Autoradiographic localization of δ opioid receptors in the rat brain using a highly selective bis-penicillamine cyclic enkephalin analog (original) (raw)
Related papers
Journal of Medicinal Chemistry, 1992
Opioid Receptor Binding. Radioreceptor binding assays were carried out as described previously using guinea pig brain homogenate.12 The radioligand used was [3H]bremazocine (New England Nuclear) (37.0 Ci/mmol) at a concentration of 0.6 nM for determination of total opioid binding sites. For p-binding s i b 1.0 nM [3H]DAG0 [~-Ala~-NMePhe'-Gly-ol~-enkephalin] (Ameraham) (60.0 Ci/mmol) was used. For r-binding sites, 0.5 nM [3H]bremezocine was used in the presence of 100 nM unlabeled DAG0 and 100 nM unlabeled DPDPE to block p and S sites, respectively. For &binding si-1.0 nM [SH]DPDPE [D-Pen2-~-Pen~-enkephalin] (New England Nuclear) (34.3 Ci/mmol) was used. Nonspecific binding was determined using naloxone (10 pM). Stock solutions of each test compound were prepared immediately prior to the assay by dissolving the free amine in 50% acetic acid (200 pL) and then were serially diluting with water. Nine concentrations of each ligand to be tested were examined in competition experiments with each radioligand. The samples were incubated in 50 mM Tris-HC1 buffer (pH 7.4) at 25 "C for 429.1848, obed 429.1836. Anal. (CBHBN204S.HCl) C, H, N. 1 h and then rapidly filtered through Whatman GF/B filters, which were rinsed twice with cold buffer (2 mL each) and aftsr standing overnight in Aquasol II scintillation fluid (10 mL) were counted in a scintillation counter. ICw values were determined using log-probit analysis. Irreversibility and Protection Studies. The studies were carried out as degcribed previously.12 Membrane preparations were incubated with drug to be tested for 1 h at 25 "C. For protection studiea, naloxone was added at a concentration of 1 pM (rewvery was checked with naloxone alone). Aft8r incubation$ the samples were diluted 4-fold with buffer and centrifuged for 15 min at 20000g. The supernatant was removed, and the pellet waa resuspended in 3 times the original volume of buffer and incubated at 37 "C for 15 min, centrifuged again, and reeuepBILded in the original volume of buffer. A binding assay using [sH]bremezocine, [3H]DAG0, or [3H]DPDPE waa carried out as described above. Acknowledgment. We gratefully acknowledge support of this work by the National Institute on Drug Abuse through research grants DA-03933 and DA-06675.
Proceedings of the National Academy of Sciences, 1985
Specific binding properties of the tritium-labeled 6 opiate receptor agonist [3H][2-D-penicillamine, 5-Dpenicillaminelenkephalin ([3H][D-Pen7, D-Pen5]enkephalin) were characterized in the rat brain and in a mouse neuroblastoma-rat glioma hybrid cell line (NG 108-15). Saturation isotherms of [3H[D-Pen2, D-Pen ]enkephalin binding to rat brain and NG 108-15 membranes gave apparent Kd values of 1-6 nM. These values are in good agreement with the Kd value obtained from the kinetic studies. The Bmax value in NG 108-15 membranes was 235.3 fmol/mg of protein. An apparent regional distribution of (3HJ[D-Pen2, D-Pen ]enkephalin binding was observed in the rat brain. A number of enkephalin analogues inhibited [3H][D-Pen2, D-Pen ]enkephalin binding with high affinity (IC5o values of 0.5-5.0 nM) in both NG 108-15 and rat brain membranes. However, putative ,. receptorselective ligands such as morphine, [D-Ala2, MePhe4, Gly5ollenkephalin, [MePhe3, D-Pro4]morphiceptin, and naloxone were less effective inhibitors of [3H][D-Pen2, D-Pen5]enkephalin binding in both systems tested. These data suggest that (3H][D-Pen2, D-Pen ]enkephalin is a potent and selective ligand for the 6 opioid receptor.
Subcellular Compartmentation of Opioid Receptors: Modulation by Enkephalin and Alkaloids
Journal of Neurochemistry, 1986
A subclone of NG 108-15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes P-glucuronidase, galactosyltransferase, 5'nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 1 I% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a ki-netic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [3H]u-AlaZ-~-Leu5-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [3H]diprenorphine, chased with various unlabeled opiates, and the distribution of 3H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [3H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells. Key Words: Opioid receptors-Enkephalin-Golgi-Receptosomes-Down regulation-Tolerance.
Peptides, 1998
peptide receptor studies. 9. Identification of a novel non--non-␦-like opioid peptide binding site in rat brain. PEPTIDES 19(6) 1079 -1090, 1998. Quantitative binding studies resolved two high-affinity [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin binding sites in rat brain membranes depleted of binding sites by pretreatment with the irreversible agent BIT. -The two binding sites had lower (␦ ncx-2 , Ki ϭ 96.6 nM) and higher (␦ ncx-1 , Ki ϭ 1.55 nM) affinity for DPDPE. The ligand-selectivity profile of the ␦ ncx-1 site was that of a classic ␦ binding site. The ligand-selectivity profile of the ␦ ncx-2 site was neitheror ␦-like. The Ki values of selected agents for the ␦ ncx-2 site were: [pCl]DPDPE (3.9 nM), DPLPE (140 nM), and DAMGO (2.6 nM). Under these assay conditions, [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin binding to the cells expressing the cloned receptor is very low and pretreatment of cell membranes with BIT almost completely inhibits [ 3 H]DAMGO and [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin binding. Intracerebroventricular administration of antisense DNA to the cloned delta receptor selectively decreased [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin binding to the ␦ ncx-1 site. Administration of buprenorphine to rats 24 h prior to preparation of membranes differentially affected , ␦ ncx-1 , and ␦ ncx-2 binding sites. Viewed collectively, these studies have identified a novel non--non-␦-like binding site in rat brain.
Journal of Pharmacology and Experimental Therapeutics, 2004
Among the different mechanisms underlying opioid tolerance, receptors desensitization would represent a major cellular adaptation process in which the role of receptor internalization is still a matter of debate. In the present study, we examined desensitization of the human delta opioid receptor (hDOR) produced by endogenous opioid peptides, Leu-(Tyr-Gly-Gly-Phe-Leu) and Met-enkephalin (Tyr-Gly-Gly-Phe-Met), and the contribution of internalization in this process. Results obtained with natural peptides were compared to those produced by a synthetic opioid agonist, SNC-80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide). After a 30 min-treatment, we observed a different regulation of hDOR between agonists. SNC-80 produced a stronger and faster desensitization and was associated with a loss of opioid binding sites by 50 %. SNC-80 caused also a marked hDOR down-regulation by 30 % as observed by western-blot. Immunocytochemistry revealed that SNC-80 induced a complete redistribution of hDOR from cell surface into intracellular compartments while a partial internalization was visualized upon enkephalins exposure. In constrast, a stronger hDOR recycling and resensitization were measured after enkephalins treatment compared to SNC-80. These data strongly suggested a differential sorting of the internalized receptors caused by enkephalins and SNC-80 that was further confirmed by chloroquine as a lysosomal degradation blocker and monensin as a recycling endosome inhibitor. Finally, by preventing hDOR internalization with 0.5 M sucrose, we demonstrated that hDOR internalization contributes partially to desensitization. In conclusion, hDOR desensitization depends both on its internalization and its sorting either to the recycling pathway or to lysosomes.
Pharmacological properties of a proenkephalin A-derived opioid peptide: BAM 18
European Journal of Pharmacology, 1987
BAM 18 is a derivative of the opioid precursor proenkephalin A. Although it exists in rat and guinea-pig brain in relatively high concentrations, its physiological function is presently unknown. In the present study we have determined the opioid receptor selectivity of this peptide using radioligand binding and peripheral tissue bioassay. When selective binding conditions were used, BAM 18 bound to the # opioid receptor with an affinity three times that of the r opioid receptor and over 10 times that of the 8 opioid receptors (K i = 0.29, 0.75, and 3.2 nM respectively). BAM 18 also displayed mixed receptor selectivity in in vitro bioassay. K e values for naloxone antagonism of BAM 18 agonist activity in the electrically stimulated guinea-pig ileum and the mouse vas deferens were 4.3 and 9.9 nM, respectively. These data indicate that BAM 18 binds to all three opioid receptor subtypes with a selectivity profile of #>x>& BAM 18; Proenkephalin A; Metorphamide; Opioid peptides; Opioid receptors
Affinity Labeling of δ Opioid Receptors by an Enkephalin-Derivative Alkylating Agent, DSLET-Mal
Biochemical and Biophysical Research Communications, 1999
[Ile 5,6 ]deltorphin II-stimulated [ 35 S]GTP␥S binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [ 35 S]GTP␥S binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLET-Mal can serve as an affinity label for further studies of the delta-opioid receptors.