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1.
Br J Pharmacol ; 172(2): 571-82, 2015 Jan.
Article in English | MEDLINE | ID: mdl-24666365

ABSTRACT

BACKGROUND AND PURPOSE: Single-prolonged stress (SPS), a rat model of post-traumatic stress disorder (PTSD), also induces long-lasting hyperalgesia associated with hypocortisolism and elevated nociceptin/orphanin FQ (N/OFQ) levels in serum and CSF. Here, we determined the effect of JTC-801 (N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl) benzamide monohydrochloride), a nociceptin/orphanin FQ peptide (NOP) receptor antagonist, on symptoms of pain and anxiety in rats after SPS exposure, and examined N/OFQ-NOP receptor system changes. EXPERIMENTAL APPROACH: Male Sprague Dawley rats received JTC-801 (6 mg kg(-1) i.p., once daily) during days 7-21 of SPS. The ability of JTC-801 to inhibit N/OFQ-stimulated [(35) S]-GTPγS binding was confirmed in rat brain membranes. Anxiety-like behaviour and pain sensitivity were monitored by changes in elevated plus maze performance and withdrawal responses to thermal and mechanical stimuli. Serum corticosterone and N/OFQ content in CSF, serum and brain tissues were determined by radioimmunoassay; NOP receptor protein and gene expression in amygdala, hippocampus and periaqueductal grey (PAG) were examined by immunoblotting and real-time PCR respectively. KEY RESULTS: JTC-801 treatment reversed SPS-induced mechanical allodynia, thermal hyperalgesia, anxiety-like behaviour and hypocortisolism. Elevated N/OFQ levels in serum, CSF, PAG and hippocampus at day 21 of SPS were blocked by JTC-801; daily JTC-801 treatment also reversed NOP receptor protein and mRNA up-regulation in amygdala and PAG. CONCLUSION AND IMPLICATIONS: JTC-801 reversed SPS-induced anxiety- and pain-like behaviours, and NOP receptor system up-regulation. These findings suggest that N/OFQ plays an important role in hyperalgesia and allodynia maintenance after SPS. NOP receptor antagonists may provide effective treatment for co-morbid PTSD and pain. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.


Subject(s)
Aminoquinolines/therapeutic use , Anxiety/drug therapy , Benzamides/therapeutic use , Narcotic Antagonists/therapeutic use , Pain/drug therapy , Receptors, Opioid/metabolism , Stress Disorders, Post-Traumatic/drug therapy , Aminoquinolines/pharmacology , Amygdala/drug effects , Amygdala/metabolism , Animals , Anxiety/metabolism , Benzamides/pharmacology , Disease Models, Animal , Hippocampus/drug effects , Hippocampus/metabolism , Hot Temperature , Hyperalgesia/drug therapy , Hyperalgesia/metabolism , Male , Narcotic Antagonists/pharmacology , Opioid Peptides/blood , Opioid Peptides/cerebrospinal fluid , Opioid Peptides/metabolism , Pain/metabolism , Periaqueductal Gray/drug effects , Periaqueductal Gray/metabolism , Rats, Sprague-Dawley , Receptors, Opioid/genetics , Stress Disorders, Post-Traumatic/metabolism , Nociceptin Receptor , Nociceptin
2.
Neuropharmacology ; 43(6): 979-90, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12423667

ABSTRACT

The molecular mechanism(s) underlying cross-tolerance between mu and opioid receptor-like 1 (ORL1) receptor agonists were investigated using two human neuroblastoma cell lines endogenously expressing these receptors and G protein-coupled receptor kinases (GRKs). Prolonged (24 h) activation of the mu receptor desensitized both mu and ORL1 receptor-mediated inhibition of forskolin-stimulated cAMP accumulation and upregulated GRK2 levels in SH-SY5Y and BE(2)-C cells. Prolonged ORL1 activation increased GRK2 levels and desensitized both receptors in SH-SY5Y cells. Upregulation of GRK2 correlated with increases in levels of transcription factors Sp1 or AP-2. PD98059, an upstream inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2), reversed all these events. Pretreatment with orphanin FQ/nociceptin (OFQ/N) also upregulated GRK3 levels in both cell lines, and desensitized both receptors in BE(2)-C cells. Protein kinase C (PKC), but not ERK1/2, inhibition blocked OFQ/N-mediated GRK3 induction and mu and ORL1 receptor desensitization in BE(2)-C cells. Antisense DNA treatment confirmed the involvement of GRK2/3 in mu and ORL1 desensitization. Here, we demonstrate for the first time a role for ERK1/2-mediated GRK2 induction in the development of tolerance to mu agonists, as well as cross-tolerance to OFQ/N. We also demonstrate that chronic OFQ/N-mediated desensitization of ORL1 and mu receptors occurs via cell-specific pathways, involving ERK1/2-dependent GRK2, or PKC-dependent and ERK1/2-independent GRK3 induction.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/biosynthesis , Protein Serine-Threonine Kinases/biosynthesis , Receptor Cross-Talk/physiology , Receptors, Opioid, mu/physiology , Receptors, Opioid/physiology , Analysis of Variance , Cell Membrane/metabolism , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/physiology , Dose-Response Relationship, Drug , Drug Interactions , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , G-Protein-Coupled Receptor Kinase 3 , Humans , Mitogen-Activated Protein Kinases/physiology , Morphine/agonists , Morphine/pharmacology , Neuroblastoma , Oligodeoxyribonucleotides, Antisense/pharmacology , Opioid Peptides/pharmacology , Protein Serine-Threonine Kinases/physiology , Receptors, Opioid/agonists , Receptors, Opioid, mu/agonists , Transcription Factors/drug effects , Transcription Factors/metabolism , Tumor Cells, Cultured , beta-Adrenergic Receptor Kinases , Nociceptin Receptor , Nociceptin
3.
J Comp Neurol ; 431(4): 424-36, 2001 Mar 19.
Article in English | MEDLINE | ID: mdl-11223812

ABSTRACT

Vesicle associated membrane protein (VAMP; also known as synaptobrevin) is a key component of the core complex needed for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Recent work indicates that the precise complement of presynaptic proteins associated with transmitter release and their isoforms vary among synapses, presumably conferring specific functional release properties. The retina contains two types of vesicular synapses with distinct morphologic, functional, and biochemical characteristics: ribbon and conventional synapses. Although the precise complement of presynaptic proteins is known to differ between conventional and ribbon synapses and among conventional synapses, the distribution of VAMP isoforms among retinal synapses has not been determined. The expression and localization of VAMP isoforms in the salamander retina, a major model system for studies of retinal circuitry, was examined by using immunocytochemical and immunoblotting methods. Both methods indicated that at least two VAMP isoforms were expressed in salamander retina. One isoform, recognized by an immunoglobulin M antibody that recognizes both mammalian VAMP-1 and VAMP-2, was associated with photoreceptor and bipolar cell terminals as well as many conventional synapses, and probably corresponds to mammalian VAMP-2. A different VAMP isoform associated with a subset of amacrine cells, was recognized only by antibodies directed against the N-terminus of mammalian VAMP-2. An antiserum directed against the N-terminus of mammalian VAMP-1 did not specifically recognize any salamander VAMPs in either immunocytochemical or immunoblotting experiments. Heterogeneous distribution of VAMP isoforms among conventional retinal synapses was confirmed by double labeling for synapsin I, a marker for conventional synapses. These studies indicate that VAMP isoforms are expressed heterogeneously among retinal synapses but cannot account for the differences in transmitter release characteristics at ribbon and conventional synapses. These results also corroborate previous studies in Xenopus indicating that the N-terminus of nonmammalian VAMP isoforms differs from their mammalian counterparts.


Subject(s)
Membrane Proteins/analysis , Nerve Tissue Proteins/analysis , Photoreceptor Cells, Vertebrate/cytology , Retina/chemistry , Retina/cytology , Synapses/ultrastructure , Ambystoma , Animals , Immunoblotting , Immunohistochemistry , Larva , Nerve Endings/ultrastructure , R-SNARE Proteins
4.
Neurosci Lett ; 299(3): 173-6, 2001 Feb 23.
Article in English | MEDLINE | ID: mdl-11165763

ABSTRACT

kappa(3) opioid receptors have a unique binding and analgesic profile, as originally defined by naloxone benzoylhydrazone (NalBzoH). Although antisense studies demonstrated the close relationship between kappa(3) opioid and Orphan opioid receptor-like receptor (ORL1) and implied they were generated from the same gene, these studies also revealed differences in the sensitivity profiles of NalBzoH and orphanin FQ/nociceptin (OFQ/N), indicating that they were not identical. To help define the relationship between kappa(3) and ORL1 receptors, we utilized BE(2)-C human neuroblastoma cells that natively express functional ORL1 and kappa(3) opioid receptors. (125)I-[Tyr(14)]OFQ/N binds to a single population of receptors in BE(2)-C cells. Competition binding and adenylyl cyclase studies clearly illustrated marked selectivity differences between the ORL1 and the kappa(3) sites. Furthermore, antisense DNA targeting ORL1 blocked the inhibition of cAMP by OFQ/N, but not by NalBzoH. Thus, the receptor mechanisms mediating the activity of OFQ/N and NalBzoH in BE(2)-C cells are distinct.


Subject(s)
Naloxone/pharmacology , Opioid Peptides/pharmacology , Receptors, Opioid, kappa/drug effects , Receptors, Opioid/drug effects , Tumor Cells, Cultured/drug effects , Antisense Elements (Genetics)/pharmacology , Binding Sites/drug effects , Binding Sites/physiology , Binding, Competitive/drug effects , Binding, Competitive/physiology , Cell Membrane/drug effects , Cell Membrane/metabolism , Cyclic AMP/metabolism , Humans , Naloxone/analogs & derivatives , Neuroblastoma , Opioid Peptides/metabolism , Radioligand Assay , Receptors, Opioid/chemistry , Receptors, Opioid/metabolism , Receptors, Opioid, kappa/chemistry , Receptors, Opioid, kappa/metabolism , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , Tumor Cells, Cultured/metabolism , Nociceptin Receptor , Nociceptin
5.
Brain Res ; 876(1-2): 62-75, 2000 Sep 08.
Article in English | MEDLINE | ID: mdl-10973594

ABSTRACT

Although morphine and its active metabolite, morphine-6beta-glucuronide (M6G), each induce mu-opioid receptor-sensitive feeding, different antisense oligodeoxynucleotide (AS ODN) probes directed against the MOR-1 clone produce distinct effects. Thus, MOR-1 AS ODN probes directed against exons 1 or 4 reduce morphine-, but not M6G-induced feeding, whereas probes directed against exons 2 or 3 reduce M6G-, but not morphine-induced feeding. AS ODN probes directed against different G-protein alpha-subunits differentially reduced morphine (G(ialpha2)) and M6G (G(ialpha1))-induced analgesia. The present study evaluated the ability of AS ODN probes directed against G-protein alpha-subunits to reduce feeding induced by morphine and M6G in rats. The AS ODN probes (25 microg, i.c.v.) were administered once 24 h prior to morphine (5 microg, i.c.v.) or M6G (250 ng) and spontaneous free feeding was assessed 1, 2 and 4 h thereafter. In agreement with analgesic studies, morphine-induced feeding was significantly reduced by the G(ialpha2) AS ODN probe. Morphine-induced feeding was unaffected by AS ODN probes directed against either G(ialpha1), G(ialpha3), G(oalpha), G(x/zalpha), G(qalpha) or a nonsense control probe, and was significantly enhanced by pretreatment with the G(salpha) probe. In contrast, M6G-induced feeding was significantly reduced by AS ODN probes directed against either G(ialpha1), G(ialpha3) or G(x/zalpha), whereas AS ODN probes targeting G(ialpha2), G(oalpha), G(salpha), G(qalpha) or a nonsense control probe were ineffective. When M6G-induced feeding was assessed at a dose (500 ng) which was sensitive to MOR-1 AS ODN effects, none of the G-protein alpha-subunit AS ODN probes were effective. These data indicate that morphine and M6G-induced feeding are mediated through different G-protein alpha-subunits, and provide further evidence for separate and distinct molecular mechanisms mediating these functional responses through different opioid receptors. This strongly suggests that M6G may act through a novel opioid receptor displaying a distinct pharmacological mechanism.


Subject(s)
Eating/drug effects , GTP-Binding Proteins/genetics , Morphine Derivatives/pharmacology , Morphine/pharmacology , Oligonucleotides, Antisense/pharmacology , Animals , Body Weight/drug effects , Male , Protein Isoforms/genetics , Rats , Rats, Sprague-Dawley
6.
Eur J Pharmacol ; 402(1-2): R1-37, 2000 Aug 18.
Article in English | MEDLINE | ID: mdl-10940375

ABSTRACT

The potential interactions of natively expressed mu-opioid and opioid receptor-like (ORL1) receptors were studied by exposing intact BE(2)-C cells to agonists or antagonists for 1 h. Pretreatment with the mu-opioid receptor agonist, [D-Ala(2), N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), or the ORL1 receptor agonist, orphanin FQ/nociceptin desensitized both mu-opioid and ORL1 receptor responses. beta-Funaltrexamine (beta-FNA) pretreatment also blocked both mu-opioid and ORL1 receptor responses, but only mu-opioid receptor binding was reduced. Moreover, beta-FNA (1 microM) failed to inhibit specific ORL1 receptor binding.


Subject(s)
Brain Neoplasms/metabolism , Naltrexone/analogs & derivatives , Narcotic Antagonists/pharmacology , Neuroblastoma/metabolism , Cyclic AMP/metabolism , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/metabolism , Humans , Morphine/pharmacology , Naltrexone/pharmacology , Narcotics/pharmacology , Opioid Peptides/pharmacology , Receptors, Opioid/agonists , Receptors, Opioid, mu/antagonists & inhibitors , Tumor Cells, Cultured , Nociceptin Receptor , Nociceptin
7.
Vis Neurosci ; 17(1): 11-21, 2000.
Article in English | MEDLINE | ID: mdl-10750823

ABSTRACT

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the retina and also appears to act as a trophic factor regulating photoreceptor development and regeneration. Although the tiger salamander is a major model system for the study of retinal circuitry and regeneration, our understanding of GABA receptors in this species is almost exclusively based on the results of physiological studies. Therefore, we have examined the pharmacological binding properties of GABA(A) receptors and their anatomical localization in the tiger salamander retina. Radioligand-binding studies showed that specific 3H-GABA binding to GABA(A) receptors was dominated by a single high-affinity binding site (Kd = 15.6+/-6.9 nM). Specific binding of 3H-GABA was almost completely eliminated by muscimol (Ki = 105+/-62 nM) and bicuculline (Ki = 14.3+/-2.2 microM); however, SR-95531 only displaced about 40% of specific 3H-GABA binding (Ki = 35.0+/-3.8 nM). These data indicate that there are at least two subtypes of GABA(A) receptors present in the salamander retina that can be distinguished by their antagonist binding properties: one sensitive to both bicuculline and SR-95531, and one sensitive to bicuculline but insensitive to SR-95531. Because localization of GABA receptors in the salamander retina by immunocytochemistry is problematic, GABA(A) receptors were localized by fluorescent ligand binding combined with immunocytochemical labeling for cell specific markers. Binding of fluorescently labeled muscimol to GABA(A) receptors was present in both plexiform layers and on photoreceptor cell bodies. GABA(A) receptors in the outer plexiform layer were localized to both photoreceptor terminals and horizontal cell processes.


Subject(s)
Ambystoma/metabolism , Receptors, GABA-A/metabolism , Retina/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Bicuculline/pharmacology , Binding Sites , Binding, Competitive/drug effects , Fluorescent Antibody Technique, Indirect , GABA Antagonists/pharmacology , Muscimol/pharmacology , Pyridazines/pharmacology , Radioligand Assay
9.
J Pharmacol Exp Ther ; 282(3): 1291-7, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9316837

ABSTRACT

Morphine-6beta-glucuronide (M6G) is a potent morphine metabolite. In an effort to further explore its mechanisms of action, we synthesized 3H-M6G of high specific activity and examined its binding. Although its affinity toward traditional mu receptors is similar to morphine in binding assays in brain and in Chinese hamster ovary cells stably transfected with MOR-1, M6G is >100-fold more potent than morphine in analgesic assays. This apparent discrepancy cannot be explained by differing intrinsic activities of the two drugs because both agents are partial agonists with similar efficacies in adenylyl cyclase assays in the transfected cell lines. Behavioral studies have implied the possibility of a distinct M6G receptor. Detailed binding studies in brain tissue reveal evidence for heterogeneity. Nonlinear regression analysis of 3H-M6G saturation studies reveals two components. The lower-affinity component (K(D) = 1.93 +/- 0.6 nM) corresponds to labeling of traditional mu receptors. In addition, 3H-M6G labels another site of low abundance with very high affinity (K(D) = 68 +/- 7 pM). Competition studies indicate that both sites are relatively mu selective. However, several compounds clearly distinguish between the two sites. These binding studies support the concept of a unique M6G receptor responsible for its analgesic activity.


Subject(s)
Brain/metabolism , Morphine Derivatives/metabolism , Receptors, Opioid, mu/physiology , Animals , Binding Sites , Binding, Competitive , CHO Cells , Cricetinae , Receptors, Opioid, mu/genetics , Transfection
10.
Cell Signal ; 9(3-4): 237-48, 1997.
Article in English | MEDLINE | ID: mdl-9218123

ABSTRACT

Most opioid receptor-mediated functions appear to be mediated through G protein interactions, therefore an understanding of opioid signalling requires knowledge of those interactions. This review chronicles the studies examining these interactions for all the opioid receptor subtypes, both in vivo and in vitro.


Subject(s)
GTP-Binding Proteins/metabolism , Receptors, Opioid/metabolism , Signal Transduction , Animals , Forecasting , Humans , Receptors, Opioid/classification
11.
Neurochem Res ; 21(11): 1309-14, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8947921

ABSTRACT

Behaviorally, sigma1 agents modulate opioid analgesia. To examine possible mechanisms responsible for these interactions, we have identified a cell line containing both sigma1 and opioid receptors. [3H](+)-pentazocine binding in BE(2)-C human neuroblastoma cells is high affinity (KD 3.4 +/- 0.7 nM) and high density (Bmax 2.98 +/- 0.14 pmol/mg protein). Competition studies reveal a selectivity profile similar to that of sigma1 sites in guinea pig brain. (+)-Pentazocine has no effect upon either basal or forskolin-stimulated cyclase in the BE(2)-C cells, but cAMP accumulation is inhibited by the morphine, DPDPE and naloxone benzoylhydrazone. (+)-Pentazocine at concentrations as high as 10 microM does not affect this opioid effect, implying that sigma1/opioid interactions are not mediated at the level of the cell. This suggest that their behavioral interactions result from interacting neural circuits. Although (+)-pentazocine is without effect in the cyclase system, it does block carbachol-stimulated phosphoinositol turnover (IC50 6.5 +/- 1.14 microM). The specificity of the effect is confirmed by the ability of haloperidol (1 microM) to shift the IC50 value of (+)-pentazocine 2-fold to the right.


Subject(s)
Analgesics/pharmacology , Enkephalins/pharmacology , Morphine/pharmacology , Naloxone/analogs & derivatives , Pentazocine/metabolism , Receptors, Opioid/metabolism , Receptors, sigma/metabolism , Adenylyl Cyclases/metabolism , Analysis of Variance , Animals , Binding, Competitive , Brain/metabolism , Carbachol/pharmacology , Cell Line , Colforsin/pharmacology , Cyclic AMP/metabolism , Enkephalin, D-Penicillamine (2,5)- , Guanylyl Imidodiphosphate/pharmacology , Guinea Pigs , Haloperidol/pharmacology , Humans , Kinetics , Naloxone/pharmacology , Neuroblastoma , Pentazocine/pharmacology , Phosphatidylinositols/metabolism , Trypsin/pharmacology , Tumor Cells, Cultured
12.
Synapse ; 24(2): 193-201, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8890461

ABSTRACT

Naloxone benzoylhydrazone (NalBzoH) has proved a valuable tool in the investigation of opioid receptor subtypes. In the present study, we have examined a series of derivatives of NalBzoH in which substitutions have been made on the benzoyl ring. Overall, we see dramatic effects on the binding affinities of derivatives against the various opioid receptor subtypes. Although the range of affinities against the mu receptors is quite modest, ranges of the others vary almost 30-fold for kappa 3, 50-fold for kappa 1 and 100-fold for delta and kappa 2 binding. Few substituted derivatives display greater affinity than NalBzoH for any of the receptors, except for delta sites where several derivatives have affinities almost tenfold greater than NalBzoH. Along with the wide variations in affinity, the compounds also appear to exhibited widely divergent activities in traditional bioassays.


Subject(s)
Binding, Competitive , Naloxone/analogs & derivatives , Naloxone/metabolism , Receptors, Opioid/drug effects , Animals , Cattle , Guinea Pigs
13.
Mol Pharmacol ; 50(2): 293-8, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8700136

ABSTRACT

Antisense oligodeoxynucleotides directed against various G protein alpha subunits differentially block the analgesic actions of mu-, delta-, and kappa-opioid agonists in mice. Intracerebroventricular administration of oligodeoxynucleotides targeting Gi alpha 2, G(o) alpha, and Gs alpha block supraspinal mu-opioid analgesia, whereas Gi alpha 2 and Gx/z alpha antisense probes block spinal mu analgesia. Although supraspinal and spinal morphine-6 beta-glucuronide (M6G) analgesia also is sensitive to these antisense treatments, its sensitivity profile differs from that of morphine, implying the existence of a different analgesic system. Gi alpha 1 and Gx/z alpha antisense probes block supraspinal M6G analgesia, whereas Gi alpha 1, Gi alpha 3, G(o) alpha, and Gx/z alpha antisense probes block spinal M6G analgesia. Spinal delta-opioid analgesia is blocked by antisense probes to all of the G protein alpha subunits tested, whereas kappa 1-opioid analgesia is sensitive to only Gq alpha. The kappa 3 agonist naloxone benzoylhydrazone produces its analgesia through supraspinal mechanisms and is blocked by Gi alpha 1, Gi alpha 3, Gs alpha, Gq alpha, and Gx/z alpha antisense oligodeoxynucleotides. Together, these results support the presence of seven different analgesic systems for these various opioid agonists.


Subject(s)
Analgesia , Analgesics, Opioid/antagonists & inhibitors , GTP-Binding Proteins/antagonists & inhibitors , Oligonucleotides, Antisense/pharmacology , Animals , Base Sequence , GTP-Binding Proteins/physiology , Male , Mice , Molecular Sequence Data , Receptors, Opioid/physiology
14.
Synapse ; 22(3): 247-52, 1996 Mar.
Article in English | MEDLINE | ID: mdl-9132993

ABSTRACT

To help characterize kappa3 receptors and establish their relationship to traditional mu and delta receptors, we have generated a kappa3-selective monoclonal antibody. Monoclonal antibodies were raised against BE(2)-C cells, a human neuroblastoma cell line containing mu, kappa3, and delta opioid receptors. Of the 5,000 hybridoma cell lines screened, approximately 2,000 hybridomas tested positive against BE(2)-C membranes by ELISA, but only 98 of these were negative against a different neuroblastoma cell line lacking opioid receptors. Supernatants from one hybridoma, 8D8, inhibited up to 90% of 3H-NalBzoH (kappa3) binding without affecting 3H-DAMGO (mu) or 3H-naltrindole (delta) binding in BE(2)-C membranes. The selectivity of the antibody was further demonstrated by its blockade of the inhibition of cAMP accumulation in BE(2)-C cells by the kappa3 agonist NalBzoH but not the mu agonist morphine. Monoclonal antibody 8D8 (mAb8D8) also recognizes kappa3 receptors from mouse, rat, and calf brain. Administered intracerebroventricularly, mAb8D8 blocked kappa3 but not morphine (mu) analgesia in vivo. On Western blots, mAb8D8 recognized a protein with a molecular mass of approximately 70 kilodaltons in BE(2)-C. These studies demonstrate the selectivity of mAb8D8 for kappa3 receptors and provide additional support for the existence of this unique opioid receptor subtype.


Subject(s)
Antibodies, Monoclonal , Receptors, Opioid, kappa/drug effects , Analgesics, Opioid/pharmacology , Animals , Binding, Competitive/drug effects , Blotting, Western , Brain Neoplasms/immunology , Cyclic AMP/metabolism , Enzyme-Linked Immunosorbent Assay , Male , Mice , Mice, Inbred Strains , Neuroblastoma/immunology , Opioid Peptides/metabolism , Pain Measurement/drug effects , Rats , Receptors, Opioid, kappa/immunology , Tumor Cells, Cultured
15.
J Neurochem ; 65(5): 1981-7, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7595481

ABSTRACT

Phosphodiester antisense oligodeoxynucleotides (ODNs) directed against various domains of the cloned mouse delta receptor DOR-1 reduce delta-opioid receptor binding in vivo and in vitro. The present study examines the stability of an antisense ODN (275 nM) directed against the delta-opioid receptor and its effect on DOR-1 mRNA in cultured neuroblastoma cells and in vivo. When added to NG108-15 cells, much of the antisense ODN is degraded. However, > 1% is intact, associated with cells, and stable for at least 72 h. Northern blot analysis demonstrates that treatment of NG108-15 cells with the antisense ODN reduces the levels of a species of DOR-1 mRNA by approximately 25%. Similarly, intrathecal administration of the antisense ODN results in the accumulation of intact ODN within the spinal cord, which is stable for at least 72 h, although the levels of accumulation in vivo are lower than in vitro after either 4 or 72 h. Antisense ODN treatment lowers DOR-1 mRNA levels by approximately 25%. The loss of mRNA both in vivo and in vitro corresponds quite well to the decreases in receptor binding previously observed by our laboratory and is consistent with reduction of delta-opioid receptor protein in vitro as determined by western blot with a monoclonal antibody selective for the delta-opioid receptor. In conclusion, these studies indicate that a small, but significant, proportion of ODN is taken up by cells and remains intact for up to 72 h. This appears to be sufficient to down-regulate mRNA levels of delta-opioid receptors and their expression.


Subject(s)
Cloning, Molecular , Gene Expression Regulation , Oligonucleotides, Antisense , Receptors, Opioid, delta/genetics , Animals , Antibodies, Monoclonal , Base Sequence , Blotting, Northern , Blotting, Western , Drug Stability , Male , Mice , Mice, Inbred Strains , Molecular Sequence Data , Nucleic Acid Hybridization , Oligonucleotide Probes/genetics , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacokinetics , Tumor Cells, Cultured
16.
Trends Pharmacol Sci ; 16(10): 344-50, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7491712

ABSTRACT

Understanding the pharmacology of opioid receptors took a major step forward with the cloning of genes that encode four members of the opioid receptor family. Gavril Pasternak and Kelly Standifer show how strategies that use antisense oligodeoxynucleotides can provide a selective approach to correlate the properties of the cloned receptors with their in vivo pharmacological effects, and have confirmed the association of the delta-, mu- and kappa 1-opioid receptor clones with analgesia mediated through delta-, mu- and kappa 1-opioid receptors, respectively. Approaches that use antisense oligodeoxynucleotides provide an opportunity to characterize the pharmacology of short cDNA fragments without necessitating the cloning of the entire cDNA and can assess the role of the products of specific exons in a receptor, providing an approach for the study of alternative splicing as demonstrated with the mu- and kappa 3-opioid receptor clones.


Subject(s)
Oligonucleotides, Antisense , Peptide Mapping/methods , Receptors, Opioid/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Humans
17.
Neurosci Lett ; 198(2): 99-102, 1995 Sep 29.
Article in English | MEDLINE | ID: mdl-8592651

ABSTRACT

An antisense oligodeoxynucleotide directed against the 5'-untranslated region of MOR-1 blocks the analgesic actions of the mu 1 analgesics morphine and [D-Ala2,D-Leu5]enkephalin (DADL) when they are microinjected into the periaqueductal gray. In contrast, morphine-6 beta-glucuronide (M6G) analgesia is unaffected by this treatment. Antisense oligodeoxynucleotides directed against distinct Gi alpha subunits also distinguish between morphine and M6G analgesia. A probe targeting Gi alpha 2 blocks morphine analgesia, as previously reported, but is inactive against M6G analgesia. Conversely, an antisense oligodeoxynucleotide against Gi alpha 1 inhibits M6G analgesia without affecting morphine analgesia. The antisense oligodeoxynucleotide directed against G(o)alpha is ineffective against both compounds. These results confirm the prior association of Gi alpha 2 with morphine analgesia and strongly suggests that M6G acts through a different opioid receptor, as revealed by its insensitivity towards the MOR-1 antisense probe and differential sensitivity towards G-protein alpha subunit antisense oligodeoxynucleotides.


Subject(s)
Analgesics, Opioid/antagonists & inhibitors , GTP-Binding Proteins/genetics , Morphine Derivatives/antagonists & inhibitors , Morphine/antagonists & inhibitors , Oligonucleotides, Antisense/pharmacology , Receptors, Opioid, mu/genetics , Analgesics/pharmacology , Analgesics, Opioid/pharmacology , Animals , Base Sequence , Enkephalin, Ala(2)-MePhe(4)-Gly(5)- , Enkephalin, Leucine-2-Alanine/antagonists & inhibitors , Enkephalin, Leucine-2-Alanine/pharmacology , Enkephalins/antagonists & inhibitors , Enkephalins/pharmacology , GTP-Binding Proteins/drug effects , Male , Microinjections , Molecular Sequence Data , Morphine/pharmacology , Morphine Derivatives/pharmacology , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Oligonucleotides, Antisense/administration & dosage , Periaqueductal Gray/physiology , Rats , Rats, Sprague-Dawley , Receptors, Opioid, mu/drug effects
18.
J Neurochem ; 65(1): 170-5, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7790858

ABSTRACT

In addition to the mu- and delta-opioid receptors previously reported, the SH-SY5Y human neuroblastoma cell line has high levels of kappa 3 receptors, accounting for 40% of total opioid binding, as measured with [3H]-diprenorphine binding. Competition studies reveal binding profiles for all three receptor classes that are similar to those observed in brain membranes. Differentiation with retinoic acid increases the levels of opioid receptor binding in the cell line, with the largest elevations in kappa 3 binding. Fully 75% of the increased binding corresponds to kappa 3 sites, which represent 50% of total opioid receptor binding in differentiated cells. Morphine inhibits forskolin-stimulated cyclic AMP accumulation, and this effect is readily blocked by the mu antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). Naloxone benzoylhydrazone, a kappa 3 agonist, inhibits forskolin-stimulated cyclic AMP accumulation more potently than morphine and is not reversed by CTAP. These studies indicate that SH-SY5Y cells contain high levels of functional kappa 3 receptors.


Subject(s)
Neuroblastoma/metabolism , Receptors, Opioid, kappa/metabolism , Binding Sites/drug effects , Binding, Competitive , Cell Differentiation , Cyclic AMP/antagonists & inhibitors , Diprenorphine/metabolism , Humans , Morphine/pharmacology , Naloxone/analogs & derivatives , Naloxone/pharmacology , Neuroblastoma/pathology , Tretinoin/pharmacology , Tumor Cells, Cultured/drug effects
19.
Mol Pharmacol ; 47(6): 1180-8, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7603458

ABSTRACT

We have identified a putative opioid receptor from mouse brain (KOR-3), belonging to the G protein-coupled receptor family, that is distinct from the previously cloned mu, delta, and kappa 1 receptors. Assignment of the clone to the opioid receptor family derives from both structural and functional studies. Its predicted amino acid sequence is highly homologous to that of the other opioid receptors, particularly in many of the transmembrane regions, where long stretches are identical to mu, delta, and kappa 1 receptors. Both cyclazocine and nalorphine inhibit cAMP accumulation in COS-7 cells stably expressing the clone. Northern analysis shows that the mRNA is present in brain but not in a number of other organs. Southern analysis suggests a single gene encoding the receptor. A highly selective monoclonal antibody directed against the native kappa 3 receptor recognizes, in Western analysis, the clone expressed in COS-7 cells. The in vitro translation product is also labeled by the antibody. Additional clones reveal the presence of several introns, including one in the second extracellular loop and another in the first transmembrane region. Antisense studies with an oligodeoxynucleotide directed against a region of the second extracellular loop reveal a selective blockade of kappa 3 analgesia in vivo that is not observed with a mismatch oligodeoxynucleotide based upon the antisense sequence. The mu, delta, and kappa 1 analgesia is unaffected by this antisense treatment. Antisense mapping of the clone downstream from the splice site in the first transmembrane region reveals that six different antisense oligodeoxynucleotides all block kappa 3 analgesia. In contrast, only one of an additional six different antisense oligodeoxynucleotides directed at regions upstream from this splice site is effective. This strong demarcation between the two regions raises the possibility of splice variants of the receptor. An additional clone reveals an insert in the 3' untranslated region. In conclusion, the antibody and antisense studies strongly associate KOR-3 with the kappa 3-opioid receptor, although it is not clear whether it is the kappa 3 receptor itself or a splice variant.


Subject(s)
Nerve Tissue Proteins/genetics , Oligonucleotides, Antisense , Receptors, Opioid, kappa/genetics , Receptors, Opioid , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Blotting, Western , Cell Line , Cloning, Molecular , Male , Mice , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Receptors, Opioid, kappa/metabolism , Sequence Homology, Amino Acid , Nociceptin Receptor
20.
Neurosci Lett ; 181(1-2): 47-9, 1994 Nov 07.
Article in English | MEDLINE | ID: mdl-7898768

ABSTRACT

TIPP[psi] is a new delta-selective opioid peptide antagonist. In the current study, we have explored its selectivity against the mu and kappa receptor subtypes. Against [3H]DPDPE binding, TIPP[psi] is quite potent, with a Ki value of < 1 nM, confirming its potent activity at delta receptors. In contrast, its Ki values against mu 1, mu 2, kappa 1, kappa 2 and kappa 3 binding sites are all > 5 microM. DPDPE also is delta-selective. It labels delta sites > 25-fold more potently than mu 1 receptors and is even more selective against the other subtypes. However, this selectivity does not compare to the delta/mu 1 selectivity of TIPP[psi] which exceeds 15,000. This far higher selectivity, coupled with its antagonist properties, gives TIPP[psi] a number of advantages over previously reported delta-selective compounds. We have utilized these advantages to develop an improved mu 1 binding assay using TIPP[psi].


Subject(s)
Oligopeptides/metabolism , Receptors, Opioid, delta/metabolism , Animals , Binding, Competitive , Cattle , Corpus Striatum/metabolism , Enkephalin, D-Penicillamine (2,5)- , Enkephalins/metabolism , Guinea Pigs , Ligands , Narcotic Antagonists/metabolism , Receptors, Opioid, kappa/metabolism , Receptors, Opioid, mu/metabolism , Thalamus/metabolism
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