Diane M.P. Lawrence

κ-Opioid binding sites on a murine lymphoma cell line

Abstract 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 κ-selective opioids, but neither μ nor δ opioids, inhibited [3H]naloxone binding. Saturation binding studies with the κ-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°C exhibited properties consistent with a single class of binding sites. Low concentrations of κ-selective opioids, including dynorphin peptides, inhibited [3H]U69,593 binding, while high concentrations of μ opioids were needed to inhibit binding, and the δ-selectivc ligands were ineffective at concentrations up to 10 μM. Stereosclectivity 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, α-neo-endorphin, and dynorphin B, the κ opioid binding site on R1.1 cell membranes belongs to the k1b subtype. As observed with brain κ 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 κ opioid binding sites that are very similar to brain κ opioid binding sites.

Kappa opioid receptors expressed on three related thymoma cell lines: Differences in receptor-effector coupling

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.

Fluorescent staining of κ opioid receptors using naltrexamine derivatives and phycoerythrin

An immunofluorescent technique that is more sensitive than radioligand binding was developed in order to detect opioid receptors expressed on leukocytes. The current study was designed to optimize the method for fluorescently labeling kappa opioid receptors. For these experiments, the opioid antagonist naltrexamine was conjugated to either fluorescein (FITC-NTXamine) or biotin (biotin-NTXamine). One-step, two-step, and three-step protocols were compared to determine which procedure resulted in optimal staining of the kappa opioid receptor expressed on intact, unfixed R1E/TL8x.1.OUAr.1(R1EGO) cells, a thymoma known to express kappa opioid receptors. The one-step method involved incubating cells with FITC-NTXamine, and the fluorescein intensity was measured by flow cytometry. In the two-step method, cells were incubated with biotin-NTXamine, followed by extravidin-conjugated phycoerythrin, and the phycoerythrin fluorescence was measured. Finally, in the three-step protocol, cells were incubated with FITC-NTXamine, followed by biotin-conjugated anti-fluorescein IgG, then extravidin-phycoerythrin. The one-step protocol stained the cells, but the signal was not diminished in the presence of opioid competitors. The two-step approach did not stain cells significantly above background levels. Only the three-step approach yielded staining that was displaced by the kappa-selective antagonist nor-binaltorphimine. Thus, the addition of a secondary biotinylated antibody, resulting in the amplification of binding, which was detected using phycoerythrin as a fluorophore, was required to detect low levels of opioid receptor expression on leukocytes.

Kappa Opioid Receptors on Immune Cells as Studied by Fluorescent Ligands

Despite both functional (1–4) and molecular biological (5–8) evidence for the presence of opioid receptors on lymphocytes, the search for opioid binding sites on leukocytes has been difficult. Opioid binding sites possessing all the characteristics of the classical brain opioid receptors had not been detected until the identification of the κ opioid receptor on the mouse Rl.l thymoma cell line (9,10). Possible reasons why opioid receptors have been difficult to detect on mixed cell populations from the immune system include: 1) opioid receptors may be expressed on only a small population of lymphocytes; 2) the expression of opioid receptors on lymphocyte changes during lymphocyte maturation; and 3) radioreceptor binding methodology is not sufficiently sensitive to detect opioid receptors present at a low density. While the R1.1 cell line was useful in establishing that a lymphocyte could express an opioid receptor, interest still lies in determining if lymphocytes from a mixed cell population, such as thymus and spleen, express opioid receptors. To address this question, we developed an indirect immunofluorescent method that is more sensitive than radioreceptor binding assays (11,12).

Kappa Opioid Receptors on Three Related Thymoma Cell Lines

The search for opioid binding sites on leukocytes has been difficult. The presence of opioid binding sites has been reported on human and murine leukocytes (1–3) as well as on some cell lines (4,5). However, in contrast to brain opioid receptors, these sites do not exhibit all of the classical opioid binding characteristics, such as stereoselectivity and high affinity for both alkaloids and peptides (6). In addition, agonist-induced functional responses have not been correlated with these binding sites. It seems likely that only a small population of leukocytes actually express opioid receptors, and possibly only under certain conditions. If this is the case, binding studies with mixed cell populations may not show a detectable signal-to-noise ratio, even when receptors are present.

Identification of Opioid Receptors in the Immune System Using a Novel Combination of Selective Opioid Ligands and Indirect Phycoerythrin Immunofluorescence

Although there is substantial evidence for direct, in vitro opioid effects on the immune system, radioligand binding studies have not definitively described brain opioid receptors on these cells (1). It is possible that the density of receptors required for immunomodulation is too low to detect with radioligands, or that only a small percentage of cells may express opioid receptors in heterogeneous leukocyte populations. In addition, the level of receptor expression in leukocytes may depend on the developmental stage or the activation state of the cells. The use of fluorescent opioid ligands would allow visualization of opioid receptors by microscopy, analysis of cell subpopulations for opioid receptor expression, or separation of cells expressing opioid receptors by flow cytometry. Several groups have synthesized fluorescent opioid probes, using fluorophores such as 1-dimethylaminonaphthalene-5-sulfonic acid (dansyl) (2, 3), rhodamine (4, 5), fluorescein (5), pyrene (6), and nitrobenzodiazole (7). Although these compounds showed high affinity and biological activity at opioid receptors, the peak emission wavelengths of some may overlap with autofluorescence (3). In addition, irradiation at wavelengths near the excitation peak for dansyl compounds can greatly reduce opioid receptor affinity by irreversibly altering the structures of opioid receptors and ligands (8). The longer excitation and emission wavelengths of the rhodamine-and fluorescein-labeled opioid probes (4, 5) avoid the problems of irradiation-induced receptor alteration and tissue autofluo-rescence, but specific histological labeling of opioid receptors with these fluorescent ligands has not been demonstrated.

Chronic opioid treatment of R1.1 cells results in the downregulation of the kappa opioid receptor without desensitization of adenylyl cyclase activity

The mouse thymoma R1.1 cell line expresses a κ opioid receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive G i protein. This study addressed whether chronic exposure of R1.1 cells to the κ agonist U50,488 would alter the receptor binding properties or opioid-induced inhibition of adenylyl cyclase activity. Culturing R1.1 cells in the presence of U50,488 for 3 hr and longer produced a time- and concentration-dependent decrease in the binding of the κ-selective ligand [ 3 U]69,593 to membranes prepared from these cells. Unlike the decrease in receptor binding, opioid imbibition of adenylyl cyclase activity was not altered by culturing cells in the presence of U50,488