved in the adaptation of a neuronal system during chronic exposure to a drug. However, initial studies of opioid receptor regulation following chronic in vivo administration of narcotic agonists, such as morphine, reported no down-regulation in the number of opioid receptors in the brain. Subsequent studies, employing in vitro preparations, have reported evidence of opioid receptor down-regulation under specific conditions. It remains to be determined whether the in vitro phenomena of opioid receptor plasticity is relevant to the intact mammalian central nervous system. The data in this report shows that chronic in vivo administration the opioid peptide methionine enkephalin, results in a significant, regionally specific down-regulation of delta opioid receptors in rat brain: 30% decrease in receptor density in the striatum; no change in hypothalamus.     

Structure selectivity relationship studies of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl)carboxamido]morphinan derivatives toward the development of the mu opioid receptor antagonists

Mu opioid receptor antagonists have been applied to target a variety of diseases clinically. The current study is designed to explore the structure selectivity relationship (SSR) of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl)carboxamido]morphinan (NAP), a lead compound identified as a selective mu opioid receptor antagonist based on the previous study. Among a series of NAP derivatives synthesized, compounds 6 (NMP) and 9 (NGP) maintained comparable binding affinity, selectivity and efficacy to the lead compound. Particularly, the mu opioid receptor selectivity over kappa opioid receptor of NGP was considerably enhanced compared to that of NAP. Overall, the preliminary SSR supported our original hypothesis that an alternate 'address' domain may exist in the mu opioid receptor, which favors the ligands carrying a hydrogen bond acceptor and an aromatic system to selectively recognize the mu opioid receptor.     

3-Aryl pyridone derivatives. Potent and selective kappa opioid receptor agonists.

A new series of 3-aryl pyridone based kappa opioid receptor agonists was designed and synthesised, based on an understanding of the classical kappa opioid receptor pharmacophore. The most potent of the new compounds were comparable to U-69,593 in receptor affinity, selectivity and functional agonist effect at the cloned human kappa opioid receptor.     

Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-type Calcium Channel Regulation

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, μ-, δ-, and κ-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The μ-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of μ-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.     

背根神经节神经元阿片受体和离子通道的研究进展

阿片及阿片受体与外周神经系统镇痛机制的研究,随着分子生物学技术的发展,已在受体的分子结构、形态学、分子药理学、离子通道和细胞内信号转导系统等方面取得了显著进展。μ、δ、κ阿片受体分子结构上的部分差异决定了它们各自的功能特征。三种受体在初级感觉神经元分布的比例不同,但都能介导细胞Ｃａ＾２＋通道的抑制和Ｋ＾＋电流增加及减少。阿片受体和通道之间由多种第二信使系统偶联。分子药理学研究表明它们还存在亚型受体     

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory G_i proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-x_L, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells.     

NPY-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats

The ability of neuropeptide Y to potently stimulate food intake is dependent in part upon the functioning of mu and kappa opioid receptors. The combined use of selective opioid antagonists directed against mu, delta or kappa receptors and antisense probes directed against specific exons of the MOR-1, DOR-1, KOR-1 and KOR-3/ORL-1 opioid receptor genes has been successful in characterizing the precise receptor subpopulations mediating feeding elicited by opioid peptides and agonists as well as homeostatic challenges. The present study examined the dose-dependent (5-80 nmol) cerebroventricular actions of general and selective mu, delta, and kappa1 opioid receptor antagonists together with antisense probes directed against each of the four exons of the MOR-1 opioid receptor gene and each of the three exons of the DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor genes upon feeding elicited by cerebroventricular NPY (0.47 nmol, 2 ug). NPY-induced feeding was dose-dependently decreased and sometimes eliminated following pretreatment with general, mu, delta, and kappa1 opioid receptor antagonists. Moreover, NPY-induced feeding was significantly and markedly reduced by antisense probes directed against exons 1, 2, and 3 of the MOR-1 gene, exons 1 and 2 of the DOR-1 gene, exons 1, 2, and 3 of the KOR-1 gene, and exon 3 of the KOR-3/ORL-1 gene. Thus, whereas the opioid peptides, beta-endorphin and dynorphin A(1-17) elicit feeding responses that are respectively more dependent upon mu and kappa opioid receptors and their genes, the opioid mediation of NPY-induced feeding appears to involve all three major opioid receptor subtypes in a manner similar to that observed for feeding responses following glucoprivation or lipoprivation.     

Mu opioid receptor-like sequences are present throughout vertebrate evolution

The sequence of the mu opioid receptor is highly conserved among human, rat, and mouse. In order to gain insights into the evolution of the mu opioid receptor, polymerase chain reaction (PCR) was used to screen genomic DNA from a number of different species using degenerate oligonucleotides which recognize a highly conserved region. DNA was assayed from representative species of both the protostome and deuterostome branches of the metazoan phylogenetic tree. Mu opioid receptor-like sequences were found in all vertebrate species that were analyzed. These species included bovine, chicken, bullfrog, striped bass, thresher shark, and Pacific hagfish. However, no mu opioid receptor-like sequences were detected from protostomes or from any invertebrates. The PCR results demonstrate that the region of the mu opioid receptor gene between the first intracellular loop and the third transmembrane domain (TM3) has been highly conserved during evolution and that mu opioid receptor-like sequences are present in the earliest stages of vertebrate evolution. Additional opioid receptor-like sequence was obtained from mRNA isolated from Pacific hagfish brain using rapid amplification of cDNA ends (RACE). The sequence of the Pacific hagfish was most homologous with the human mu opioid receptor (72% at the amino acid level between intracellular loop 1 and transmembrane domain 6) although over the same region high homology was also observed with the delta opioid receptor (69%), the kappa receptor (63%), and opioid receptor-like (ORL1) (59%). The hagfish sequence showed low conservation with the mammalian opioid receptors in the first and second extracellular loops but high conservation in the transmembrane and intracellular domains. Received: 5 January 1996 / Accepted: 7 March 1996     

Endogenous opioids and feeding behavior: A decade of further progress (2004–2014). A Festschrift to Dr. Abba Kastin

Functional elucidation of the endogenous opioid system temporally paralleled the creation and growth of the journal, Peptides, under the leadership of its founding editor, Dr. Abba Kastin. He was prescient in publishing annual and uninterrupted reviews on Endogenous Opiates and Behavior that served as a microcosm for the journal under his stewardship. This author published a 2004 review, “Endogenous opioids and feeding behavior: a thirty-year historical perspective”, summarizing research in this field between 1974 and 2003. The present review “closes the circle” by reviewing the last 10 years (2004–2014) of research examining the role of endogenous opioids and feeding behavior. The review summarizes effects upon ingestive behavior following administration of opioid receptor agonists, in opioid receptor knockout animals, following administration of general opioid receptor antagonists, following administration of selective mu, delta, kappa and ORL-1 receptor antagonists, and evaluating opioid peptide and opioid receptor changes in different food intake models.     

Opioid antagonist naltrexone disrupts feedback interaction between mu and delta opioid receptors in splenocytes to prevent alcohol inhibition of NK cell function

Naltrexone, an opioid antagonist, has been used in clinical trials to treat alcoholism. As the opioid peptides beta-endorphin and enkephalin increase splenic NK cell function in laboratory animals, it is anticipated that naltrexone treatment will cause immunosuppression. However, we report in this study that chronic naltrexone administration in laboratory rats increases the cytolytic activity of NK cells. It also prevents alcohol's suppressive effect on these cells. We identified that, in the splenocytes, delta opioid receptor expression is tightly controlled by negative feedback regulation of micro opioid receptors. Naltrexone disrupts this feedback control by reducing micro opioid receptor function, thereby up-regulating delta opioid receptor binding, which results in an enhanced NK cell cytolytic response to delta opioid receptor ligands. We conclude that naltrexone, which has been shown to be a promising agent for the clinical management of alcoholism, may have potential use in the treatment of immune deficiency in alcoholic and nonalcoholic patients.     

Drug design and synthesis of epsilon opioid receptor agonist: 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821) inducing antinociception mediated by putative epsilon opioid receptor

Here we report the new drug design and synthesis of a series of 6,14-endoethenomorphinan-7-carboxamide derivatives as a putative epsilon opioid receptor agonist. One of these compounds, 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821), showed agonistic activity for a putative epsilon opioid receptor (IC(50) = 71.71nM) in the rat vas deferens (RVD) preparations. TAN-821 stimulated the binding of the nonhydrolyzable guanosine 5'-triphosphate analog, guanosine 5'-(gamma-thio)-triphosphate (GTPgammaS), to the mouse pons/medulla membrane via the activation of putative epsilon opioid receptor. Moreover, TAN-821 given intracerebroventricularly (i.c.v.) produced a marked antinociception in the tail-flick test (ED(50) = 1.73 microg) and the hot-plate test (ED(50) = 2.05 microg) in a dose-dependent manner. The antinociception induced by TAN-821 administered i.c.v. was blocked by the i.c.v.-pretreatment with a putative epsilon opioid receptor partial agonist beta-endorphin [1-27], but not a mu opioid receptor antagonist beta-FNA, a delta opioid receptor antagonist NTI, or a kappa opioid receptor antagonist nor-BNI. The present results suggest that TAN-821 may be a useful tool for the investigation on the pharmacological properties of the putative epsilon opioid receptor.