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Major depressive disorder is a highly common disorder, with a lifetime prevalence in the United States of approximately 21%. Traditional antidepressant treatments are limited by a delayed onset of action and minimal efficacy in some patients. Ketamine is effective and fast-acting, but there are concerns over its abuse liability. Thus, there is a need for safe, fast-acting antidepressant drugs. The opioid buprenorphine shows promise but also has abuse liability due to its mu-agonist component. Preclinical evidence indicates that the delta-opioid system contributes to mood disorders, and delta-opioid agonists are effective in preclinical models of depression- and anxiety-like states. In this study, we test the hypothesis that the mu-opioid antagonist diprenorphine by virtue of its partial delta opioid agonist activity may offer a beneficial profile for an antidepressant medication without abuse liability. Diprenorphine was confirmed to bind with high affinity to all three opioid receptors, and functional experiments for G protein activation verified diprenorphine to be a partial agonist at delta- and kappa-opioid receptors and a mu-antagonist. Studies in C57BL/6 mice demonstrated that an acute dose of diprenorphine produced antidepressant-like effects in the tail suspension test and the novelty-induced hypophagia test that were inhibited in the presence of the delta-selective antagonist, naltrindole. Diprenorphine did not produce convulsions, a side effect of many delta agonists but rather inhibited convulsions caused by the full delta agonist SNC80; however, diprenorphine did potentiate pentylenetetrazole-induced convulsions. Diprenorphine, and compounds with a similar pharmacological profile, may provide efficient and safe rapidly acting antidepressants. SIGNIFICANCE STATEMENT: The management of major depressive disorder, particularly treatment-resistant depression, is a significant unmet medical need. Here we show that the opioid diprenorphine, a compound with mu-opioid receptor antagonist activity and delta- and kappa-opioid receptor partial agonist activities, has rapid onset antidepressant-like activity in animal models. Diprenorphine and compounds with a similar pharmacological profile to diprenorphine should be explored as novel antidepressant drugs., (U.S. Government work not protected by U.S. copyright.)

Oxytocin (OT) influences various physiological functions such as uterine contractions, maternal/social behavior, and analgesia. Opioid signaling pathways are involved in one of the analgesic mechanisms of OT. We previously showed that OT acts as a positive allosteric modulator (PAM) and enhances μ-opioid receptor (MOR) activity. In this study, which focused on other opioid receptor (OR) subtypes, we investigated whether OT influences opioid signaling pathways as a PAM for δ-OR (DOR) or κ-OR (KOR) using human embryonic kidney-293 cells expressing human DOR or KOR, respectively. The CellKey TM results showed that OT enhanced impedance induced by endogenous/exogenous KOR agonists on KOR-expressing cells. OT did not affect DOR activity induced by endogenous/exogenous DOR agonists. OT potentiated the KOR agonist-induced Gi/o protein-mediated decrease in intracellular cAMP, but did not affect the increase in KOR internalization caused by the KOR agonists dynorphin A and (-)-U-50488 hydrochloride (U50488). OT did not bind to KOR orthosteric binding sites and did not affect the binding affinities of dynorphin A and U50488 for KOR. These results suggest that OT is a PAM of KOR and MOR and enhances G protein signaling without affecting β-arrestin signaling. Thus, OT has potential as a specific signaling-biased PAM of KOR.

Aerial translocation of captured black rhinoceroses (Diceros bicornis) has been accomplished by suspending them by their feet. We expected this posture would compromise respiratory gas exchange more than would lateral recumbency. Because white rhinoceroses (Ceratotherium simum) immobilized with etorphine alone are hypermetabolic, with a high rate of carbon dioxide production (VCO2), we expected immobilized black rhinoceroses would also have a high VCO2. Twelve (nine male, three female; median age 8 yr old [range: 4-25]; median weight 1,137 kg [range: 804-1,234] body weight) wild black rhinoceroses were immobilized by aerial darting with etorphine and azaperone. The animals were in lateral recumbency or suspended by their feet from a crane for approximately 10 min before data were collected. Each rhinoceros received both treatments sequentially, in random order. Six were in lateral recumbency first and six were suspended first. All animals were substantially hypoxemic and hypercapnic in both postures. When suspended by the feet, mean arterial oxygen pressure (PaO2) was 42 mm Hg, 4 mm Hg greater than in lateral recumbency (P=0.030), and arterial carbon dioxide pressure (PaCO2) was 52 mm Hg, 3 mm Hg less than in lateral recumbency (P=0.016). Tidal volume and minute ventilation were similar between postures. The mean VCO2 was 2 mL/kg/min in both postures and was similar to, or marginally greater than, VCO2 predicted allometrically. Suspension by the feet for 10 min did not impair pulmonary function more than did lateral recumbency and apparently augmented gas exchange to a small degree relative to lateral recumbency. The biological importance in these animals of numerically small increments in PaO2 and decrements in PaCO2 with suspension by the feet is unknown. Black rhinoceroses immobilized with etorphine and azaperone were not as hypermetabolic as were white rhinoceroses immobilized with etorphine., (© Wildlife Disease Association 2021.)

Ligand efficiency indices are widely used to guide chemical optimization in drug discovery, due to their predictive value in the early steps of optimization. At later stages, however, as more complex properties become critical for success, indices relying on calculated, rather than experimental, parameters become less informative. This problem is particularly acute when developing positron emission tomography (PET) imaging agents, for which nonspecific binding (NSB) to membranes and non-target proteins is a frequent cause of failure. NSB cannot be predicted using in silico parameters. To address this gap, we explored the use of the experimentally determined chromatographic hydrophobicity index on immobilized artificial membranes, CHI(IAM), to guide the optimization of NSB. The ligand specific efficiency (LSE) index was defined as the ratio between affinity (pIC50 or pKd ) and the logarithmic value of CHI(IAM). It allows for quantification of binding affinity to the target of interest, relative to NSB. Its use was illustrated by the optimization of PET tracer candidates for the prostacyclin receptor., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Opiate receptors are found in the brain, the spinal cord, some peripheral sensory neurons, and the gastrointestinal tract. Naturally occurring and synthetic opiate ligands exert their influence on a wide variety of processes including analgesia, euphoria, dysphoria, sedation, respiratory depression, and miosis and are frequently topics for discussions on addiction and physical dependence. This review looks at the history of positron emission tomography radioligands for probing this receptor system., (Copyright © 2013 John Wiley & Sons, Ltd.)

Opioids that stimulate the μ-opioid receptor (MOR1) are the most frequently prescribed and effective analgesics. Here we present a structural model of MOR1. Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop that couples with the G protein complex. These simulations likewise identified residues that form frequent contacts with ligands. We validated the binding residues using site-directed mutagenesis coupled with radioligand binding and functional assays. The model was used to blindly screen a library of ∼1.2 million compounds. From the 34 compounds predicted to be strong binders, the top three candidates were examined using biochemical assays. One compound showed high efficacy and potency. Post hoc testing revealed this compound to be nalmefene, a potent clinically used antagonist, thus further validating the model. In summary, the MOR1 model provides a tool for elucidating the structural mechanism of ligand-initiated cell signaling and for screening novel analgesics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Buprenorphine is a partial μ-opioid receptor agonist used for the treatment of opioid dependence that has several advantages over methadone. The principal route of buprenorphine disposition has been well established; however, little is known regarding the potential for buprenorphine to influence the metabolism and clearance of other drugs by affecting the expression of drug-metabolizing enzymes (DMEs). Here, we investigate the effects of buprenorphine on the activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), as well as the induction of DMEs, in both HepG2 cells and human primary hepatocytes (HPHs). In HepG2 cells, buprenorphine significantly increased human PXR-mediated CYP2B6 and CYP3A4 reporter activities. CYP2B6 reporter activity was also enhanced by buprenorphine in HepG2 cells cotransfected with a chemical-responsive human CAR variant. Real-time reverse transcription-polymerase chain reaction analysis revealed that buprenorphine strongly induced CYP3A4 expression in both PXR- and CAR-transfected HepG2 cells. However, treatment with the same concentrations of buprenorphine in HPHs resulted in literally no induction of CYP3A4 or CYP2B6 expression. Further studies indicated that buprenorphine could neither translocate human CAR to the nucleus nor activate CYP2B6/CYP3A4 reporter activities in transfected HPHs. Subsequent experiments to determine whether the differential response was due to buprenorphine's metabolic stability revealed a dramatically differential rate of elimination for buprenorphine between HPHs and HepG2 cells. Taken together, these studies indicate that metabolic stability of buprenorphine defines the differential induction of DMEs observed in HepG2 and HPHs, and the results obtained from PXR and CAR reporter assays in immortalized cell line require cautious interpretation.

This longitudinal, case-control pilot study examined amygdala growth in rhesus macaque infants receiving the complete US childhood vaccine schedule (1994-1999). Longitudinal structural and functional neuroimaging was undertaken to examine central effects of the vaccine regimen on the developing brain. Vaccine-exposed and saline-injected control infants underwent MRI and PET imaging at approximately 4 and 6 months of age, representing two specific timeframes within the vaccination schedule. Volumetric analyses showed that exposed animals did not undergo the maturational changes over time in amygdala volume that was observed in unexposed animals. After controlling for left amygdala volume, the binding of the opioid antagonist [(11)C]diprenorphine (DPN) in exposed animals remained relatively constant over time, compared with unexposed animals, in which a significant decrease in [(11)C]DPN binding occurred. These results suggest that maturational changes in amygdala volume and the binding capacity of [(11)C]DPN in the amygdala was significantly altered in infant macaques receiving the vaccine schedule. The macaque infant is a relevant animal model in which to investigate specific environmental exposures and structural/functional neuroimaging during neurodevelopment.

Conclusions based on either in vitro or in vivo approach to evaluate the P-gp affinity status of opioids may be misleading. For example, in vitro studies indicated that fentanyl is a P-gp inhibitor while in vivo studies indicated that it is a P-gp substrate. Quite the opposite was evident for meperidine. The objective of this study was to evaluate the P-gp affinity status of methadone, buprenorphine and diprenorphine to predict P-gp-mediated drug-drug interactions and to determine a better candidate for management of opioid dependence. Two in vitro (P-gp ATPase and monolayer efflux) assays and two in vivo (tissue distribution and antinociceptive evaluation in mdr1a/b (-/-) mice) assays were used. Methadone stimulated the P-gp ATPase activity only at higher concentrations, while verapamil and GF120918 inhibited its efflux (p < 0.05). The brain distribution and antinociceptive activity of methadone were enhanced (p < 0.05) in P-gp knockout mice. Conversely, buprenorphine and diprenorphine were negative in all assays. P-gp can affect the PK/PD of methadone, but not buprenorphine or diprenorphine. Our report is in favor of buprenorphine over methadone for management of opioid dependence. Buprenorphine most likely is not a P-gp substrate and concerns regarding P-gp-mediated drug-drug interaction are not expected., (2009 Wiley-Liss, Inc. and the American Pharmacists Association)

Opioid efficacy on mu-receptor may be influenced by various Gi/o-G-protein subunits interacting with intracellular face of receptor. Pertussis toxin-insensitive Galphai1 and Galphai2 subunits tethered with mu-receptor were stably transfected into AtT20 cells to (i) determine coupling of different alpha-subunits on opioid efficacy, and (ii) determine coupling to downstream effectors, for example, calcium and potassium channels. After pertussis toxin, stimulation of [35S]GTP-gamma-S incorporation persisted. Both constructs were able to couple to native calcium and potassium channels, with endomorphins 1 and 2 equally effective. However, pertussis toxin abolished opioid actions on calcium and potassium channels suggesting strong coupling to endogenous G-proteins, and that differences in coupling efficacy to Galphai1 and Galphai2 previously observed are restricted to initial step of signaling cascade.

We previously demonstrated that D3.49(164)Y or T6.34(279)K mutation in the rat mu opioid receptor (MOPR) resulted in agonist-independent activation. Here, we identified the cysteine(s) within the transmembrane domains (TMs) of the D3.49(164)Y mutant that became accessible in the binding-site crevice by use of methanethiosulfonate ethylammonium (MTSEA) and inferred conformational changes associated with receptor activation. While the C7.38(321)S mutant was insensitive to MTSEA, the D3.49(164)Y/C7.38(321)S mutant showed similar sensitivity as the D3.49(164)Y, suggesting that, in the D3.49(164)Y mutant, C7.38(321) becomes inaccessible while other cysteines are accessible in the binding-site crevice. Each of the other seven cysteines in the TMs was mutated to serine on the background of D3.49(164)Y/C7.38(321)S, and the resulting triple mutants were evaluated for [3H]diprenorphine and [d-Ala2,NMe-Phe4,Gly5-ol]-enkephalin (DAMGO) binding and effect of MTSEA on [3H]diprenorphine binding. The D3.49(164)Y/C7.38(321)S mutant and the triple mutants, except the C6.47(292)S triple mutant, retained similar affinities for [3H]diprenorphine and DAMGO as the D3.49(164)Y mutant. The second-order rate constants for MTSEA reactions showed that C3.44(159)S, C4.48(190)S, C5.41(235)S, and C7.47(330)S significantly reduced sensitivity to MTSEA, compared with the D3.49(164)Y/C7.38(321)S. These results suggest that the four cysteines may be rotated and/or tilted to become accessible. While the D3.49(164)Y/C7.38(321)S was similarly sensitive to MTSEA as the D3.49(164)Y mutant, the T6.34(279)K/C7.38(321)S was much less sensitive to MTSEA than the T6.34(279)K mutant, suggesting that the two constitutively active mutants assume different conformations and/or possess different dynamic properties. Molecular models of the MOPR monomer and homodimer, using the crystal structures of rhodopsin, the beta2-adrenergic receptor, and the ligand-free opsin, which contains several features characteristic of the active state, were employed to analyze these experimental results in a structural context.

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR*) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR* is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR* was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.

Ubiquitination of the human kappa opioid receptor (hKOR) expressed in Chinese hamster ovary (CHO) cells was observed in the presence of the proteasomal inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and enhanced by the agonists (-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny) cyclohexyl] benzeneacetamide (U50,488H) and dynorphin A (Dyn A). The dominant-negative (DN) mutants GRK2-K220R and beta-arrestin (319-418), but not dynamin I-K44A, reduced Dyn A-stimulated hKOR ubiquitination, and a phosphorylation-defective hKOR mutant (hKOR-S358N) did not undergo Dyn A-stimulated ubiquitination, indicating that hKOR ubiquitination is enhanced by receptor phosphorylation but not by receptor internalization. A hKOR mutant (hKOR-10 KR) in which all 10 intracellular Lys residues were changed to Arg showed greatly reduced basal and agonist-promoted receptor ubiquitination and substantially decreased Dyn A-induced receptor down-regulation, without changing ligand binding affinity, receptor-G protein coupling, or receptor internalization or desensitization. The ubiquitination sites were further determined to be the three Lys residues in the C-terminal domain. The K63R ubiquitin mutant decreased Dyn A-induced hKOR ubiquitination and down-regulation, but the K48R mutant did not. Expression of HN-CYLD, a DN mutant of deubiquitination enzyme cylindromatosis tumor suppressor gene (CYLD) that breaks Lys63-linked polyubiquitin chain, increased Dyn A-induced hKOR ubiquitination and down-regulation. These results indicate that ubiquitinated hKOR after agonist treatment contains predominantly Lys63-linked polyubiquitin chains and ubiquitination of the hKOR involved in agonist-induced down-regulation.

Ligand-induced regulation of the rat kappa-opioid receptor (rKOR) was investigated in human embryonic kidney 293 cells stably expressing the FLAG-tagged rKOR. Incubation of rKOR cells with naltrexone for 24 h increased the B(max) >3-fold, with no change in the affinity of [(3)H]diprenorphine. Two immunoreactive receptor species were present in cell lysates: naltrexone treatment caused a >3-fold increase in the 52-kDa species while decreasing the level of the 42-kDa species. Dynorphin(1-13), U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)benzeneacetamide], or salvinorin A [2S,4aR,6aR,7R,9S,10aS, 10bR)-9-(acetyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1c]pyran-7-carboxylic acid methyl ester] treatment did not alter the level of immunoreactive rKOR protein, whereas etorphine, cyclazocine, naloxone, and naloxone methiodide increased the 52-kDa and decreased the 42-kDa rKOR bands. Receptor up-regulation was associated with an increase in the number of cell surface receptors and a 2-fold increase in the E(max) for guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Glycosidase digestion indicated that the 52- and 42-kDa receptors contained complex and high-mannose N-glycans, respectively, Pulse-chase analysis and glycosidase digestion sensitivities suggested that the 42-kDa rKOR species was a precursor of the 52-kDa species. Naltrexone did not alter rKOR mRNA levels or translational efficiency, and rKOR up-regulation was not inhibited by cycloheximide. Brefeldin A caused accumulation of intracellular rKOR intermediates, and coincubation with naltrexone increased the levels of the brefeldin-induced species significantly. These results suggest that select opioid ligands up-regulate rKOR by enhancing the rate of receptor folding and maturation and by protecting the receptor from degradation, resulting in an increase in the number of rKOR binding sites, immunoreactive protein, and functional receptors.

We investigated the effects of morphine and other agonists on the human mu opioid receptor (MOP) expressed in M2 melanoma cells, lacking the actin cytoskeleton protein filamin A and in A7, a subclone of the M2 melanoma cells, stably transfected with filamin A cDNA. The results of binding experiments showed that after chronic morphine treatment (24 h) of A7 cells, MOP-binding sites were down-regulated to 63% of control, whereas, unexpectedly, in M2 cells, MOP binding was up-regulated to 188% of control naive cells. Similar up-regulation was observed with the agonists methadone and levorphanol. The presence of antagonists (naloxone or CTAP) during chronic morphine treatment inhibited MOP down-regulation in A7 cells. In contrast, morphine-induced up-regulation of MOP in M2 cells was further increased by these antagonists. Chronic morphine desensitized MOP in A7 cells, i.e., it decreased DAMGO-induced stimulation of GTPgammaS binding. In M2 cells DAMGO stimulation of GTPgammaS binding was significantly greater than in A7 cells and was not desensitized by chronic morphine. Pertussis toxin treatment abolished morphine-induced receptor up-regulation in M2 cells, whereas it had no effect on morphine-induced down-regulation in A7 cells. These results indicate that, in the absence of filamin A, chronic treatment with morphine, methadone or levorphanol leads to up-regulation of MOP, to our knowledge, the first instance of opioid receptor up-regulation by agonists in cell culture.

We examined glycosylation of FLAG-hKOR expressed in CHO cells and determined its functional significance. FLAG-hKOR was resolved as a broad and diffuse 55-kDa band and a less diffuse 45-kDa band by immunoblotting, indicating that the receptor is glycosylated. Endoglycosidase H cleaved the 45-kDa band to approximately 38 kDa but did not change the 55-kDa band, demonstrating that the 45-kDa band is N-glycosylated with high-mannose or hybrid-type glycan. Peptide-N-glycosidase F digestion of solubilized hKOR or incubation of cells with tunicamycin resulted in two species of 43 and 38 kDa, suggesting that the 43-kDa band is O-glycosylated. FLAG-hKOR was reduced to lower Mr bands by neuraminidase and O-glycosidase, indicating that the hKOR contains O-linked glycan. Mutation of Asn25 or Asn39 to Gln in the N-terminal domain reduced the Mr by approximately 5 kDa, indicating that both residues were glycosylated. The double mutant hKOR-N25/39Q was resolved as a 43-kDa (mature form) and a 38-kDa (intermediate form) band. When transiently expressed, hKOR-N25/39Q had a lower expression level than the wild type. In CHO cells stably expressing the hKOR-N25/39Q, pulse-chase experiments revealed that the turnover rate constants (ke) of the intermediate and mature forms were approximately 3 times those of the wild type. In addition, the maturation rate constant (ka) of the 43-kDa form of hKOR-N25/39Q was 6 times that of the mature form of the wild type. The hKOR-N25/39Q mutant showed increased agonist-induced receptor phosphorylation, desensitization, internalization, and downregulation, without changing ligand binding affinity or receptor-G protein coupling. Thus, N-glycosylation of the hKOR plays important roles in stability and trafficking along the biosynthesis pathway of the receptor protein as well as agonist-induced receptor regulation.

Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5M Na(2)CO(3) buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30min shifted approximately 25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-beta-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [(35)S]GTPgammaS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, G(alphai) co-immunoprecipitated with caveolin-1, which was shown to inhibit G(alphai/o), and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor.

Opioids, acting via G-protein coupled membrane receptors, induce analgesia. However their role is not limited to their anti-nociceptive action. They are found in several peripheral tissues acting as negative regulators of cellular processes. Even though that is not fully elucidated, it becomes obvious that opioids exert their effects in close relation to other neuropeptides such as somatostatin. Hepatocellular carcinoma is one tumor, among others, which secrete bioactive peptides while somatostatin analogs exert an inhibitory effect. We have used the human hepatocyte-derived cancer cell line HepG2, in order to examine the effect of opioids on cell growth and their possible mode of action. Our results show that the opioid ethylketocyclazocine (EKC) inhibits cell proliferation and induces apoptosis. This inhibitory effect is not exerted via opioids receptors since it was not reversed by the opioid antagonist diprenorphine and functional opioid receptors were not found on HepG2 cells. On the contrary, we show that EKC binds to somatostatin receptors, and activates a PTP signalling cascade. In this respect, the interaction of opioids with somatostatin receptors on hepatocellular carcinoma cells, and the fact that they are widely used for pain control, may provide some additional clues for the discrepancies during treatment with somatostatin analogues.

The pharmacological profile of opioid-binding sites in zebrafish brain homogenates has been studied using radiolabeled binding techniques. The nonselective antagonist [(3)H]diprenorphine binds with high affinity (K(D) = 0.27 +/- 0.08 nM and a B(max) = 212 +/- 14.3 fmol/mg protein), displaying two different binding sites with affinities of K(D1) = 0.08 +/- 0.02 nM and K(D2) = 17.8 +/- 9.18 nM. The nonselective agonist [(3)H]bremazocine also binds with high affinity to zebrafish brain membranes but only displays one single binding site with a K(D) = 1.1 +/- 0.09 nM and a B(max) = 705 +/- 19.3 fmol/mg protein. Competition binding assays using [(3)H]diprenorphine and several unlabeled ligands were performed. The synthetic selective agonists for mammalian opioid receptors DPDPE ([DPen(2),D-Pen(5)]-enkephalin), DAMGO ([D-Ala(2),NMe-Phe(4),Gly(5)-ol]-enkephalin), and U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] failed to effectively displace [(3)H]diprenorphine binding, whereas nonselective ligands and the endogenous opioid peptides such as dynorphin A showed good affinities in the nanomolar range, although several of the endogenous peptides only displaced approximately 50% of the specifically bound [(3)H]diprenorphine. Our results provide evidence that, although the selective synthetic compounds for mammalian receptors do not fully recognize the opioid-binding sites in zebrafish brain, the activity of the endogenous zebrafish opioid system might not significantly differ from that displayed by the mammalian opioid system. Hence, the study of zebrafish opioid activity may contribute to an understanding of endogenous opioid systems in higher vertebrates.

Matrix metalloproteinases (MMP) are ubiquitous enzymes involved in extracellular matrix remodeling, and as a consequence in a number of physiological and pathological states, including development, wound healing and cancer. A crucial feature of cancer progression and metastasis is the disruption of extracellular matrix, and spreading of proliferating cancer cells. Modulation of MMP is a main target of cancer research. Using the mouse fibrosarcoma cell line WEHI 164, producing high amounts of MMP-2, we investigated whether we could modulate its production. We report that MMP-2 is under the control of nitric oxide (NO)/nitric oxide synthase (NOS) system. In addition, we show that NOS activity is controlled by opioids in a non-opioid receptor-related manner. Finally, we provide evidence that morphine, when administrated at low, non-toxic concentrations (<10(-9) M) attenuates MMP-2 activity. We conclude that, as morphine is able to decrease metalloproteinase activity via the NO/NOS system, it may have a place in the treatment of several sarcomas including fibrosarcoma.

Unlabelled: The tracer 6-O-(2-(18)F-fluoroethyl)-6-O-desmethyldiprenorphine (18F-FDPN) provides enhanced flexibility to PET studies of the opioidergic system because the label has a longer half-life than the label of 11C-diprenorphine. Here we evaluated the ideal length of PET studies with 18F-FDPN., Methods: 18F-FDPN binding kinetics were quantified with protocols of different lengths by use of a 1-tissue or a 2-tissue compartment model for different volumes of interest. Furthermore, the effects of scanning duration were assessed by parametric analyses., Results: A 90-min protocol resulted in less than 10% bias in distribution volume (DV) relative to the full-length protocol. Correlation analyses of the DV estimates for the full-length protocol and the shortened protocols showed good replication of DV estimates for regions with both low and high levels of binding at schedules of up to 90 min., Conclusion: Data sampling in dynamic 18F-FDPN PET acquisitions should not be shorter than 90 min to maintain reliable estimates of DV.

The purpose of the present study is to compare the capacity of opioid antagonists to elicit withdrawal jumping in mice following two acute pretreatment doses of the opioid agonist morphine. Antagonists that precipitate vigorous withdrawal jumping across both morphine treatment doses are hypothesized to be strong inverse agonists at the mu-opioid receptor, whereas antagonists that elicit withdrawal jumping in mice treated with the high but not the low dose of morphine are hypothesized to be weak inverse agonists. Male, Swiss-Webster mice (15-30 g) were acutely treated with 56 or 180 mg kg(-1) morphine 4 h prior to injection with naloxone, naltrexone, diprenorphine, nalorphine, or naloxonazine. Vertical jumping, paw tremors, and weight loss were recorded. Naloxone, naltrexone, and diprenorphine produced withdrawal jumping after 56 and 180 mg kg(-1)morphine pretreatment. Nalorphine and naloxonazine produced moderate withdrawal jumping after 180 mg kg(-1) morphine pretreatment, but failed to elicit significant withdrawal jumping after 56 mg kg(-1) morphine pretreatment. Nalorphine and naloxonazine blocked the withdrawal jumping produced by naloxone. All antagonists produced paw tremors and weight loss although these effects were generally not dose-dependent. Taken together, these findings reveal a rank order of negative intrinsic efficacy for these opioid antagonists as follows: naloxone=naltrexone> or =diprenorphine>nalorphine=naloxonazine. Furthermore, the observation that nalorphine and naloxonazine blocked the naloxone-induced withdrawal jumping provides additional evidence that nalorphine and naloxonazine are weaker inverse agonists than naloxone.

The discriminative stimulus effects of acute morphine followed by naltrexone have been described previously in nonhuman primates. The purposes of this study were to 1) extend the pharmacological characterization of the discrimination by testing mu-opioid agonists other than morphine and opioid-like compounds other than naltrexone and 2) to examine further the relationship between agonist pretreatment time and manifestation of the cue produced by morphine followed by naltrexone. Subjects were trained to discriminate 1.7 mg/kg morphine --> 0.1 mg/kg naltrexone (MOR --> NTX) versus saline followed by 0.1 mg/kg naltrexone. When combined with 0.1 mg/kg naltrexone, all agonists tested, save buprenorphine, meperidine, and nalbuphine, produced dose-dependent increases in MOR --> NTX-appropriate responding, culminating in criterion levels of responding. Comparing agonist ED50 values revealed a rank order of potency of etorphine >> fentanyl >> levorphanol > heroin > or = methadone > or = nalbuphine > or = morphine. ED50 values for buprenorphine and meperidine could not be calculated. MOR --> NTX-appropriate responding after doses of agonist that produced criterion or near criterion levels of responding was also a function of naltrexone dose. After morphine pretreatment, diprenorphine and nalorphine, but not buprenorphine, dose-dependently substituted for naltrexone. The MOR --> NTX discrimination also depended upon the interval between morphine and NTX administration. Finally, 1-h pretreatment with morphine and etorphine, but not buprenorphine, followed by naltrexone generalized to 4 h MOR --> NTX. These results suggest a minimum efficacy requirement of acutely administered agonists together with the naltrexone training dose for stimulus control of behavior. However, in some cases this requirement can be overcome with higher doses of naltrexone.

Lepidopteran cell lines have been engineered to constitutively express high levels of mouse delta opioid receptors either alone or in combination with human Galpha16 protein. Biochemical and pharmacological studies demonstrate that these lines contain all the mediator G proteins and downstream effectors required for opioid receptor function, including phospholipase C, and that expression of exogenous Galpha16 does not contribute significantly to increased receptor responses upon activation. The activation of the phospholipase C pathway in the transformed cells upon stimulation with known receptor ligands results in easily and quantitatively measurable increases in free intracellular calcium, which can be monitored by automated fluorescent methods, while the addition of specific antagonists blocks the agonist-induced responses. Therefore, the transformed lepidopteran cell lines can be used as sensitive high-throughput screening platforms for fast detection of delta opioid receptor ligand mimetics (agonists and antagonists) in collections of natural products and synthetic compounds.

Arylacetamide analgesics that stimulate kappa opioid receptors in the central nervous system mediate dysphoria and psychosis as well as analgesia. However, the naturally occurring peptide agonist, dynorphin A, is analgesic in the absence of dysphoria and psychosis, indicating that the therapeutic effects of kappa opioid agonists may be separated from their side effects. As part of our effort to discover kappa opioid receptor analgesics lacking side effects, we designed and constructed two mu/kappa chimeric receptors, composed primarily of amino acid residues derived from the mu opioid receptor, that were expected to bind dynorphin A with high affinity. In one, extracellular loop 2 and transmembrane domain 4 were derived from the kappa opioid receptor and in the other, only extracellular loop 2 was derived from the kappa opioid receptor. Most competitors of [(3)H]diprenorphine binding from a variety of structural classes bound to the chimeras with affinities similar to those with which they bound to the mu opioid receptor. In contrast, dynorphin A analogs bound to the chimeras with affinities similar to those with which they bound to the kappa opioid receptor. Pharmacological characterization of [(35)S]GTPgammaS binding mediated by the chimera with extracellular loop 2 derived from the kappa opioid receptor showed that it behaved as if it were mu opioid receptor with high affinity for dynorphin A analogs. These chimeras may be useful in identifying novel kappa receptor agonists that bind to the second extracellular loop of the receptor and share the desirable therapeutic profile of dynorphin A.

Substitute methadone prescribing is one of the main modes of treatment for opioid dependence with established evidence for improved health and social outcomes. However, the pharmacology underpinning the effects of methadone is little studied despite controversies about dosing in relation to outcome. We therefore examined the relationship between methadone dose and occupation of opioid receptors in brain using the positron emission tomography (PET) radioligand [(11)C]diprenorphine in humans and rats. Eight opioid-dependent subjects stable on their substitute methadone (18-90 mg daily) had an [(11)C]diprenorphine PET scan at predicted peak plasma levels of methadone. These were compared with eight healthy controls. No difference in [(11)C]diprenorphine binding was found between the groups, with no relationship between methadone dose and occupancy. Adult male Sprague-Dawley rats that had been given an acute i.v. injection of methadone hydrochloride (0.35, 0.5, 0.7, or 1.0 mg kg(-1)) before [(11)C]diprenorphine showed a dose-dependent increase in biodistribution but no reduction in [(11)C]diprenorphine binding. We suggest that the lack of a dose-dependent relationship between methadone dose, either given chronically in human or acutely in rat, and occupancy of opioid receptor measured with [(11)C]diprenorphine PET is related to efficacy of this opioid agonist at very low levels of opioid receptor occupancy. This has implications for understanding the actions of methadone in comparison with other opioid drugs such as partial agonists and antagonists.

Differences in the anorectic activity of morphinan (e.g., naltrexone) and 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (4PP) opioid receptor antagonists have been described. In an attempt to explain these differences, the influence of Na(+) on opioid binding affinity and functional activity of 4PP antagonists was compared to other opioid antagonists. The binding affinities of neutral antagonists were unaffected by the addition of Na(+), whereas that for the peptide, inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174864) was increased. Similarly, the binding affinities of the 4PP antagonist (3R,4R)-1-((S)-3-hydroxy-3-cyclohexylpropyl)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidine (LY255582) and other 4PP antagonists were increased in the presence of Na(+) with the greatest effects at the delta opioid receptor followed by the mu and kappa opioid receptors, respectively. Similar to ICI174864, 4PP antagonists were found to inhibit basal GTPgamma[(35)S] binding at the delta opioid receptor indicating inverse agonist activity. A correlation was observed between the binding affinities in the presence of Na(+), the inverse agonist potency, and the anorectic potency of 4PP antagonists. These data suggest that 4PP antagonists differ from morphinan antagonists in their inverse agonist activity and suggest a relationship between inverse agonism and anorectic activity.

Basis pursuit denoising is a new approach for data-driven estimation of parametric images from dynamic positron emission tomography (PET) data. At present, this kinetic modeling technique does not allow for the estimation of the errors on the parameters. These estimates are useful when performing subsequent statistical analysis, such as, inference across a group of subjects or when applying partial volume correction algorithms. The difficulty with calculating the error estimates is a consequence of using an overcomplete dictionary of kinetic basis functions. In this paper, a bootstrap approach for the estimation of parameter errors from dynamic PET data is presented. This paper shows that the bootstrap can be used successfully to compute parameter errors on a region of interest or parametric image basis. Validation studies evaluate the methods performance on simulated and measured PET data ([(11)C]Diprenorphine-opiate receptor and [(11)C]Raclopride-dopamine D(2) receptor). The method is presented in the context of PET neuroreceptor binding studies, however, it has general applicability to a wide range of PET/SPET radiotracers in neurology, oncology and cardiology.

Delta- and kappa-opioid receptors (OPRs), but not micro-OPRs, are expressed in the myenteric plexus of the porcine distal small intestine. In a subpopulation of myenteric neurons, delta- and kappa-OPRs seem to be colocalized and may functionally interact. In this study, radioligand binding was used to characterize myenteric OPR populations in detail. The nonselective OPR antagonist [3H]diprenorphine bound to a single, high-affinity site in myenteric neural membrane homogenates. Naloxone displaced 65 and 59% of [3H]diprenorphine binding from this site in Na(+)-free Tris and Krebs-HEPES buffers, respectively. Naltrexone-derived delta- and kappa-OPR antagonists, including naltriben, 7-benzylidenenaltrexone, nor-binaltorphimine, and 5'-guanidinonaltrindole, displaced [3H]diprenorphine from two distinct binding sites to levels similar to that of naloxone. The selective delta-OPR ligands Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP), [D-Pen2,D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin II, and (+)-4-[(alphaR)-alpha((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl-3-methoxybenzyl)-N,N-diethylbenzamide (SNC-80) and the kappa-OPR agonist (D-(5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxoaspiro-(4,5)dec-8-yl) benzeneacetamide (U-69,593) displaced [3H]diprenorphine from three independent binding sites; these included high-affinity delta- and kappa-OPR sites, and a residual binding site. Residual [3H]diprenorphine binding was displaced by the selective kappa-OPR antagonist nor-binaltorphimine after saturation of delta and kappa sites, respectively, with DPDPE and U-69,593. The residual binding site displayed low affinity for delta- and kappa-OPR agonists and TIPP, as well as moderate affinity for naltrexone-derived ligands, properties reminiscent of delta-/kappa-OPR heterodimers.

In the intestine, opioids produce antidiarrhoeal and constipating actions that are mediated by enteric neurones. Through interactions with opioid receptors (ORs) on submucosal neurones, opioids suppress active ion transport evoked by transmural electrical stimulation (TES) in mucosa-submucosa sheets from the porcine ileum. In this study, we examined the pharmacological characteristics of the previously described OR, which is sensitive to the delta1-OR antagonist 7-benzylidenenaltrexone and modulates neurogenic transepithelial ion transport in this tissue preparation. Increases in short-circuit current (Isc, a measure of active anion transport) evoked by TES in ileal mucosa-submucosa sheets were inhibited by opioid agonists possessing high selectivity for either delta- or micro-ORs including [d-Pen2,5]enkephalin (DPDPE), [d-Ala2, Glu4]deltorphin II, and [d-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO). As determined by the Schild analysis, the actions of these agonists were competitively inhibited by 7-benzylidenenaltrexone. The nonequilibrium micro-OR antagonist beta-funaltrexamine inhibited the actions of DAMGO only at a high concentration (1 microm) but did not alter DPDPE or deltorphin II action. At concentrations up to 10 microm, the nonequilibrium delta-OR antagonist naltrindole 5'-isothiocyanate did not alter the actions of delta- or micro-OR agonists. Radioligand binding analyses of neuronal homogenates from the ileal submucosa revealed that the nonselective OR ligand [3H]diprenorphine bound to two populations of specific binding sites. One of these sites possessed binding characteristics similar to the delta-OR. In summary, neurogenic ion transport in the porcine intestine is modulated by an OR which shares pharmacological characteristics of both micro- and delta-ORs and may represent a novel receptor entity.

The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken.

Objective: To determine the affinity of new opioid receptor ligands to cloned mu opioid receptors stably expressed in CHO cell., Methods: The binding characteristics of the opioid ligand [3H] diprenorphine (3H-dip) were studied by cellular biological techniques and radioligands binding in cloned mu opioid receptors stably expressed in CHO cells in saturation binding experiments, and were followed by competition binding experiments with a variety of new synthesized opioid receptor ligands., Results: The Kd and Bmax of [3H] diprenorphine bound to mu receptors were 1.06 nmol/L and 930 fmol/mg protein, respectively. Competition binding experiments revealed that ligand 3# and 12# displayed much higher affinity than DAMGO and Morphine for the cloned mu opioid receptor. However, the affinities of ligands 2#, 6#, 8# and 9# were lower than DAMGO and Morphine., Conclusion: The present results suggest that the new ligands 3# and 12# have higher affinity to mu opioid receptors. However, ligands 2#, 6#, 8# and 9# have lower affinity to mu opioid receptors.

A series of aminoalkylazetidines has been discovered as novel ORL1 receptor ligands. Structure-activity relationships have been investigated at the azetidine N and the alkyl side chain sites. Several potent and selective analogues have been identified.

Activation of mu-opioid receptors (MORs) transfected into human embryonic kidney 293 cells, caused a multiphasic increase in cytosolic free Ca(2+) levels (Ca(2+)i). The first Ca(2+)i maximum (peak 1) between 5 and 7 s depended on the presence of extracellular Ca(2+) (Ca(2+)e). The second phase peaking at approximately 15 s (peak 2) was independent of Ca(2+)e and thus represents Ca(2+) release from intracellular stores. A decrease in temperature from 37 to 25 degrees C also caused reduction of peak 1 but not peak 2, suggesting that the two responses arise from mechanistically distinct pathways. A delayed Ca(2+)e-dependent third response phase is thought to represent capacitative Ca(2+)e influx evoked after release of Ca(2+) from internal stores. Agonists and antagonists of two major classes of opioid ligands, oxymorphinans (morphine and naloxone) and oripavines (etorphine and diprenorphine), had differential effects on Ca(2+) currents. Although morphine activated both phases with equal potency, etorphine was 20-fold less potent at stimulating peak 1 over peak 2. Similarly, the antagonists, naloxone and diprenorphine, blocked the Ca(2+) response to each agonist with greatly varying potencies. Specifically, concomitant injection of diprenorphine failed to affect peak 1 (thought to represent rapid Ca(2+)e influx) stimulated by morphine while fully blocking peak 2 (intracellular Ca(2+) release). However, diprenorphine potently inhibited peak 1 as well when added to the cells before morphine, indicating limited or slow access of diprenorphine to these morphine binding sites. The existence of multiple, functionally distinct binding site conformations could account for these findings. In conclusion, different opioid ligands can differentially affect Ca(2+) response patterns resulting from MOR activation.

The agonist (-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide [(-)U50,488H] caused desensitization of the human kappa-opioid receptor (hkor) and Flag-tagged hkor (Flag-hkor) but not the rat kappa-opioid receptor (rkor) and Flag-tagged rkor (Flag-rkor) stably expressed in CHO cells as assessed by guanosine 5'-O-(3-[35S]thiotriphosphate) binding. In addition, (-)U50,488H stimulation enhanced phosphorylation of the Flag-hkor, but not Flag-rkor. (-)U50,488H-induced phosphorylation of the Flag-hkor was reduced by expression of the dominant negative mutant GRK2-K220R, demonstrating the involvement of G protein-coupled receptor kinases (GRKs). However, expression of GRK2 and arrestin-2 or GRK3 and arrestin-3 did not result in desensitization or phosphorylation of the Flag-rkor after (-)U50,488H pretreatment. To understand the molecular basis of the species differences, we constructed two Flag-tagged chimeric receptors, Flag-h/rkor and Flag-r/hkor, in which the C-terminal domains of Flag-hkor and Flag-rkor were switched. When stably expressed in CHO cells, Flag-r/hkor, but not Flag-h/rkor, was desensitized and phosphorylated after exposure to (-)U50,488H, indicating that the C-terminal domain plays a critical role in the differences. We then generated a Flag-hkor mutant, in which S358 was mutated to N (Flag-hkorS358N) and a Flag-rkor mutant, in which N358 was substituted with S (Flag-rkorN358S). Although Flag-hkorS358N was not phosphorylated or desensitized by (-)U50,488H stimulation, Flag-rkorN358S underwent (-)U50,488H-induced desensitization with slightly increased phosphorylation. These results indicate that there are differences in (-)U50,488H-induced desensitization and phosphorylation between the hkor and the rkor. In addition, the C-terminal domain plays a crucial role in these differences and the 358 locus contributes to the differences. Our findings suggest caution in extrapolating studies on kappa-opioid receptor regulation from rats to humans.

Various mechanisms have been proposed for opioid receptor down regulation in different experimental preparations. The present study was aimed to test whether distinct mechanisms can mediate opioid receptor down regulation within the same cell. For this purpose we transfected HEK-293 cells with rat delta-opioid receptor (DOR). We exposed the cells to the opioid agonist etorphine in the absence or presence of various pharmacological agents and measured the binding of the opioid ligand [(3)H]diprenorphine to either isolated cell membranes or whole cells. We found that internalization of the receptors into the cell was mediated by clathrin coated pits and that the internalized receptors were degraded either in lysosomes or by proteosomes. Down regulation involved phosphorylation and at least two different kinases, a tyrosine kinase (TK) and MAPK kinase (MEK), mediated DOR down regulation in parallel routes. G-protein-coupled receptor kinase (GRK) was found to have only a minor role in DOR down regulation in HEK-293 cells. On the other hand, in N18TG2 cells that endogenously express delta-opioid receptors, GRK was the predominant kinase mediating DOR down regulation, with only a minor role for TK and MEK. We conclude that down regulation can take place via divers pathways within the same cell, and that in different cells down regulation is mediated by different mechanisms, depending on the kinase profile of the cells and the compartmentalization of the receptors within the cells.

The physiological effects on respiratory function of etorphine (M99, Logos Agvet) (30 microg/kg) administered intramuscularly were determined in boer goats. The goats were habituated to the experimental procedures so that respiratory function could be determined while the animals stood quietly at rest. This enabled the physiological changes induced by etorphine to be measured and compared with those obtained before administration of the immobilising drug. The effectiveness of diprenorphine (M5050, Logos Agvet) (3 mg/l mg etorphine) as an antagonist of the physiological changes induced by the etorphine treatment was also determined. Etorphine depressed respiratory function, which resulted in a decrease in PaO2 and an increase in PaCO2. These changes were limited and occurred as a result of decreases in respiratory minute volume and alveolar minute ventilation caused by a decrease in respiratory rate. The physiological shunt fraction did not change significantly but there was a significant decrease in percentage physiological dead space ventilation. It was not possible to determine how effectively diprenorphine reversed the respiratory effects due to etorphine.

We have mapped the residues in the sixth transmembrane domains (TMs 6) of the mu, delta, and kappa opioid receptors that are accessible in the binding-site crevices by the substituted cysteine accessibility method (SCAM). We previously showed that ligand binding to the C7.38S mutants of the mu and kappa receptors and the wild-type delta receptor was relatively insensitive to methanethiosulfonate ethylammonium (MTSEA), a positively charged sulfhydryl-specific reagent. These MTSEA-insensitive constructs were used as the templates, and 22 consecutive residues in TM6 (excluding C6.47) of each receptor were mutated to cysteine, 1 at a time. Most mutants retained binding affinities for [3H]diprenorphine, a nonselective opioid antagonist, similar to that of the template receptors. Treatment with MTSEA significantly inhibited [3H]diprenorphine binding to 11 of 22 mutants of the rat mu receptor and 9 of 22 mutants of the human delta receptor and 10 of 22 mutants of the human kappa receptor. Naloxone or diprenorphine protected all sensitive mutants, except the A6.42(287)C mu mutant. Thus, V6.40, F6.44, W6.48, I6.51, Y6.54, V6.55, I6.56, I6.57, K6.58, and A6.59 of the mu receptor; F6.44, I6.51, F6.54, V6.55, I6.56, V6.57, W6.58, T6.59, and L6.60 of the delta receptor; and F6.44, W6.48, I6.51, F6.54, I6.55, L6.56, V6.57, E6.58, A6.59, and L6.60 of the kappa receptor are on the water-accessible surface of the binding-site crevices. The accessibility patterns of residues in the TMs 6 of the mu, delta, and kappa opioid receptors are consistent with the notion that the TMs 6 are in alpha-helical conformations with a narrow strip of accessibility on the intracellular side of 6.54 and a wider area of accessibility on the extracellular side of 6.54, likely due to a proline kink at 6.50 that bends the helix in toward the binding pocket and enables considerable motion in this region. The wider exposure of residues 6.55-6.60 to the binding-site crevice, combined with the divergent amino acid sequences, is consistent with the inferred role of residues in this region in determining ligand binding selectivity. The conservation of the accessibility pattern on the cytoplasmic side of 6.54 suggests that this region may be important for receptor activation. This accessibility pattern is similar to that of the D2 dopamine receptor, the only other GPCR in which TM6 has been mapped by SCAM. That opioid receptors and the remotely related D2 dopamine receptor have similar accessibility patterns in TM6 suggest that these segments of GPCRs in the rhodopsin-like subfamily not only share secondary structure but also are packed similarly into the transmembrane bundle and thus have similar tertiary structure.

Met-enkephalin-arg-phe (MEAP) interrupts vagal bradycardia when infused into the systemic circulation. This study was designed to locate the opiate receptors functionally responsible for this inhibition. Previous observations suggested that the receptors were most likely located in either intracardiac parasympathetic ganglia or the pre-junctional nerve terminals innervating the sinoatrial node. In this study 10 dogs were instrumented with a microdialysis probe inserted into the sinoatrial node. The functional position of the probe was tested by briefly introducing norepinephrine into the probe producing an increase in heart rate of more than 30 beats/min. Vagal stimulations were conducted at 0.5, 1.2 and 4 Hz during vehicle infusion (saline ascorbate). Cardiovascular responses during vagal stimulation were recorded on-line. MEAP was infused directly into the sinoatrial node via the microdialysis probe. The evaluation of vagal bradycardia was repeated during the nodal application of MEAP, diprenorphine (opiate antagonist), and diprenorphine co-infused with MEAP. MEAP introduced into the sinoatrial node via the microdialysis probe reduced vagal bradycardia by more than half. Simultaneous local nodal blockade of these receptors with the opiate antagonist, diprenorphine, eliminated the effect of MEAP demonstrating the participation by opiate receptors. Systemic infusions of MEAP produced a reduction in vagal bradycardia nearly identical to that observed during nodal administration. When local nodal opiate receptors were blocked with diprenorphine, the systemic effect of MEAP was eliminated. These data lead us to suggest that the opiate receptors responsible for the inhibition of vagal bradycardia are located within the sinoatrial node with few, if any, participating extra-nodal or ganglionic receptors.

ZFOR1 is a putative opioid receptor from zebrafish brain which has 66% homology with the mammalian delta-opioid receptor. When expressed in HEK293 cells ZFOR1 bound the non-selective opioid antagonist [(3)H]diprenorphine with high affinity. However, the binding of this ligand was not readily displaced by opioids selective for mu, delta or kappa opioid receptors (affinities>1000 nM). Rather non-selective ligands showed good affinity, as did the non-peptide delta-ligand BW373U86 (Ki 69 nM), the delta-antagonist naltrindole (Ki 28 nM) and the peptide beta-endorphin (Ki 37 nM). Agonist binding to the receptor encoded by ZFOR1 receptor stimulated the binding of [(35)S]GTPgammaS confirming coupling to G proteins. Study of the receptor should contribute to understanding of the evolution of the opioid system.

The transforming growth factor beta1 (TGFbeta1) is a major regulator of human endometrial function. Human endometrium possesses specific opioid binding sites, the majority of which belong to the kappa type, for which the prodynorphin-derived opioids are the endogenous ligands. Since these two systems interact in several other tissues we postulated that opioids may affect the production of TGFbeta1 in human endometrium. We have found that kappa opioids exerted a time- and dose-dependent inhibitory effect on TGFbeta1 production from endometrial stromal and epithelial cells and from the Ishikawa human endometrial adenocarcinoma cell line. This effect was reversible by the specific opioid antagonist diprenorphine. To examine if this effect represents a paracrine endometrial response to locally produced kappa opioids we searched for the presence of the endogenous kappa opioid receptor ligands. Indeed, the prodynorphin transcript was detectable on Northern blots from normal and tumoral human endometrial cells; its size was that of the pituitary transcript, i.e. approximately 2.4 kb long. Most immunoreactive dynorphin from human endometrium had a molecular weight of 8 kDa. Finally, immunofluorescence staining of normal and tumoral human endometrial cells revealed the presence of dynorphin-positive cytoplasmic secretory granules. Taken together, our data suggest that in human endometrium, kappa opioids and the TGFbeta1 form a paracrine network which appears to be retained by the Ishikawa human endometrial adenocarcinoma cell line.

The aim of the current research has been to validate an original radioiodinated diprenorphine (DPN) derivative suitable for imaging studies of opioid receptors. [(125)I]7alpha-O-iodoallyl diprenorphine (7alpha-O-IA-DPN) was prepared by radioiododestannylation and in vitro and in vivo opioid receptor binding assays were performed with CDF1 mouse brains. In vitro binding studies showed high affinity (K(i)= 0.4 +/- 0.2 nM) for mouse brain membranes. In vivo studies showed 63% specific binding. Ex vivo autoradiography of brain sections confirmed high uptake and retention of [(125)I]7alpha-O-IA-DPN in regions rich in opioid receptors. This new radioiodinated DPN analogue appears to be a potential radioprobe for in vivo visualization of human cerebral opioid receptors with single photon emission computed tomography (SPECT)., (Copyright 1999 Harcourt Publishers Ltd.)

The cDNA coding for the bovine mu-opioid receptor has been cloned and sequenced. Conserved sequences from murine delta-receptor cDNA were used as primers in polymerase chain reaction (PCR) to amplify cDNA, prepared by reverse transcription of bovine brain mRNA. This cDNA was used to probe a bovine brain library. The partial sequence obtained was extended to provide the full length clone by PCR. The cDNA has an open reading frame of 1203 base pairs (bp) with a 3'-untranslated region of 1900 bp and a 5'-untranslated region of 265 bp. The protein contains 401 amino acids and has 94% amino acid identity with the human and 91% with the rat mu-opioid receptor. It has the putative seven transmembrane domains, characteristic of G protein-coupled receptors and contains 5 potential N-linked glycosylation sites near the N-terminus. Several potential phosphorylation sites and a putative palmitoylation site are also present. The receptor was stably expressed in HEK293 cells. The binding profile was found to be that of a typical mu receptor, i. e., mu agonists and antagonists, but not delta and kappa ligands, bound with high affinity. Functional assays, namely, opioid stimulation of [35S]GTPgammaS binding and inhibition of forskolin-activated adenylyl cyclase, were also found to be highly specific for mu-opioid agonists. The receptor was downregulated by chronic exposure to mu agonists but not delta or kappa agonists. Evidence is presented indicating that the cloned receptor is the same as the bovine mu receptor previously purified to homogeneity in our laboratory. No evidence was found for genes for multiple mu-type opioid receptors.

Aim: To characterize the internalization of delta opioid receptors (DOR) stably expressed in Chinese hamster ovary (CHO) cells and the role of the C-terminal in this process., Methods: Receptor membrane anchoring was shown by immunofluorescence microscopy. Receptor internalization was assessed by measuring the radioligand binding resistant to the acid-buffer wash., Results: Originally, all the wild-type (CHO-W) and C-truncated (CHO-T) DOR expressed were localized to the membrane. Agonist [3H] leucine-2-alanine enkephalin (LAE) but not the antagonist [3H]diprenorphine (Dip) induced rapid receptor internalization. The internalization of C-truncated DOR in CHO-T was similar to that of the wild-type in maximal level, but climbed up more slowly. DOR internalization was extracellular osmolarity- and temperature-sensitive. Pertussis toxin and universal protein kinase inhibitor staurosporine had no effect on it., Conclusion: DOR internalization is an agonist and clathrin-coated pits dependent, but post-receptor cellular signal transduction independent process; moreover, the C-terminal of DOR, not engaged in membrane anchoring, affects the initialization of DOR internalization.

The following studies were conducted to determine if the ability of the intrinsic cardiac opioid, met-enkephalin-arg-phe to interrupt vagal bradycardia can be generalized to include the disruption of vagal effects on atrial contraction and coronary blood flow. Anesthetized dogs were instrumented to measure heart rate and left atrial contractile force or heart rate and coronary blood flow. The response of each variable was recorded at rest and during vagal stimulation. During the evaluation of vagal effects on contractile activity and coronary blood flow, heart rate was maintained constant by electrically pacing the hearts above their resting heart rate. In the first protocol, vagal stimulation reduced both heart rate and atrial contractile force in a frequency dependent fashion. When met-enkephalin-arg-phe (MEAP) was infused systemically for three min at 3 nmol min(-1) kg(-1), there were no observed changes in resting heart rate or atrial contraction. However, when the vagal stimuli were reapplied during the peptide infusion, the previously observed vagal effects on rate and contractile force were reduced in magnitude by one-half to two-thirds. The ability of MEAP to interrupt the vagal control of heart rate and contractile activity involves opiate receptors since the effect was eliminated in both cases by prior opiate receptor blockade with the high affinity antagonist, diprenorphine. In the second protocol, vagal stimulation produced a transient increase in coronary blood flow and an accompanying increase in myocardial oxygen consumption. These effects were reduced by approximately 80% during the systemic infusion of MEAP. A similar increase in coronary blood flow mediated by the direct acting muscarinic agonist, methacholine, was unaltered by the infusion of peptide. In summary, these data suggest that the intrinsic cardiac enkephalin, MEAP, is capable of inhibiting the vagal control of heart rate, contractile force and coronary blood flow and probably does so through a common opiate receptor located prejunctionally on vagal nerve terminals or within nearby parasympathetic ganglia.

Using 18F-fluorodeoxyglucose and 11C-diprenorphine positron emission tomography (PET), we investigated alterations in glucose metabolism and opioid receptor binding in a patient with central poststroke pain, which developed after a small pontine hemorrhagic infarction. In comparison with normal databases, reduced 11C-diprenorphine binding was more accentuated than the hypometabolism on the lateral cortical surface contralateral to the symptoms, and a differential abnormal distribution between the tracers was seen in pain-related central structures. These results show that 11C-diprenorphine PET provides unique information for the understanding of central poststroke pain.

Phosphorylation of specific amino acid residues is believed to be crucial for the agonist-induced regulation of several G protein-coupled receptors. This is especially true for the three types of opioid receptors (mu, delta, and kappa), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of mu- and delta-opioid receptors and to potentiate agonist-induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid-activated PKC contributes to homologous receptor down-regulation and then expand our focus to include the exploration of the mechanism(s) by which mu-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin (DAMGO)-induced PKC translocation follows a time-dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control-treated cells in a process we termed "reverse translocation." The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L-type calcium channel antagonist nimodipine attenuated opioid-mediated effects on PKC and mu-receptor down-regulation, suggesting that this is a process partially regulated by Ca2+-dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+-sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down-regulation. In addition to expressing conventional (PKC-alpha) and novel (PKC-epsilon) isoforms, SH-SY5Y cells also contain a DAG- and Ca2+-independent, atypical PKC isozyme (PKC-zeta), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-zeta is similarly sensitive to activation by mu-opioids. PKC-zeta translocates from the cytosol to the membrane with kinetics similar to those of PKC-alpha and epsilon in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by mu-opioid agonists is involved in the processes that result in mu-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.

Initial studies were undertaken to examine the properties of [3H]-diprenorphine binding to Rana pipiens whole brain tissue using naltrexone for the definition of nonspecific binding. Saturation analysis demonstrated the binding of [3H]-diprenorphine to be saturable with a K(D) value of 0.65 nM and a Bmax value of 287.7 fmol/mg protein. Unlabeled diprenorphine dose-dependently displaced [3H]-diprenorphine from a single noninteractive site in competition studies which yielded a Ki of 0.22 nM. However, control studies in the absence of tissue revealed significant binding of [3H]-diprenorphine to the filter alone. Interestingly, [3H]-diprenorphine in the presence of unlabeled naltrexone as well as with unlabeled naloxone showed significantly greater binding to the filter than did [3H]-diprenorphine alone. Given this observation of increased nonspecific binding, an artificially low Bmax value would be expected. It is our hypothesis that the unlabeled nonspecific drug forms a complex with [3H]-diprenorphine preventing it from being effectively washed through the filter or the unlabeled drug itself is blocking the flow of [3H]-diprenorphine through the filter. The latter is unlikely however as other binding studies done in our lab using the radioligand [3H]-naloxone with unlabeled naltrexone do not show significant binding to the filter.

Agonist-induced down-regulation of opioid receptors appears to require the phosphorylation of the receptor protein. However, the identities of the specific protein kinases that perform this task remain uncertain. Protein kinase C (PKC) has been shown to catalyze the phosphorylation of several G protein-coupled receptors and potentiate their desensitization toward agonists. However, it is unknown whether opioid receptor agonists induce PKC activation under physiological conditions. Using cultured SH-SY5Y neuroblastoma cells, which naturally express mu- and delta-opioid receptors, we investigated whether mu-opioid receptor agonists can activate PKC by measuring enzyme translocation to the membrane fraction. PKC translocation and opioid receptor densities were simultaneously measured by 3H-phorbol ester and [3H]diprenorphine binding, respectively, to correlate alterations in PKC localization with changes in receptor binding sites. We observed that mu-opioid agonists have a dual effect on membrane PKC density depending on the period of drug exposure. Exposure for 2-6 h to [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin or morphine promotes the translocation of PKC from the cytosol to the plasma membrane. Longer periods of opioid exposure (>12 h) produce a decrease in membrane-bound PKC density to a level well below basal. A significant decrease in [3H]diprenorphine binding sites is first observed at 2 h and continues to decline through the last time point measured (48 h). The opioid receptor antagonist naloxone attenuated both opioid-mediated PKC translocation and receptor down-regulation. These results demonstrate that opioids are capable of activating PKC, as evidenced by enhanced translocation of the enzyme to the cell membrane, and this finding suggests that PKC may have a physiological role in opioid receptor plasticity.

Kinetic studies of binding of the delta-opioid antagonist [3H]DPN with receptors of mouse splenocytes are performed. Kinetic analysis of experimental data has shown that receptors of these cells possess activity toward the delta-opioid ligands. Presence of compounds that inhibit the conjugation of receptors with G-proteins, reduces receptor binding. Experimental data are computer simulated, and numerical values for various equilibrium as well as kinetic parameters of receptor binding and the G-protein cycle are obtained.