Tetrapeptides of primary sequence Tyr-X-Phe-YNH2, where X is D-Cys or D-Pen (penicillamine) and where Y is D-Pen or L-Pen, were prepared and were cyclized via the side chain sulfurs of residues 2 and 4 to disulfide or dithioether-containing analogs. These peptides are related to previously reported penicillamine-containing pentapeptide enkephalin analogs but lack the central glycine residue of the latter and were designed to assess the effect of decreased ring size on opioid activity. Binding affinities of the tetrapeptides were determined to both mu and delta opioid receptors. Binding affinity and selectivity in the tetrapeptide series were observed to be highly dependent on primary sequence. For example, L-Pen4 analogs displayed low affinity and were nonselective, while the corresponding D-Pen4 diastereomers were of variable affinity and higher selectivity. Among the latter compounds were examples of potent analogs in which selectivity shifted from delta selective to mu selective as the ring size was increased. The relatively high binding affinity and delta receptor selectivity observed with one of the carboxamide terminal disulfide analogs led to the synthesis of the corresponding carboxylic acid terminal, Tyr-D-Cys-Phe-D-PenOH. This analog displayed delta receptor binding selectivity similar to that of the standard delta ligand, [D-Pen2,D-Pen5]enkephalin (DPDPE), and was found to have a 3.5-fold higher binding affinity than DPDPE. All the tetrapeptides were further evaluated in the isolated mouse vas deferens (mvd) assay and all displayed opioid agonist activity. In general, tetrapeptide potencies in the mouse vas deferens correlated well with binding affinities but were somewhat lower. Receptor selectivity in the mvd, assessed by examining the effect of opioid antagonists on the tetrapeptide concentration-effect curves, was similar to that determined in the binding studies.
SUMMARYIn C 6 glial cells stably expressing rat -opioid receptor, opioid agonist activation is negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. H]DAMGO binding in membranes with the rank order of etorphine Ͼ DAMGO ϭ -endorphin Ͼ morphine Ͼ butorphanol, and the affinity of DAMGO in alkaloid-but not peptide-treated membranes was significantly lower in comparison with control. Pertussis toxin treatment of the cells before agonist treatment did not prevent the down-regulation by full agonists; DAMGO and etorphine exhibited ϳ80% internalization, whereas the ability of partial agonists was greatly impaired. In addition to establishing this cell line as a good model for further studies on the mechanisms of opioid tolerance, these results indicate important differences in the inactivation pathways of receptor triggered by full and partial agonists.Opioid receptors are activated by endogenous opioid peptides and alkaloids, which cause a multitude of important physiological functions. Recent cloning of -, ␦-, and -opioid receptors showed that these proteins contain seven transmembrane domains and belong to the family of GPCRs (1). The -opioid receptor is the molecular target for potent analgesics such as morphine and fentanyl, which are indispensable in the management of pain despite their abuse potential (2). The biochemical mechanisms of tolerance have been studied in many systems, including cell lines containing ␦-opioid receptors such as N4TG1 (3) and NG108 -15 (4) cells. Although studies conducted in the central nervous system often led to inconsistent results due to the heterogeneity of the system, experiments carried out in a single brain region, such as locus ceruleus, demonstrated the physiological relevance of the cellular model originally proposed by Sharma et al. (5) in NG108 -15 cells based on the alterations in the opioid/AC system. Subsequent studies using 7315c (6) and SH-SY5Y (7, 8) cells examined altered properties of -opioid receptor/effector components during tolerance; however, the exact mechanisms involved in this process are largely unknown. To study the molecular mechanisms of -opioid receptor selectively, we transfected C 6 glial cells that express many other receptors, but not opioid receptors (9), with the rat receptor cDNA. Transfected receptor in these cells is coupled to AC through PTX-sensitive G proteins (10). We characterized opioid agonist efficacies (11) and showed that this cell line exhibits sodium regulation of receptor in much the same fashion as SH-SY5Y cells (12).The major goal of the current study was to investigate the molecular changes involved in the development of tolerance by different agonists of varying efficacies. In the C 6 cell line stably expressing high levels of receptor (ϳ8 pmol/mg), tolerance to peptides and alkaloids was induced, and alterations were examined at every step of the signal transduction pathway (i.e., ligand/receptor interactions, G protein and effector functions). The diminished receptor activation of G protein, ...
The enantiomeric (-)- and (+)-N-(methyl through decyl) normetazocines (5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans) were synthesized and their in vitro and in vivo activities determined. Increasingly bulky enantiomeric N-alkyl homologs were prepared until their interaction with the sigma 1 receptor decreased and their insolubility became a hindrance to their evaluation in vivo and/or in vitro. The (-)-methyl, -pentyl, -hexyl, and -heptyl homologs were essentially as potent as, or more potent than, morphine in the tail-flick, phenylquinone, and hot-plate assays for antinociceptive activity; the (-)-propyl homolog had narcotic antagonist activity between that of nalorphine and naloxone in the tail-flick vs morphine assay, and it also displayed antagonist properties in the single-dose suppression assay in the rhesus monkey. The antinociceptively potent (-)-heptyl homolog did not substitute for morphine in monkeys but did show morphine-like properties in a primary physical-dependence study in continuously infused rats. All five potent compounds showed high affinity for the mu opioid receptor from both rat and monkey preparations and the kappa opioid receptor (< 0.05 microM), and all except the (-)-methyl homolog interacted reasonably well at the delta receptor (K(i) < 0.1 microM). The (-)-propyl compound was equipotent (K(i) 1.5-2.0 nM) at mu and kappa receptors. The pattern of interaction of the (-)-enantiomeric homologs with mu receptors from rat and monkey preparations was similar, but not identical. The enantioselectivity of the homologs for mu receptors was greater in the rat than in the monkey preparation for all but the N-H and butyl compounds, and the enantioselectivity of the lower homologs (methyl through butyl) for the mu (monkey) receptor was greater than for the kappa or delta receptors. However, bulkier homologs (hexyl through decyl) displayed higher enantioselectivity at kappa or delta receptors than at the mu (monkey) receptor. The (+)-butyl through (+)-octyl homologs were essentially equipotent with, or more potent than, (+)-pentazocine at the sigma receptor. Only the (+)-H and (+)-methyl homologs had high affinity (< 0.05 microM) at PCP binding sites.
In membranes from SH-SY5Y human neuroblastoma cells differentiated with retinoic acid, the ,u-selective agonist Tyr-D-Ala-Gly-N-Me,-Phe-Gly-ol (DAMGO) inhibited cAMP formation with an IC5o of 26 nM. Two separate antibodies raised against distinct regions of the Go. sequence attenuated the effect of DAMGO by 50-60%, whereas antibodies to Gial,2 or G1a3 reduced the ,u-opioid signal insignificantly or to a lesser extent. In contrast, inhibition of adenylyl cyclase by the 8-opioid agonist Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE; Pen = peniciliamine) was very sensitive to the Gial,2 antibody. In membranes from rat brain striatum, coupling of the 1t opioid receptor to adenylyl cyclase was also maximally blocked by antibodies to G... After long-term treatment of the cells with DAMGO, the content of Goa was reduced by 26%, whereas the levels of G1.1,2, G1.3, and Gsa were unaltered.
Phencyclidine (PCP) displaceable binding of 3H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced 3H-PCP in both rat and pigeon brain membranes with an EC50 of 1 microM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of 3H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound 3H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing 3H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of 3H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60-70 nM and 300-400 nM, respectively.
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