Morphinans and isoquinolines: Acetylcholinesterase inhibition,pharmacophore modeling,and interaction with opioid receptors

Following indications from pharmacophore-based virtual screening of natural product databases, morphinan and isoquinoline compounds were tested in vitro for acetylcholinesterase (AChE) inhibition. After the first screen, active and inactive compounds were used to build a ligand-based pharmacophore model in order to prioritize compounds for biological testing. Among the virtual hits tested, the enrichment of actives was significantly higher than in a random selection of test compounds. The most active compounds were biochemically tested for their activity on μ, δ, and κ opioid receptors.     

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.     

Complex functional interaction between integrin receptors and ion channels

Integrin receptors mediate adhesion of the cell to the extracellular matrix and thereby regulate cell motility, proliferation, differentiation and apoptosis. These processes are frequently accompanied by alterations in ion flow. Recent evidence suggests that integrins can regulate ion channels and form macromolecular complexes, thus contributing to the localization of the channel onto the plasma membrane. The integrin-channel complex regulates downstream signaling proteins, such as tyrosine kinases and GTPases. This process could occur in plasma membrane microdomains, such as caveolae. It seems that ion channels sometimes transmit their signals through conformational coupling, instead of change in ion fluxes. Finally, the channel protein is not merely a final target, because it often feeds back by controlling integrin activation and/or expression. These findings have important implications for the physiology of normal and neoplastic cells and suggest interesting perspectives for studies of synaptic plasticity.     

Structure-activity relationships of bifunctional cyclic disulfide peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors

Prolonged opioid exposure increases the expression of cholecystokinin (CCK) and its receptors in the central nervous system (CNS), where CCK may attenuate the antinociceptive effects of opioids. The complex interactions between opioid and CCK may play a role in the development of opioid tolerance. We designed and synthesized cyclic disulfide peptides and determined their agonist properties at opioid receptors and antagonist properties at CCK receptors. Compound 1 (Tyr-c[d-Cys-Gly-Trp-Cys]-Asp-Phe-NH(2)) showed potent binding and agonist activities at delta and mu opioid receptors but weak binding to CCK receptors. The NMR structure of the lead compound displayed similar conformational features of opioid and CCK ligands.     

Trafficking of central opioid receptors and descending pain inhibition

The delta-opioid receptor (DOR) belongs to the superfamily of G-protein-coupled receptors (GPCRs) with seven transmembrane domains, and its membrane trafficking is regulated by intracellular sorting processes involving its C-tail motifs, intracellular sorting proteins, and several intracellular signaling pathways. In the quiescent state, DOR is generally located in the intracellular compartments in central neurons. However, chronic stimulation, such as chronic pain and sustained opioid exposure, may induce membrane trafficking of DOR and its translocation to surface membrane. The emerged functional DOR on cell membrane is actively involved in pain modulation and opioid analgesia. This article reviews current understanding of the mechanisms underlying GPCRs and DOR membrane trafficking, and the analgesic function of emerged DOR through membrane trafficking under certain pathophysiological circumstances.     

Drosophila gustatory receptors: from gene identification to functional expression

Recent years have seen long-awaited progress in understanding of the molecular mechanisms of taste perception in insects. The breakthrough came in the early 2000 with the identification of a novel family of candidate gustatory receptor (Gr) genes in the first release of the Drosophila melanogaster genome sequence. The 60 Gr genes are expressed in the subsets of gustatory neurons in the fly's taste organs and, without exception, encode heptahelical G protein-coupled receptors (GPCRs). Here I review our current knowledge about Gr genes and their products focusing on the newly emerging information regarding the function of the Gr-encoded proteins.     

Interactions between opioid and chemokine receptors: heterologous desensitization

The opioid and chemokine receptors are both members of the seven transmembrane G protein-coupled receptor (GPCR) superfamily. Desensitization is believed to be a major element of the regulation of the function of these receptors, and recent findings suggest that both agonist-dependent (homologous) desensitization and heterologous desensitization can control receptor activity. The cross-desensitization between opioid and chemokine receptors has significant implications for our understanding of both the regulation of leukocyte trafficking, as well as the regulation of chemokine receptor function in inflammatory disease states. We also review findings which suggest that pro-inflammatory chemokine receptor-induced heterologous desensitization of opioid receptors has important implications for the regulation of opioid receptor function in the nervous system.     

Types of opioid receptors: relation to antinociception

The endogenous opioid peptides are derived from three large precursors. Pro-opiocortin and proenkephalin yield [Met]enkephalin, carboxy-extended [Met]enkephalins and [Leu]enkephalin. The fragments of prodynorphin are all carboxy-extended [Leu]enkephalins. Three approaches are of importance for an analysis of the physiological functions of the different endogenous opioid peptides. First, since these peptides interact with more than one of the mu-, delta- and kappa-binding sites and thus with their receptors, it is necessary to synthesize peptides or non-peptides, which bind to only one of the sites. As far as narcotic analgesics are concerned, morphine fulfils these conditions since it interacts almost exclusively with the mu-receptor. Secondly, antagonists are required that are selective for only one of the opioid receptors, even when used in high concentrations. Finally, it is important to find circumscribed areas in the nervous system that possess only one type of opioid receptor. It is now known that in the rabbit cerebellum the opioid receptors are almost exclusively of the mu-type whereas in the guinea-pig cerebellum they are almost exclusively of the kappa-type.     

Synaptogenesis: a balancing act between excitation and inhibition

Recent studies have implicated a number of membrane-associated proteins, including the signaling pair neuroligin and beta-neurexin, in synapse formation, suggesting that they govern the ratio of inhibitory and excitatory synapses on CNS neurons. These findings, together with data indicating that the genes encoding neuroligin and PSD95 are altered in autism patients, suggest that a molecular understanding of complex neurological diseases is within reach.     

Subcellular distribution of the inositol 1,4,5-trisphosphate receptors: functional relevance and molecular determinants

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel that is for the largest part expressed in the endoplasmic reticulum. Its precise subcellular localization is an important factor for the correct initiation and propagation of Ca2+ signals. The relative position of the IP3Rs, and thus of the IP3-sensitive Ca2+ stores, to mitochondria, nucleus or plasma membrane determines in many cases the physiological consequences of IP3-induced Ca2+ release. Most cell types express more than one IP3R isoform and their subcellular distribution is cell-type dependent. Moreover, it was recently demonstrated that depending on the physiological status of the cell redistribution of IP3Rs and/or of IP3-sensitive Ca2+ stores could occur. This indicates that the cell must be able to regulate not only IP3R expression but also its distribution. The various proteins potentially determining IP3R localization and redistribution will therefore be discussed.     

Kentsin: tetrapeptide from hamster embryos produces naloxone-sensitive effects without binding to opioid receptors

The opioid nature of kentsin (Thr-Pro-Arg-Lys) and its ability to alter pain perception and intestinal transit were examined. Kentsin (30,000 nM) did not inhibit electrically stimulated contractions of the guinea pig ileum (GPI) or mouse vas deferens (MVD), nor did it cause a rightward displacement of the inhibitory concentration-response curves of the mu-selective opioid agonist PL017 in the GPI or the delta-selective agonist DPDPE in the MVD. Kentsin (10,000 nM) did not displace [3H] naloxone from rat brain homogenates. These results indicate that kentsin lacks opioid agonist and mu and delta opioid antagonist properties and does not bind to opioid receptors. In vivo, kentsin produced dose-dependent analgesia in both the hotplate and abdominal stretch tests when administered intracerebroventricularly (ICV) and intrathecally but not intravenously. The central analgesic effect of kentsin was partially antagonized by the opioid antagonist naloxone. Kentsin inhibited intestinal transit in a dose-dependent manner after ICV administration only. The intestinal antitransit effect of kentsin was not blocked by pretreatment with naloxone. These results suggest that kentsin acts centrally to produce both opioid and non-opioid effects. Further, the opioid-mediated analgesic effects of kentsin involve mechanisms other than direct interaction with opioid receptors.     

Molecular interaction in the mouse PAG between NMDA and opioid receptors in morphine-induced acute thermal nociception

Previous evidence demonstrates that low dose morphine systemic administration induces acute thermal hyperalgesia in normal mice through μOR stimulation of the inositol signaling pathway. We investigated the site of action of morphine and the mechanism of action of μOR activation by morphine to NMDA receptor as it relates to acute thermal hyperalgesia. Our experiments show that acute thermal hyperalgesia is blocked in periaqueductal gray with the μOR antagonist CTOP, the NMDA antagonist MK801 and the protein kinase C inhibitor chelerythrine. Therefore, a site of action of systemically administered morphine low dose on acute thermal hyperalgesic response appears to be located at the periaqueductal gray. At this supraspinal site, μOR stimulation by systemically morphine low dose administration leads to an increased phosphorylation of specific subunit of NMDA receptor. Our experiments show that the phosphorylation of subunit 1 of NMDA receptor parallels the acute thermal hyperalgesia suggesting a role for this subunit in morphine-induced hyperalgesia. Protein kinase C appears to be the key element that links μOR activation by morphine administration to mice with the recruitment of the NMDA/glutamatergic system involved in the thermal hyperalgesic response.     

μ Receptors and opioid cardiovascular effects in the NTS of rat

In order to assess the potential role of mu (μ) and delta (δ) opiate receptors in the central regulation of the cardiovascular and respiratory systems, the cardiovascular and respiratory effects of the relatively selective μ-opioid agonist D-Ala², MePhe⁴, Gly-ol⁵ enkephalin (DAGO) and relatively selective δ-agonist D-Ala²-D-Leu⁵ enkephalin (DADL) were compared following microinjection of these compounds into the nucleus tractus solitarius of pentobarbital-anesthetized rats. Both opioid agonists produced dose dependent increases in systolic and diastolic blood pressure as well as heart rate; but DAGO was nearly ten times more potent in eliciting these changes. Respiratory rate was increased by DADL and by lower doses of DAGO, but was depressed by higher doses of DAGO. Tidal volume was depressed by both peptides. These data support the concept that the cardiovascular pressor responses and tachycardia as well as the respiratory effects of opioids in the rat NTS are mediated by μ receptors.     

The molecular structure of opiate receptors

Examples are given which demonstrate that the opiate receptor can be separted from and subtypes by their physical parameters. When the subunit composition of the subtypes are compared, no definite differences are encountered. The data from the literature are also contradictory. This may in part be explained by the fact that the different receptors appear to contain a structurally common high affinity binding site. A possible hypothesis would be that the subtypes differ from each other by the number of subunits.Abbreviations CHAPS 3/cholamidopropyl-dimethylammonio 1-propansulfonate - DAGO D-Ala ²-Me-Phe⁴-Gly-ol⁵-enkephalin - DALA d-Ala²-Leu⁵-enkephalin - DALECK d-Ala²-Leu⁵-enkaphalin chloromethyl ketone - EGF Epidermal growth factor - EKC ethylketocyclazocine - SDS-PAGE Sodiumdodecylsulfate polyacrylamide gel elecctrophoresis Special Issue Dedicated to Dr. Abel Lajtha.     

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.     

Autoradiographic evidence for two classes of mu opioid binding sites in rat brain using [I]FK33824

Previous studies demonstrated that pretreatment of brain membranes with the irreversible mu antagonist, beta-funaltrexamine (beta-FNA), partially eliminated mu binding sites [25,35], consistent with the existence of two mu binding sites distinguished by beta-FNA. This paper tests the hypothesis that the FNA-sensitive and FNA-insensitive mu binding sites have different anatomical distributions in rat brain. Prior to autoradiographic visualization of mu binding sites, [³H]oxymorphone, [³H]D-ala²-MePhe⁴, Gly-ol⁵-enkephalin (DAGO), and [¹²⁵I]D-ala²-Me-Phe⁴-met(o)-ol]enkephalin (FK33824) were shown to selectively label mu binding sites using slide mounted sections of molded minced rat brain. As found using membranes, beta-FNA eliminated only a portion of mu binding sites. Autoradiographic visualization of mu binding sites using the mu-selective ligand [¹²⁵I]FK33824 in control and FNA-treated sections of rat brain demonstrated that the proportion of mu binding sites sensitive to beta-FNA varied across regions of the brain, particularly the dorsal thalamus, ventrobasal complex and the hypothalamus, providing anatomical data supporting the existence of two classes of mu binding sites in rat brain.     

The effect of the stimulation of opioid receptors on lymphocyte functional activity in vivo

With the aid of lymphocytes' spontaneous adhesion test and the reaction of blast transformation with phytohemagglutinin (PHA) the influence of met-enkephaline upon the functional activity of lymphocytes of healthy donors was studied in vitro. It was shown, that met-enkephaline stimulate lymphocyte spontaneous adhesion and the lymphocyte PHA-induced proliferative activity. The stimulating effects of met-enkephalin revealed in vitro were blocked by the opioid receptor blocker naloxone.     

Ethanol induced conformational changes of the peptide ligands for the opioid receptors and their relevance to receptor interaction

The FT-IR (Fourier Transform Infrared) Spectrum of [Met 5]-enkephalinamide in aqueous solution shows the presence of both the beta-turn and beta-sheet conformations. The beta-turn and beta-sheet conformations of enkephalins have been proposed to play a role in receptor selectivity. Addition of ethanol alters these secondary structural features and hence the effect of ethanol on ligand-receptor interaction may be mediated primarily through conformational changes of the ligand rather than those of the receptor.