u-50488 and Thymoma

Kappa opioid agonists alter some immune functions of macrophages, and T- and B-lymphocytes. The mouse thymoma cell lines R1.G1 and R1EGO express only kappa-opioid receptors and these kappa-opioid receptors are coupled to an inhibitory GTP-binding regulatory protein. Binding of kappa-opioid agonists to the opioid receptor leads to the inhibition of adenylyl cyclase activity in these cells. In this study, an acute (15 min) and chronic (24 h) treatment of R1.G1 and R1EGO cell with a potent kappa-opioid agonist (-)U50,488 (100 nM) was studied to determine if a kappa-opioid agonist altered receptor number and/or desensitization of adenylyl cyclase activity in these two cell lines. Chronic treatment of both R1.G1 and R1EGO cells with (-)U50,488 lead to down-regulation of the kappa-opioid receptor, measured as a decrease of approximately 50% in the Bmax value for the binding of [3H]U69,593. The binding affinity (Kd value) was not affected after chronic treatment either in R1.G1 or R1EGO cells. There was no difference in the magnitude of inhibition of adenylyl cyclase activity by (-)U50,488 between the acute (15 min) and chronic (24-h) treatment in both cell lines R1.G1 and R1EGO. This study indicates that chronic opioid treatment of mouse thymoma R1.G1 and R1EGO cell lines leads to down-regulation of the receptor, without desensitization. This phenomenon was observed in R1.1 parent mouse thymoma cell line and recently in CHO cells expressing kappa-opioid receptor. This study demonstrates that unlike some neuronal preparations, chronic opioid treatment of the thymoma cell lines resulted in receptor down-regulation without desensitization.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Benzeneacetamides; Down-Regulation; Enzyme Inhibitors; Kinetics; Mice; Pyrrolidines; Receptors, Opioid, kappa; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured

In this paper, the effect of the synthetic kappa-opioid agonist (-)U50,488 on 45Ca2- transport into R1.1 mouse thymoma cells is presented. This thymoma cell line expresses selectively the kappa-opioid class of receptors. 45Ca2+ transport into R1.1 cells was not affected by the kappa-opioid agonist (-)U50,488 (10(-10) M-10(-4) M) alone, or in the presence of the plant lectins: PHA (250 microg/ml) and Con A (800 microg/ml), after a 60 min treatment. The plant lectins PHA and Con A stimulated 45Ca2+ transport into R1.1 cells, in high concentrations (100-800 microg/ml) and (200-1000 microg/ml) respectively, after a 60 min treatment. Thus, 45Ca2+ transport was not affected in R1.1 cells by the kappa-opioid agonist (-)U50,488 alone, or in the presence of mitogens after a 60 min treatment. This negative result does not indicate the lack of calcium channels on R1.1 cells, since the plant lectins PHA and Con A were able to stimulate 45Ca2+ transport into R1.1 cells.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Calcium; Concanavalin A; Ion Transport; Mice; Phytohemagglutinins; Receptors, Opioid, kappa; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured

The mouse thymoma R1.1 cell line was shown previously to express a single high-affinity kappa 1 opioid receptor that is negatively coupled through a pertussis toxin-sensitive G-protein to adenylyl cyclase. This study compared opioid receptor binding and inhibition of adenylyl cyclase activity in three unique derivatives of the R1.1 cell line. Membranes from the R1.G1 and R1E/TL8x.1.G1.OUAr.1 (R1EGO) cell lines bound both [3H]U69,593 and [3H](-)-bremazocine with similar affinities compared with R1.1 membranes, whereas membranes from the R1E/TL8x.1 (R1E) cell line did not possess any opioid binding sites, detected by radioreceptor binding. The Bmax values for [3H]U69,593 and [3H]-(-)-bremazocine binding to R1.G1 and R1EGO cell membranes were, respectively, 3- and 6-fold greater than those obtained with the parent R1.1 cell line. GTP and its nonhydrolyzable analog, Gpp(NH)p, inhibited [3H]U69,593 binding to all three cell lines. Stimulation of low-Km GTPase activity by the kappa-selective agonist (-)U50,488 was greatest in R1.G1 membranes, followed by R1EGO and R1.1. The maximal inhibition of forskolin-stimulated adenylyl cyclase activity by (-)U50,488 was 66 +/- 2% in R1.G1 and 49 +/- 2% in R1EGO, compared with 37 +/- 1% in R1.1 membranes. Whereas maximal inhibition of adenylyl cyclase activity did not correlate with receptor number among cell lines, the inhibition of cyclic AMP production did correlate with stimulation of low-Km GTPase activity. The R1.1 cell line and its derivatives, R1.G1 and R1EGO, express a similar type of kappa opioid receptor, which exhibits differences in coupling to G-proteins and to adenylyl cyclase among cell lines. These cell lines provide an excellent model system for studying the regulation of opioid receptor-adenylyl cyclase coupling efficiency.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Analgesics; Animals; Benzeneacetamides; Benzomorphans; Cell Membrane; GTP Phosphohydrolases; Kinetics; Mice; Pyrrolidines; Receptors, Opioid, kappa; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured

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)

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Benzeneacetamides; Cell Membrane; Down-Regulation; In Vitro Techniques; Mice; Naloxone; Naltrexone; Pertussis Toxin; Pyrrolidines; Receptors, Opioid, kappa; Thymoma; Tumor Cells, Cultured; Virulence Factors, Bordetella

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.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Analgesics; Animals; Cell Membrane; Cyclic AMP; Dynorphins; GTP-Binding Proteins; Mice; Naltrexone; Pertussis Toxin; Pyrrolidines; Receptors, Opioid, kappa; Sensitivity and Specificity; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured; Virulence Factors, Bordetella