Identification of opioid receptors in the immune system using a novel combination of selective opioid ligands and indirect phycoerythrin immunofluorescence.
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Animals↗
Biomedical subjects
J M Bidlack
Publications and source records attributed to J M Bidlack.
Kappa opioid receptors on three related thymoma cell lines. Differences in receptor-effector coupling.
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Adenylyl Cyclases↗
Preventing morphine antinociceptive tolerance by irreversible mu opioid antagonists before the onset of their antagonism.
Irreversible opioid antagonists, when administered at small doses, require several hours to display their antagonism of antinociception mediated by opioid receptors. However, most opioid affinity ligands only need a few minutes to produce wash-resistant inhibition of opioid binding to brain membranes. Our study investigated whether the irreversible antagonists, beta-funaltrexamine (beta-FNA), 14 alpha, 14'beta-[dithiobis[(2-oxo-2,1-ethanediyl)imino]]-7,8-dihydro-N- (cyclopropylmethyl)normorphine (N-CPM-TAMO), and N-cyclopropylmethyl-5 beta-methyl- beta-(p-nitrocinnamoylamino)-7,8-dihydromorphinone (N-CPM-MET-CAMO) had any effect on morphine-induced antinociceptive tolerance before the appearance of their antagonism in the mouse tail-flick assay. All opioids were given by i.c.v. administration. The irreversible antagonists, beta-FNA (20 nmol), N-CPM-TAMO (0.5 nmol) and N-CPM-MET-CAMO (1 nmol) did not produce any antagonism of morphine-induced analgesia until at least 8 hr after administration. Pretreatment with morphine (3 nmol, -140 min) produced acute antinociceptive tolerance as demonstrated by a 45-fold rightward shift of the morphine dose-response curve. When coadministered with morphine, beta-FNA, N-CPM-TAMO and N-CPM-MET-CAMO completely prevented the development of morphine tolerance 140 min after administration in a dose-dependent manner. This preventive effect lasted for up to 420 min, during which time, morphine was given repeatedly up to four times. This antinociception produced by morphine after coadministration with irreversible antagonists was antagonized by naloxone, demonstrating that morphine-induced analgesia was still mediated by opioid receptors. The kappa- and delta-selective opioid antagonists, nor-binaltorphimine and ICI 174,864, respectively, did not block the preventive effect produced by the irreversible antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line down-regulates without desensitizing during chronic opioid exposure.
The R1.1 mouse thymoma cell line expresses a single class of kappa opioid receptors that is negatively coupled to adenylyl cyclase through a Bordetella pertussis toxin-sensitive inhibitory guanine nucleotide-binding protein. The aim of the present study was to determine whether chronic opioid treatment of R1.1 cells altered either the binding properties or the functional response associated with the kappa opioid receptor. Culturing of R1.1 cells with the kappa-selective agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U50,488) for 3 hr and longer, followed by extensive washing of R1.1 cell membranes, produced a concentration- and time-dependent reduction in the binding of the kappa-selective ligand (5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1- oxaspiro(4,5)dec-8-yl) benzeneacetamide ([3H]U69,593). Culturing of R1.1 cells with 100 nM U50,488 for 24 hr produced approximately a 50% reduction in the Bmax value for [3H]U69,593 and [3H]naloxone binding. In contrast to the reduction in binding, there was no change in the inhibition of adenylyl cyclase activity by (-)-U50,488. To determine whether kappa opioid receptor function was maintained by spare receptors after agonist-induced down-regulation, membranes from untreated R1.1 cells were incubated with 400 nM of the irreversible opioid antagonist beta-chlornaltrexamine (beta-CNA) followed by extensive washing. beta-CNA produced a 50% reduction in the [3H]U69,593 binding and a 6-fold increase in the IC50 value for (-)-U50,488 inhibition of adenylyl cyclase activity, with no change in the maximal inhibition of cyclic AMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
An all D-amino acid opioid peptide with central analgesic activity from a combinatorial library.
A synthetic combinatorial library containing 52,128,400 D-amino acid hexapeptides was used to identify a ligand for the mu opioid receptor. The peptide, Ac-rfwink-NH2, bears no resemblance to any known opioid peptide. Simulations using molecular dynamics, however, showed that three amino acid moieties have the same spatial orientation as the corresponding pharmacophoric groups of the opioid peptide PLO17. Ac-rfwink-NH2 was shown to be a potent agonist at the mu receptor and induced long-lasting analgesia in mice. Analgesia produced by intraperitoneally administered Ac-rfwink-NH2 was blocked by intracerebroventricular administration of naloxone, demonstrating that this peptide may cross the blood-brain barrier.
Amino Acid Sequence↗
14 alpha,14' beta-[Dithiobis[(2-oxo-2,1-ethanediyl)imino]]bis (7,8-dihydromorphinone) and 14 alpha,14' beta-[dithiobis[(2-oxo-2,1- ethanediyl)imino]]bis[7,8-dihydro-N-(cyclopropylmethyl)normorphinone]: chemistry and opioid binding properties.
14 alpha,14' beta-[Dithiobis[(2-oxo-2,1-ethanediyl)imino]] bis(7,8-dihydromorphinone) (TAMO) (13) was synthesized by condensing 14 beta-amino-7,8-dihydromorphine (4) with acetylthioglycolyl chloride and hydrolyzing the resulting ester with mild base to give a mixture of the thiol 9 and the disulfide 13. Chromatography of the mixture resulted in conversion of the bulk of the thiol 9 to the disulfide 13 by air oxidation. The disulfide 13 was also prepared by condensing the tert-butyldimethylsilyl ether of 4 with the dithiodiglycolyl chloride and treating the resulting product with F- to give the desired product. The pure thiol 9 free of contamination with the disulfide was prepared by treating 13 with excess N-acetyl-L-cysteine and processing the reaction mixture without resorting to chromatography for purification. The corresponding N-(cyclopropylmethyl) nor compound 15 was prepared from the silyl ether 6 and acetylthioglycolyl chloride followed by hydrolysis, treatment with F-, and air oxidation. Incubation of bovine striatal membranes with 13 and 15 resulted in wash-resistant inhibition of the binding of the mu-selective peptide [3H][D-Ala2,(Me)Phe4,Gly(ol)5]-enkephalin (DAMGO). Incubation of membranes with mu but not kappa or delta ligands protected the mu binding sites from alkylation by 13 and 15. The wash-resistant inhibition of mu opioid binding was partially reversed by the addition of the reducing reagent dithiothreitol (DTT). A Scatchard plot of the effect of 13 and 15 on [3H]DAMGO binding showed that these affinity ligands caused a marked decrease in the Bmax value without affecting the Kd value. The wash-resistant inhibition of binding, the reduction in the number of binding sites, the partial reversal of wash-resistant inhibition of binding by DTT, and previously observed long-term antagonism of mu opioid receptors in vivo support the conclusion that 13 and 15 bind covalently to the mu opioid receptor.
Alkylation↗
The kappa-opioid receptor expressed on the mouse lymphoma cell line R1.1 contains a sulfhydryl group at the binding site.
Studies were directed at determining whether the kappa-opioid receptor expressed on the mouse R1.1 thymoma cell line contained either a disulfide bond or a sulfhydryl group at the opioid binding site. The binding of the kappa-opioid receptor agonist [3H](-)-bremazocine to R1.1 cell membranes was unchanged following treatment with the disulfide bond-reducing reagent dithiothreitol at concentrations up to 130 mM. However, treatment of membranes with the sulfhydryl-alkylating reagent N-ethylmaleimide, followed by extensive washing, reduced [3H](-)-bremazocine binding by as much as 90%. Inhibition of [3H](-)-bremazocine binding by N-ethylmaleimide was concentration- and time-dependent. When R1.1 cell membranes were treated with 1 mM N-ethylmaleimide for 10 min at 24 degrees C, the Bmax value for [3H](-)-bremazocine binding decreased by 50%, with no change in receptor affinity. N-Ethylmaleimide-induced reduction of [3H](-)-bremazocine binding was attenuated by pretreatment of membranes with the kappa-selective opioids U50,488 and U69,593. The results indicate that a sulfhydryl group is present at or near the binding site on the kappa-opioid receptor expressed by the R1.1 thymoma cell line.
Alkylation↗
5 beta-Methyl-14 beta-(p-nitrocinnamoylamino)-7,8-dihydromorphinone and its corresponding N-cyclopropylmethyl analog, N-cyclopropylmethylnor-5 beta-methyl-14 beta-(p-nitrocinnamoylamino)- 7,8-dihydromorphinone: mu-selective irreversible opioid antagonists.
5 beta-Methyl-14 beta-(p-nitrocinnamoylamino)-7,8-dihydromorphinone (MET-CAMO) and its corresponding N-cyclopropylmethyl analog, N-cyclopropylmethylnor-5 beta-methyl-14 beta-(p-nitrocinnamoylamino)- 7,8-dihydromorphinone (N-CPM-MET-CAMO) were tested in opioid receptor binding assays and in the mouse tail-flick test in order to characterize the affinity, selectivity and antinociceptive properties of these two compounds. Incubating bovine striatal membranes with either MET-CAMO or N-CPM-MET-CAMO produced a wash-resistant, concentration- and time-dependent inhibition of the binding of the mu-selective ligand, [3H]-[D-Ala2,MePhe4,Gly(ol)5]enkephalin, but with no change in delta or kappa binding. Preincubating membranes with N-CPM-MET-CAMO decreased the maximum binding value for [3H]-[D-Ala2,MePhe4,Gly(ol)5]enkephalin binding without changing the Kd value. In the mouse tail-flick assay, MET-CAMO and N-CPM-MET-CAMO did not produce any antinociception up to a dose of 100 nmol after i.c.v. administration. However, pretreatment of mice with either compound produced a time- and dose-dependent antagonism of morphine-induced antinociception. Analgesia mediated by delta or kappa opioids was not altered by either MET-CAMO or N-CPM-MET-CAMO at a dose of up to 100 nmol. The mu antagonistic effect of 1 nmol of MET-CAMO and N-CPM-MET-CAMO appeared at 8 hr and lasted up to 72 hr, with a maximal effect at 16 to 24 hr after i.c.v. administration. Pretreatment of mice with 1 nmol of MET-CAMO or N-CPM-MET-CAMO, given by i.c.v. administration at -24 hr, produced a rightward and downward shift of dose-response line of i.c.v. morphine.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
14 beta-[(p-nitrocinnamoyl)amino]morphinones, 14 beta-[(p-nitrocinnamoyl)amino]-7,8-dihydromorphinones, and their codeinone analogues: synthesis and receptor activity.
A series of 14 beta-[(nitrocinnamoyl)amino]codeinones and morphinones, some of which contain a 5 beta-methyl group, were prepared from 14 beta-aminocodeinones and 14 beta-[N-(cyclopropylmethyl)-amino]norcodeinones. The affinities of the target compounds for the mu, delta, and kappa opioid receptors were determined by radiolabeled binding experiments using bovine brain membranes. An analogous series of 7,8-dihydrocodeinones and morphinones was prepared and assayed in the same systems. The 3-methoxy derivatives 3 and 4 were more selective than the corresponding morphinones for the mu receptor. The 5 beta-methylcodeinones 25 and 27 had lower affinity at all receptors than the corresponding morphinones, but the 5 beta-methylmorphinones had affinities similar to the morphinones 5 and 6. A similar pattern was observed in the 7,8-dihydro series. Two compounds, 5 beta-methyl-14 beta-[(p-nitrocinnamoyl)amino]-7,8-dihydromorphinone, 20 (MET-CAMO), and N-(cyclopropylmethyl)-14 beta-[(p-nitrocinnamoyl)amino]-7,8-dihydronormorphinone, 22 (N-CPM-MET- CAMO), acted as nonequilibrium ligands in antinociception and membrane binding studies. In mice after icv administration, neither ligand showed any agonist activity but 8-24 h after administration both compounds acted as potent mu antagonists. A Scatchard plot of the effect of N-CPM-MET-CAMO on [3H]DAMGO ([3H]D-Ala2, (Me)-Phe4, Gly(ol)5] enkephalin) binding to bovine striatal membranes showed that there was a significant decrease in the Bmax value and a marginal effect on the Kd value suggesting that the number of binding sites was reduced. When taken together, these results support the view that 20 and 22 bind covalently to the mu receptor. On the other hand, when N-acetylcysteine and 22 were allowed to react in a buffered solution, 22 was recovered unchanged. Under these conditions no Michael reaction was observed.
Animals↗
Antinociceptive evaluation of 14 beta-(bromoacetamido)-7,8-dihydro- N(cyclopropylmethyl)-normorphinone in mice.
This study evaluated the supraspinal opioid effects of 14 beta-(bromoacetamido)-7,8-dihydro-N(cyclopropylmethyl)-normorphinone+ ++ (N-CPM-H2BAMO) in the mouse acetic acid-induced writhing and tail-flick assays. In the writhing test, N-CPM-H2BAMO produced a time- and dose-dependent antinociception after i.c.v. administration, with a 50% antinociceptive response being obtained with 0.28 (0.19-0.39) nmol when given 10 min before testing. The antinociceptive effect of N-CPM-H2BAMO was antagonized in a dose-dependent manner by the kappa-selective opioid receptor antagonist, nor-binaltorphimine. In the mouse tail-flick assay, N-CPM-H2BAMO failed to produce any antinociception after i.c.v. administration. N-CPM-H2BAMO produced a dose-dependent antagonism of morphine-induced antinociception but not antinociception induced by the delta-opioid receptor agonist [D-Pen2,D-Pen5]enkephalin. Nor-binaltorphimine (0.3 nmol) at dose that completely antagonized N-CPM-H2BAMO-induced antinociception in the writhing assay did not prevent the antagonistic effect of N-CPM-H2BAMO on morphine-induced antinociception. Therefore, these data indicate that N-CPM-H2BAMO produces antinociception by acting at supraspinal kappa-opioid receptors in the writhing assay, and also acts as a mu-opioid receptor antagonist.
Acetates↗
Affinity labeling of the mu opioid receptor in bovine striatal membranes with [3H]-14 beta-(bromoacetamido)-7,8-dihydromorphine.
[3H]-14 beta-(Bromoacetamido)-7,8-dihydromorphine ([3H]H2BAM) was synthesized and tested for its ability to selectively label mu opioid receptors in bovine striatal membranes. Incubating membranes with N-tosyl-L-phenylalanine chloromethyl ketone and dithiothreitol before the addition of [3H]H2BAM reduced nonspecific [3H]H2BAM binding so that [3H]H2BAM binding to opioid receptors was up to 70% of the total [3H]H2BAM binding and was dependent on [3H]H2BAM concentration, incubation time, and pH of the reaction. At pH 7.5, [3H]H2BAM bound selectively to the mu opioid receptor, but mainly noncovalently. After the initial binding of [3H]H2BAM to the receptor, membranes were washed and then incubated at 37 degrees C in 50 mM Tris-HCl, pH 8.5, for 3 h, a time that resulted in greater than 80% of the [3H]H2BAM associated with the receptor becoming covalently bound to the opioid receptor. The mu-selective peptide [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin inhibited [3H]H2BAM labeling of membranes, while delta- or kappa-selective compounds were ineffective. Both NaCl and the nonhydrolyzable guanine nucleotide analog guanylyl 5'-imidodiphosphate reduced the incorporation of [3H]H2BAM into membranes. When [3H]H2BAM-labeled striatal membranes were separated under reducing conditions on a sodium dodecyl sulfate-polyacrylamide gel, two proteins with molecular weights of 54,000 and 44,000 were specifically labeled. The 54-kDa protein was present in a greater amount than the 44-kDa protein. Both proteins bound to wheat germ agglutinin-Sepharose and concanavalin A-Sepharose, suggesting that both proteins contain multiple carbohydrate moieties. Despite the inclusion of protease inhibitors, the 44-kDa protein may be a proteolytic fragment of the 54-kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity Labels↗
5 beta-Methyl-14 beta-(p-nitrocinnamoylamino)-7,8-dihydromorphinone: a long-lasting mu-opioid receptor antagonist devoid of agonist properties.
5 beta-Methyl-14 beta-(p-nitrocinnamoylamino)-7,8-dihydromorphinone (MET-CAMO) suppressed morphine-induced antinociception but had no effect on antinociception mediated by delta- or kappa-opioid receptors after a single i.c.v. 1-nmol injection from 8 to 72 h before testing. MET-CAMO had no agonist effects in the mouse tail-flick assay in doses up to 100 nmol. MET-CAMO is the first N-methylated morphine derivative which shows such long-lasting mu-selective opioid receptor antagonism with no agonistic properties.
Analgesics↗
Pharmacological study of 14 beta-(thioglycolamido)-7,8-dihydro-N(cyclopropylmethyl)-normor phinone (N-CPM-TAMO).
Opioid effects of 14 beta-(thioglycolamido)-7,8-dihydro-N(cyclopropylmethyl)- normorphinone (N-CPM-TAMO) were studied in the mouse tail-flick and acetic acid writhing assays. In the tail-flick test, N-CPM-TAMO failed to produce any antinociception after i.c.v. administration of up to 300 nmol. However, pretreatment of mice with N-CPM-TAMO produced a time- and dose-dependent antagonism of morphine-induced antinociception. The antagonism by N-CPM-TAMO lasted up to 48 hr, with a maximal effect at 24 hr after i.c.v. administration. Similarly, pretreatment of mice with N-CPM-TAMO at 24 hr also produced a dose-dependent antagonism of kappa-mediated antinociception, induced by U50,488 However, the antagonistic potency of N-CPM-TAMO against U50,488 was 100-fold less than against morphine. Pretreatment with N-CPM-TAMO had no effect on delta opioid receptor-mediated antinociception, as measured with [D-Pen2,D-Pen5]enkephalin. In the writhing assay, N-CPM-TAMO produced a time- and dose-dependent antinociception after i.c.v. administration, with a value of the dose producing 50% analgesia of 18.4 (10.6-31.9) nmol. The antinociceptive effect lasted up to 3 hr after administration. N-CPM-TAMO-induced antinociception was antagonized by coadministration of the kappa-selective antagonist, norbinaltorphimine. Pretreatment of mice with N-CPM-TAMO also produced a time- and dose-dependent antagonism of U50,488-induced antinociception, which lasted up to 72 hr, with a maximal effect at 24 hr after administration. These data indicate that N-CPM-TAMO is a mu-selective, long-term antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line is coupled to adenylyl cyclase through a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein.
The R1.1 mouse thymoma cell line expresses a high-affinity kappa opioid binding site. Opioid binding to this site is inhibited by guanine nucleotides, suggesting that the receptor is coupled to a guanine nucleotide-binding protein. Here, we present evidence that the kappa opioid binding site on R1.1 cell membranes is negatively coupled to adenylyl cyclase. The kappa-selective agonists (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide methane-sulfonate hydrate [(-)-U50,488], (5 alpha,7 alpha, 8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxas- piro(4,5)dec-8-yl)benzeneacetamide (U69,593) and several dynorphin peptides inhibited basal and forskolin-stimulated cyclic AMP production by up to 40% in R1.1 cell membranes. The order of potency for the inhibition of adenylyl cyclase activity by opioid agonists correlated with their Ki values for the inhibition of [3H]U69,593 binding. Opioid-mediated inhibition of adenylyl cyclase activity was stereoselective, as (-)-U50,488 was more potent than the (+) isomer, and the inhibition was blocked by the kappa-selective antagonist nor-binaltorphimine. The opioid-mediated inhibition of adenylyl cyclase activity was also completely blocked by incubating R1.1 cells with Bordetella pertussis toxin (PTX). Incubation of R1.1 cell membranes with PTX and [adenylate-32P]NAD+ resulted in the exclusive labeling of a 41-kDa protein, as determined by separating the membrane proteins under reducing conditions on a SDS polyacrylamide gel, followed by autoradiography. These results suggest that a PTX-sensitive inhibitory guanine nucleotide-binding protein mediates the link between the thymoma kappa opioid receptor and adenylyl cyclase.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
kappa-Opioid binding sites on a murine lymphoma cell line.
As a first step in determining whether any subset of lymphocytes expresses opioid receptors, membranes prepared from mouse lymphoma cell lines were screened for [3H]naloxone binding sites. Membranes from the R1.1 cell line specifically bound [3H]naloxone. The Hill coefficient for [3H]naloxone binding was 0.93 +/- 0.18, and nonlinear regression analysis indicated that a one-site model was the best fit of the [3H[naloxone saturation binding data. Low concentrations of kappa-selective opioids, but neither mu nor delta opioids, inhibited [3H]naloxone binding. Saturation binding studies with the kappa-selective compound [3H]U69,593 revealed a single binding site with a KD value of 0.204 +/- 0.039 nM and a Bmax value of 31.7 +/- 3.1 fmol/mg of membrane protein. The Hill coefficient for [3H]U69,593 binding was 1.03 +/- 0.11, indicative of a single site. Time courses for the association and dissociation of [3H]U69,593 binding at 25 degrees C exhibited properties consistent with a single class of binding sites. Low concentrations of kappa-selective opioids, including dynorphin peptides, inhibited [3H]U69,593 binding, while high concentrations of mu opioids were needed to inhibit binding, and the delta-selective ligands were ineffective at concentrations up to 10 microM. Stereoselectivity of the binding site was demonstrated by the finding that the Ki value for (-)-pentazocine in inhibiting [3H]U69,593 binding was 25 times less than for the (+)-isomer. Based on its high affinity for U69,593, alpha-neo-endorphin, and dynorphin B, the kappa opioid binding site on R1.1 cell membranes belongs to the kappa 1b subtype. As observed with brain kappa opioid binding sites, sodium inhibited [3H]U69,593 binding to R1.1 cell membranes in a concentration-dependent manner. These data demonstrate that the murine lymphoma cell line R1.1 expresses kappa opioid binding sites that are very similar to brain kappa opioid binding sites.
Animals↗
Kappa opioid binding sites on the R1.1 murine lymphoma cell line: sensitivity to cations and guanine nucleotides.
The present study describes the characterization of an opioid binding site on membranes prepared from the R1.1 cell line, a murine thymoma. Specific (-)[3H]bremazocine binding was saturable, stereoselective, and limited to a single high affinity binding site with a Kd value of 15.2 +/- 1.6 pM and a Bmax value of 54.8 +/- 6.0 fmol/mg of protein. The kappa-selective alkaloids and dynorphin peptides inhibited (-)[3H]bremazocine binding with Ki values of less than 1 nM, in contrast to mu- and delta-selective ligands. The high affinity of this site for alpha-neo-endorphin and U50,488 suggests that this kappa opioid binding site resembles the kappa 1b subtype. NaCl, as well as other mono- and divalent cations, inhibited (-)[3H]bremazocine binding. In the presence of NaCl, the nucleotides GTP, GDP, and the nonhydrolyzable analog guanylyl-5'-imidodiphosphate (Gpp(NH)p) also decreased (-)[3H]bremazocine binding, suggesting that this kappa opioid binding site is coupled to a G-protein. In summary, R1.1 cells possess a single high affinity kappa opioid receptor that shares many properties with brain kappa 1b opioid receptors.
Animals↗
Effects of beta-endorphin on mu and delta opioid receptor-coupled G-protein activity: low-Km GTPase studies.
Human beta-endorphin 1-31 (beta-END) stimulated low-Km GTPase activity in a concentration-dependent and saturable manner in membranes prepared from the delta opioid receptor-containing hybrid cell line NG108-15 and from the mu opioid receptor-enriched human neuroblastoma cell line SK-N-SH. Naloxone and the delta-selective antagonist, ICI 174,864, blocked the stimulation of the GTPase activity produced by beta-END in NG108-15 cell membranes, whereas only naloxone inhibited the beta-END-induced stimulation in SK-N-SH cell membranes, suggesting that beta-END was acting through both mu and delta opioid receptors. Treatment of the cells with Bordetella pertussis toxin before the preparation of membranes blocked the stimulation of low-Km GTPase by beta-END in both cell lines. Activation of NG108-15 and SK-N-SH low-Km GTPase by beta-END was sodium-dependent, and lithium and potassium were poor promoters of this activation. These results demonstrate that beta-END stimulates the interaction of both mu and delta opioid receptors with B. pertussis toxin-sensitive G-proteins in SK-N-SH and NG108-15 cell membranes, respectively.
Cell Membrane↗
Protein kinase C activation increases the rate and magnitude of agonist-induced delta-opioid receptor down-regulation in NG108-15 cells.
Protein kinase C (PKC) activation was examined for its role in delta-opioid receptor down-regulation in the neuroblastoma X glioma hybrid cell line NG108-15. Incubation of NG108-15 cells for 2 hr at 37 degrees with up to 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), a phorbol ester that activates PKC, had no effect on opioid binding to membranes prepared from these cells. However, as little as 3 nM PMA incubated with an opioid agonist and NG108-15 cells potentiated the decrease and the rate of decrease of opioid binding, compared with agonist alone. Scatchard analysis of [3H][D-Ala2,D-Leu5]enkephalin (DADLE) binding revealed that NG108-15 cells incubated for 3 hr with 1 nM DADLE and 30 nM PMA displayed a > 50% reduction in the number of [3H]DADLE binding sites with no affinity change at the remaining sites, compared with cells treated with DADLE alone. The antagonist naloxone blocked both DADLE-induced and PMA-enhanced DADLE-induced down-regulation. The agonists morphine and cyclazocine, which alone were unable to induce delta receptor down-regulation, did so in the presence of PMA. The PKC inhibitor staurosporine and down-regulation of PKC by chronic PMA treatment blocked PMA potentiation of DADLE-induced down-regulation, but not "normal" DADLE-induced down-regulation. The enhancement of down-regulation by PMA was unaffected by either metabolic inhibitor or incubations at 20 degrees, conditions that blocked down-regulation by DADLE alone. NG108-15 cells incubated with [3H]DADLE and PMA retained more [3H]DADLE than cells incubated with [3H]DADLE alone, suggesting that PMA enhanced receptor internalization instead of merely inhibiting membrane binding. The diacylglycerol 1-oleoyl-2-acetyl-glycerol and bradykinin substituted for PMA but not carbachol, indicating that PKC activated physiologically may play a role in delta receptor down-regulation.
Alkaloids↗