Opioid receptors in human neuroblastoma SH-SY5Y cells: Evidence for distinct morphine (μ) and enkephalin (δ) binding sites (original) (raw)
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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.
Characterization of the δ-opioid receptor found in SH-SY5Y neuroblastoma cells
European Journal of Pharmacology, 1997
The d-opioid receptor found in SH-SY5Y cells was characterized in terms of binding profile and ability to mediate the inhibition of Žw 2 5 x . forskolin-stimulated cAMP accumulation. Both DPDPE D-Pen ,D-Pen enkephalin and deltorphin II, compounds reported to be selective for the d -and d -opioid receptor respectively, were potent agonists in these cells. Binding studies indicated that naltrindole 1 2 Ž . Ž . benzofuran NTB had significantly higher affinity than 7-benzylidenenaltrexone BNTX ; however, both compounds have high affinity for the d-opioid receptor found in SH-SY5Y cells. Naltrindole benzofuran was found to be a potent antagonist, with an IC of less than 1 50 nM, while 7-benzylidene naltrexone was found to be a relatively weak antagonist, requiring greater than 100 nM to inhibit 50% of agonist activity. Binding to intact SH-SY5Y cells was compared to binding to cell membranes and guinea-pig brain membranes. In each case, binding affinities were very similar. These studies suggest that the receptor found in SH-SY5Y cells could probably be classified as a d -opioid receptor. However, the very similar binding characteristics of SH-SY5Y cells and guinea-pig brain membranes call into 2 question the ability to label d -opioid receptors. q Elsevier Science B.V. All rights reserved. 1
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.
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.
Characterization of Opioid Receptor Subtypes in Solution
Journal of Neurochemistry, 1986
Stable opioid receptor binding activity that retains distinct subtype specificities (p, 6, and K) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization. The dependence on Mg2+ ions for receptor activity is also expressed in the soluble state, where the presence of Mg2+ leads to high-affinity and high-capacity opioid peptide binding to the 6, p, and K sites (the latter subtype measured by the binding of [3H]dynorphin,-,). Binding of opiate alkaloids to soluble receptor sites is less dependent on Mg2+ than is opioid peptide binding. Soluble opioid binding activity shows the same sensitivity to Na+ ions and guanine nucleotides as the membrane-bound receptor. The ligand-receptor interactions give evidence of strong positive cooperativity, which is interpreted in terms of association-dissociation of receptor subunits on ligand binding in solution. Binding of enkephalin peptides is associated with the largc macromolecules present (apparent Stokes radii > 60 A), where$s both those and several small species present (<60 A) bind opiate alkaloids and dynorphh-,. Key Words: Opioid receptor subtypes k, 6, K-Digitonin-Mg2+ dependence. Demoliou-Mason C. D. and Barnard E. A. Characterization of opioid receptor subtypes in solution.
Novel opioid binding sites associated with nuclei of NG108-15 neurohybrid cells
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1993
Nuclear opioid binding sites have been discovered in NG108-15 neurohybrid cells. Marker enzyme analyses as well as electron and fluorescence microscopy studies attested to the high degree of purity of the nuclear preparations. Immunohistochemical studies on cryostat sections of NG108-15 cells with an antibody to the opioid receptor corroborated a nuclear localization. 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE), 3H-[D-Ala2,D-Leu5]enkephalin (3H-DADLE), and 3H-diprenorphine binding parameters, Kd and Bmax values, and heterologous competition binding and stereospecificity data satisfied criteria for the presence of delta-opioid sites in purified nuclear preparations. Neither mu-([D-Ala2,mephe4,gly-ol5] enkephalin), dihydromorphine, nor kappa-(U69593) specific binding was detectable in purified nuclear preparations. Rates of association and dissociation of 3H-[D-Ser2,L-Leu5]enkephalyl-Thr were comparable to values obtained previously for opioid receptors. Opioid binding was also shown in s...
A monoclonal antibody directed against an opiate binding site in rat neural membranes
Neuropeptides, 1984
A monoclonal antibody, capable of inhibiting opiate binding to rat neural membranes, has been obtained using hybridoma technology. This IgM cryo#obulin, 0R-989.2.4, inhibited opiate binding to rat neural membranes in a titrable and saturable manner. The immunoglobulin partially inhibited the binding of all opiate agonists and antagonists tested. With regard to opiate 980111srt=, ORd89.2.4 inhibited p greayr gumber of moles of H-dihydromorphine sites than H_[D-Ala -Leu 1 enkephalin or 3Hethylketoeyclaeocine sites. In addition to being able to block opiate binding to neural membranes, the antibody could also displace opiates from membranes equilibrated with ligand. Analysis of binding data indicates that the immunoglobulin is acting as a competitive inhibitor at a mu type opiate binding site.
Analysis of inverse agonism at the δ opioid receptor after expression in Rat 1 fibroblasts
Biochemical Journal, 1996
A cDNA encoding the mouse δ opioid receptor was expressed stably in a Rat 1 fibroblast cell line. Expression of this receptor was demonstrated both in ligand binding studies and by reverse transcriptase-PCR. In membranes of clone D2 cells the opioid peptide [-Ala#]-leucine enkephalin (DADLE) produced a robust, concentration-dependent, stimulation of basal high-affinity GTPase activity ; the prototypic opioid antagonist naloxone and the highly selective and potent δ opioid ligands H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-Tic[CH # -NH]Phe-Phe-OH (TIPP[ψ]) had little effect but N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI174864) caused a marked dose-dependent inhibition of this activity (Tic, 1,2,3,4-tetrahydroisoquinolin-2-yl-carbonyl ; Aib, α-aminobutyric acid). This effect of ICI174864 was reversed by TIPP[ψ] and attenuated after treatment of the cells with pertussis toxin. No stimulation by DADLE or inhibition by ICI174864 was observed in Rat 1 fibroblasts that did not express the δ opioid receptor. Basal binding of [$&S]guanosine 5h-O-(3-thiotriphosphate) to membranes of clone D2 cells was also stimulated by DADLE and inhibited by ICI174864 ; both of these effects were reversed by co-incubation with TIPP[ψ]. When cholera