0013-7227/90/1266-3006$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 6 Printed in U.S.A.
/?-Endorphin Binding to Naloxone-Insensitive Sites on a Human Mononuclear Cell Line (U937): Effects of Cations and Guanosine Triphosphate* NAHID A. SHAHABI, PHILLIP K. PETERSON, AND BURT SHARP Endocrine-Neuroscience Research Laboratory, Minneapolis Medical Research Foundation (N.A.S., P.K.P., B.SJ, and the Departments of Medicine, Hennepin County Medical Center and University of Minnesota (P.K.P., B.S.), Minneapolis, Minnesota 55415 phin. Increasing concentrations of mono (Na+, K+)- and divalent (Ca2+, Mg*+, Mn2+) cations reduced the binding of /S-[125I]endorphin to U937 membrane; /3-[125I]endorphin binding to rat brain membrane showed similar cation sensitivity. GTP7-sulfate (GTP7S; 10"4 M) alone reduced binding to U937 membrane by 25%. In the presence of Na+ (100 or 150 mM) or Mg2"1" (10 mM), GTP7S reduced binding by an additional 50%. Moreover, GTP7S (10"8-10~4 M) in the presence of Na+ (100 mM) reduced binding in a dose-dependent manner, whereas GMP was ineffective. In conclusion, 0-endorphin binds to sites on human U937 cells similar to those observed on normal murine splenocytes. Although naloxone insensitive, these sites exhibit properties, such as size, salt sensitivity, and coupling to a GTPbinding protein, that are similar to those observed for agonist binding to brain opiate receptors. {Endocrinology 126: 30063015,1990)
ABSTRACT. Some of the functional effects of /3-endorphin on immune cells are resistant to inhibition by naloxone. To further characterize the /3-[125I]endorphin-binding site mediating these effects and its response to cations and GTP, the human monocyte-like cell line U937 was used. Incubation of intact cells and 0-[125I]endorphin for 60 min at 4 C demonstrated a saturable, high affinity binding site [Kd = 1.2 ± 0.5 x 10~8 M (mean ± SE; n = 4] competed by equimolar /3-endorphin and iV-acetyl (Ac)/3-endorphin but not by naloxone, morphine, or selective opiate receptor agonists. Competition studies showed that /3-endorphin(6-31) and /3-endorphin-(28-31) were approximately 5- and 100fold less potent, respectively, whereas /3-endorphin-(l-16) or (1-27) was ineffective. Covalent cross-linking of |3-[125I]endorphin to intact cells and resolution by gel electrophoresis showed dominant bands at 59K and 44K and a minor band at 66K. The bands at 44K and 66K were completely displaced by increasing equivalent concentrations of /3-endorphin and iV-Ac-/3-endor-
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HE OPIATE alkyloids and /3-endorphin affect a variety of leukocyte functions that are involved in host defense and immunity. Modulation of monocyte (1) and polymorphonuclear leukocyte superoxide generation (2), inhibition of lymphocyte 7-interferon secretion (3), enhancement of natural killer cell activity (4, 5), and opioid-induced inhibition of the antibody response to sheep erythrocytes (6) are all sensitive to reversal by the opiate receptor antagonist naloxone. These effects are probably mediated by opiate receptors such as the ^-like (7) and 5-opiate receptors (8), which have been partially characterized on murine splenocytes and human peripheral mononuclear cells. The yu-like site was sensitive to displacement by naloxone or morphine and completely resistant to /3-endorphin and leucine- or methionineenkephalin. Sodium ions inhibited the binding of [3H]
Received December 29, 1989. Address all correspondence and requests for reprints to: Dr. Burt M. Sharp, Division of Endocrinology, Metabolism, and Nutrition, Department of Medicine, Hennepin County Medical Center, 701 Park Avenue South, Minneapolis, Minnesota 55415. * This work was supported by Grant DA-04196.
naloxone, as did GTP. These effects of sodium and GTP have been well described for neural opiate receptors where these agents differentially affect the binding of agonists compared to antagonists (9, 10). The recently described 5-site on immune cells was observed after covalent cross-linkage to a 5-selective radioligand (8). A 70K band, present under reducing conditions, was completely displaced by 5-ligands, partially by naloxone, and not at all by a ^-selective ligand. In contrast to opiate receptor-mediated responses, the effects of /3-endorphin on mononuclear cell proliferative responses (11-13), interleukin-2 production (14), and calcium uptake (15) are resistant to inhibition by naloxone. The biochemical characterization of the immune cell receptor(s) involved in these naloxone-insensitive effects of /3-endorphin is incomplete. Radioligand binding studies have demonstrated the presence of naloxoneresistant binding sites for /3-endorphin on murine EL4thymoma cells and transformed human mononuclear cells (16, 17). Limited characterization of this naloxoneresistant site(s) on a thymoma cell line has demonstrated a 76K band after chemical cross-linking (18), and Scat-
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EFFECTS OF CATIONS AND GTP ON 0-ENDORPHIN BINDING chard analysis showed an affinity of 65 nM (Kd). Normal murine splenocytes express a high affinity naloxoneinsensitive /3-endorphin binding site(s) (Kd = 4.1 X 10~9 M) in culture, but not on fresh whole splenic membrane or purified splenic cells taken from viral antibody-free mice (19). JV-Ac-/3-Endorphin is equipotent to /3-endorphin at this site, and the same bands, resolved on gel electrophoresis (57K and 66K), are displaced by both ligands. To further characterize this naloxone-insensitive binding site for /3-endorphin with respect to the affects of salt and guanine nucleotides, a human monocyte-like cell line (U937), was used. A descriptive biochemical profile of this site was first established from saturation, competition, and chromatographic studies to permit comparison to previous reports (16-19). Then, the effects of various cations on the binding of/3-[125I] endorphin to U937 cells were analyzed and compared to those on a rat brain preparation under the same binding conditions. Since both the classical brain opiate receptor and //-like opiate receptors on murine splenocytes appear to be coupled to GTP-binding proteins, the effect of a GTP analog, alone and in combination with cations, on /3-[125I] endorphin binding to U937 cells was determined. Our studies show that there is a naloxone-insensitive binding site for /?endorphin on U937 cells that has properties similar to those of binding sites on normal murine splenocytes (19). Moreover, this site is sensitive to cations and GTP in a way which is similar to the naloxone-responsive opiate receptors in brain and on immune cells. Materials and Methods Cell culture and preparation for radioligand binding assay The U937 cell line was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured continuously in suspension in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin, 100 tig/ml streptomycin, and 2 DIM glutamine. Cell cultures, grown as 1-1.5 X 106 cells/ml, were fed three times per week and the evening before each experiment with 20-25 ml medium. They were maintained in a humidified atmosphere of 5% CO2 in air at 37 C. Intact cells were washed three times with RPMI-1640, once with assay buffer [50 mM K2HPO4 in the presence of the protease inhibitors 50 Mg/ml bacitracin, 10 jug/ml leupeptin, 10 /xg/ml soybean trypsin inhibitor, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1 mM benzamidine, pH 7.4] and then resuspended in this buffer for use in the binding assay. Cells or cell membranes were prepared in Tris-HCl buffer (50 mM), pH 7.4, containing protease inhibitors to determine the effect of cations on radioligand binding. To study the effect of GTP in the presence or absence of cations on radioligand binding, cells were first homogenized in Tris-HCl using a Brinkmann Polytron (Westbury, NY) at a setting of 6 for 20 sec. After centrif-
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ugation at 38,000 x g for 20 min, the pellet was washed once and then resuspended in the same buffer. Membrane preparation for rat brain opiate receptor Male Holtzman rats, 2-300 g (Holtzman Co., Madison, WI), were housed at a constant temperature of 23 C with a 12-h light, 12-h dark cycle. Food and water were available ad libitum. Membrane from whole rat brain was prepared as described previously (20). Briefly, homogenization of brain was carried out in 20 vol Tris-HCl (50 mM) containing dipotassium EDTA (1 mM) at pH 7.4; a Brinkmann Polytron at a setting of 6 was used for 20 sec. After filtration through nylon gauze, the pellet obtained after centrifugation at 38,000 X g for 20 min was resuspended in a total of 6 vol sucrose (0.32 M) with dipotassium EDTA (1.0 mM) and then frozen at -80 C. The preparation was thawed once, just before the RRA. Radioligand binding assay The binding of/?-[125I]endorphin (2000 Ci/mmol; Amersham Corp., Arlington Heights, IL), monoiodinated at tyrosine-27, was determined by filtration assay as previously described (21). Briefly, 50-^1 aliquots of either rat brain membrane (100-200 Hg protein/aliquot) or a suspension of intact cells (0.4-1.0 X 107 cells) or their membrane equivalent was incubated in triplicate with £-[125I] endorphin in a total volume of 100 ix\K2HPO4 or Tris-HCl (for cation or membrane studies; 50 mM) buffer, pH 7.4, containing bacitracin (50 ^g/ml), leupeptin (10 ^g/ml), soybean trypsin inhibitor (10 pg/m\), PMSF (1 mM), and benzamidine (1 mM). Nonspecific binding was determined in the presence of 3 nM (final concentration) unlabeled /3-endorphin. After incubation for 60 min at 4 C, the receptor-bound radioactivity was isolated by filtration through glass fibers presoaked for at least 30 min at room temperature in a solution of K2HPO4 (50 mM), pH 7.4, containing 0.01% poly-L-lysine HBr (mol wt, 240,000) and 0.4% BSA. Thereafter, filters were washed three times with 3 ml cold K2HPO4 (50 mM), pH 7.4, buffer containing 0.01% Triton X-100. The filters were then counted in a Beckman y-counter with 75% efficiency. The competition of /3-endorphin binding by fragments of /?-endorphin (Peninsula Laboratories, Belmont, CA) and naloxone hydrochloride (DuPont Corp., Wilmington, DE) used /?-[125I] endorphin (0.05 nM). Scatchard analysis of the data was performed using the nonlinear least squares regression analysis of Ligand by McPherson (22). All studies were repeated at least three times except the dose response to GTP7 sulfate (GTPyS; Boehringer Mannheim, Indianapolis, IN: n = 1). Protein was measured using Bio-Rad reagents (23). Analysis of N-acetyl (Ac)-(i-endorphin by reverse phase HPLC To determine whether iV-Ac-/3-endorphin remained intact or was converted to 0-endorphin during the radioligand binding assay, HPLC (Gilson Corp., Middleton, WI) using a reverse phase C8 Ultrasphere column (Beckman Instruments, Inc., Palo Alto, CA) was performed. A gradient elution, starting with 0.1% trifluoroacetic acid and 3.0% acetonitrile (EM Science, Gibbstown, NJ) and reaching 60.0% acetonitrile over 40 min, was used to separate /?-endorphin (eluting at 21.5 min) from
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EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
JV-Ac-/3-endorphin (eluting at 22.5) which were detected by UV spectroscopy at 220 nm. In the absence of (3-[125I]endorphin, AT-Ac-/3-endorphin (5-10 jug) was added to the standard radioligand binding assay for 60 min at 4 C. After centrifugation to separate the cells, the supernatant was filtered (Centrex nylon, 0.2 /um, Schleicher and Schuell, Keene, NH), and approximately 100-150 ^1 were injected for analysis. Using this design, /?-endorphin alone and /3-endorphin plus N-Ac-f3-endorphin were also analyzed after incubation.
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Affinity labeling and electrophoretic analysis U937 cell membrane was suspended in the K2HPO4 (50 mM) buffer used for radioligand binding with the addition of PMSF (1 mM) benzamidine (1 mM), and EDTA. Membrane (1-1.5 mg protein/ml) was incubated with |8-[125I]endorphin in the presence or absence of unlabeled ligand for 60 min at 4 C. Unbound ligand was removed by centrifugation at 20,000 x g (4 C) for 20 min after the addition of 5 vol ice-cold binding buffer. After resuspension, cross-linking was initiated with the addition of JV-hydroxysulfosuccinimide and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to yield final concentrations of 5 mM and 0.1 M, respectively. The reaction was terminated by the addition of ice-cold assay buffer, followed by centrifugation. Pellets were solubilized in Laemmli sample buffer containing sodium dodecyl sulfate (SDS) and /3-mercaptoethanol, and boiled for 5 min (24). Samples were electrophoresed by the method of Laemmli using 1.5-mm SDS-polyacrylamide (10%) slab gels (SDS-PAGE). An aliquot of each sample (40-100 Mg protein) was electrophoresed at a current of 30 mamp/slab for 3-4 h. To determine the position of marker proteins (Bio-Rad), gels were stained with 0.05% Coomassie blue in 10% acetic acid plus 50% methanol and destained in 10% acetic acid plus 10% methanol. After drying, autoradiography was performed using Kodak X-Omat AR film (Rochester, NY) with intensifying screens at -70 C. All studies were performed at least two times.
Results Intact U937 cells were used to analyze the time course of /3-[125I]endorphin binding at 4, 25, or 37 C (Fig. 1). Beginning with the first data point that was obtained after 1 min of incubation, the maximal fraction of total radioligand was bound by 5 min at 37 C, 10 min at 25 C, and 30 min at 4 C. Thereafter, binding declined most rapidly at 37 C, and a nadir was achieved by approximately 120 min at 25 and 37 C. In contrast, maximal binding was stable at 4 C for greater than 200 min. Thus, studies were performed at 4 C for 60 min. Figure 2 shows a characteristic saturation study in which U937 cells were incubated with a range of /3-[125I] endorphin concentrations from 8 pM to 35 nM. Nonspecific binding (~20-30% of total binding) was linear throughout the dosage range. Scatchard analysis using Ligand showed one site with a Kd (mean ± SE of four studies) of 1.2 ± 0.5 x 10"8 M and a binding capacity (Bmax) of 4.0 ± 0.7 X 10"14 mol /3-[125I]endorphin/l x 106 cells or 26,000 receptors/cell.
120 TIME
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FIG. 1. The effects of time and temperature on /?-[126I]endorphin binding to intact U937 cells. Cells were washed and then pellets were resuspended in the radioligand binding buffer at 1.7 x 107 cells/ml, and /3-[125I]endorphin (0.1 nM) was added. Total and nonspecific binding, in triplicate, were determined for each data point. The total bound fraction (percentage) of the total count added is shown on the y-axis. For each point, the coefficient of variation was less than 15% of the mean, and specific binding was 70-80% of the total bound.
Competition studies using opiate receptor ligands and analogs of /?-endorphin-(l-31) are shown in Figs. 3 and 4. iV-Ac-j8-endorphin-(l-31), which is ineffective at binding to classical brain opiate receptors (25), was equipotent to /3-endorphin-(l-31) in the nanomolar concentration range. /3-Endorphin-(l-31) or ethylketocyclazocine displaced 7-20% of the radioligand at extremely low concentrations (femtomolar). However, a binding site with a Kd in this concentration range was not consistently demonstrable. In at least three separate experiments, morphine, naloxone, and [D-Pen2, Pen5]-enkephalin (26) or U69,593 (27) (the latter two are selective for 8- and /c-opiate receptors, respectively) failed to displace any |8-[125I] endorphin, even at concentrations as great as 10~4 M. Figure 4 shows that the C-terminal tetrapeptide i8-endorphin-(28-31) was approximately 100-fold less potent than intact /?-endorphin, and C-terminal elongation to 0-endorphin-(18-31) or 0-endorphin-(6-31) resulted in equipotent peptides that were approximately 5-fold less potent than /3-endorphin-(l-31). Addition of the natural N-terminal pentapeptide sequence to yield /?endorphin-(1-5, 16-31) restored full potency. Thus, the C-terminal of /3-endorphin-(l-31) is absolutely required for binding to this site. This was confirmed by the
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EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
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SCATCHARD
BINDING 3.42E-02
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FIG. 2. Binding isotherm and Scatchard plot of j8-[125I]endorphin binding to U937 cells. Cells were incubated for 60 min at 4 C with increasing concentrations of (3[125I]endorphin (8.0 x 10"12-8.8 x 10~8 M). The binding isotherm (left panel) shows a single site, which is confirmed by the nonlinear curve fitting analysis using the Ligand program (22), which generated a single line to fit the Scatchard data points (right panel). Kd = 9.7 X 10' 9 M; Bmax = 18,000 receptors/cell. B/F, Bound to free ratio.
/ /
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FIG. 3. Displacement of /3-[125I]endorphin binding to U937 cells by /?endorphin-(l-31), iV-Ac-/3-endorphin-(l-31), and ethylketocyclazocine (EKC). ]8-[125I]Endorphin (0.05 nM) was incubated with increasing concentrations (10~2-10~8 M) of unlabeled ligand at 4 C for 60 min.
complete failure of j8-endorphin-(l-27) or /3-endorphin(1-16) to displace the radioligand. These studies indicate that C-terminal elongation to yield /3-endorphin-(18-31) enhances potency, whereas further lengthening to yield /3-endorphin-(6-31) is unnecessary; finally, the addition of /3-endorphin-(l-5) is necessary for full potency. The integrity of iV-Ac-/3-endorphin was determined by HPLC after incubating the peptide with intact cells at 4 C for 60 min (data not shown). After incubation of NAc-/3-endorphin (5-10 i*g), analysis of a sample of supernatant showed that approximately 80-90% of the added peptide eluted at 22.5 min and none at 21.5 min (the position of /3-endorphin). The remainder of the peptide (~10-20%) appeared to be nonspecifically bound to cells, tubes, and filters, since the peak at 22.5 min was not accompanied by other novel peaks. Similarly, incubation
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FIG. 4. Displacement of j8-[ I]endorphin binding to U937 cells by fragments of/3-endorphin-(l-31). Cells were incubated for 60 min at 4 C with /3-[125I]endorphin (0.05 nM) and increasing concentrations (10~12-10"5 M) of the unlabeled ligands.
of /3-endorphin (5-10 ng) with cells showed that approximately 80-90% of the added peptide eluted at 21.5 min. Coincubation of equivalent amounts of iV-Ac-|8-endorphin (5-10 jug) and /3-endorphin (5-10 /*g) showed equivalent peaks at 21.5 and 22.5 min. Thus, iV-Ac-/?-endorphin is not degraded to /3-endorphin during the RRA. Covalent cross-linking with carbodiimide was performed after /3-[125I]endorphin was reversibly bound to U937 membrane. The reaction product was resolved by SDS-PAGE. Figure 5 shows the effect of increasing concentrations of unlabeled /?-endorphin-(l-31) (3 x 10"12-2.5 X 10"5 M) on the bands at 66K, 59K, and 44K. The 66K and 44K bands were completely displaced by increasing concentrations, whereas the 59K band was
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EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
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31 -H 22 14 FIG. 5. The effects of increasing concentrations of ]8-endorphin-(l-31) on the binding and chemical cross-linking of 0-[125I]endorphin to U937 cells. Membrane was prepared for binding assays performed at 4 C for 60 min using /3-[125I]endorphin (1.0 nM) and increasing concentrations of unlabeled j8-endorphin-(l-3l) (lane 6, 3 x 10"12 M; lane 5, 3 x 10"10 M; lane 4, 3 x 10~8 M; lane 3, 2.5 x 10"5 M). After the binding assay, chemical cross-linking and analysis using SDS-PAGE were performed, as described in Materials and Methods. Two samples were analyzed for total binding (lane 1, protein = protein of sample in lane 3; lane 2, total counts per min = total counts per min of sample in lane 3).
FIG. 6. The effects of increasing concentrations of iV-Ac-/3-endorphin(1-31) on the binding and chemical cross-linking of j8-[125I]endorphin to U937 cell membranes. The procedure was analogous to that described in Fig. 5. For competition, increasing concentrations of iV-Ac-/3-endorphin were as follows: lane 7, 3 x 10"12 M; lane 6, 3 x 10"10 M; lane 5, 3 x 10"8 M; lane 4, 3 X 10"6 M; lane 3, 2.5 X 10~6 M. Two samples were analyzed for total binding (lane 1, total counts per min = total counts per min of sample in lane 3; lane 2, protein = protein of sample in lane 3).
minimally displaced by maximum amounts of /3-endorphin-(l-31) (2.5 X 10~5 M in lane 3, compared to lane 2, which was loaded with an equivalent number of counts per min, or lane 1 which contained the protein equivalent of lane 3). Figure 6 shows a parallel experiment conducted using AT-Ac-/3-endorphin-(l-31) as the unlabeled ligand (2.5 X 10~5 M in lane 3, compared to lane 1, which was loaded with an equivalent number of counts per min, or lane 2, which contained the protein equivalent of lane 3); the results were similar. The effects of various fragments of /3-endorphin (all used at 2.5 X 10~5 M) on the appearance of the 66K, 59K, and 44K bands on SDS-PAGE were studied (Fig. 7). As expected from the aforementioned competition studies, j3-endorphin-(l-16) (lane 7) was without effect. /?-Endorphin-(28-31) (lane 6) slightly reduced the 66K and 44K bands, and /3-endorphin-(16-31) (lane 5) was more effective. 0-Endorphin-(l-5, 16-31) (lane 4) and 0-endorphin-(l-31) (lane 3) were equally effective, both completely displaced the 66K and 44K bands and partially displaced the 59K band. The coincubation of an opiate agonist and monovalent cations has been shown to reduce agonist binding to
brain homogenates (9, 28, 29). Thus, studies were performed to determine the influence of cations on the binding of /3-[125I]endorphin to intact U937 cells us. rat brain homogenates. Figure 8 shows that 100 mM Na+ or K+ reduced binding to U937 cells by approximately 35%, and inhibition to less than 95% of control occurred at a concentration of 500 mM. We also observed that 100 mM Li+ failed to affect binding, and 500 mM reduced binding by only 20% (data not shown). Figure 9 indicates that binding of /?-[125I]endorphin to rat brain membrane was more sensitive to inhibition by Na+, since concentrations of 50, 100, and 500 mM reduced binding by 23%, 63%, and 88%, respectively. U937 cells were more sensitive to inhibition by divalent than monovalent cations (Fig. 8). At 5 mM, Ca2+ or Mg2+ reduced binding by 54% and 40%, respectively; the degree of inhibition was similar at 10 mM. Mn2+ (1 or 10 mM) also inhibited binding by 35% and 70%, respectively. In contrast to the enhanced sensitivity of rat brain membrane to monovalent cations, Fig. 9 indicates that the divalent cation Mn2+ at 1 or 10 mM, which reduced binding by 30% and 75%, respectively, was equally effective on rat brain and U937 cells. To determine whether the naloxone-resistant binding
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EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
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ing studies. Na+ (100 mM) significantly reduced the Bmax from 393.7 ± 21.4 to 203.7 ± 7.9 fmol/mg protein (n = 3; P < 0.01), and Na+ (100 mM) plus GTP7S (10~4 M) further reduced the Bmax to 121.9 ± 6.2 fmol/mg protein (n = 3; P < 0.01). Kd values were not altered significantly by Na+ or Na+ plus GTP7S (control, Na+, and Na+ plus GTP7S: 2.0 ± 0.5 X 10~8, 1.0 ± 0.1 X 10"8, and 7.0 ± 1.4 X 10"9 M, respectively; n = 3).
Discussion
FIG. 7. The effect of fragments of /3-endorphin-(l-31) on the binding and chemical cross-linking of /S-[128I] endorphin to U937 cell membranes. The procedure was analogous to that described in Fig. 5. For competition, the concentration of all fragments was 25 jtM [lane 3, /3endorphin-(l-31); lane 4, (8-endorphin-(l-5, 16-31); lane 5, /3-endorphin-( 18-31); lane 6, /8-endorphin-(28-31); lane 7, /3-endorphin(1-16)]. As previously described (Fig. 6), two samples were analyzed (lanes 1 and 2) for total binding.
site for /?-endorphin on U937 is coupled to a guanine nucleotide-binding protein, the effect of a GTP analog on /3-[125I]endorphin binding to cell membrane was studied. Table 1 shows that GTP7S (10~4 M) reduced the binding of /?-[125I] endorphin by 25%. In the presence of either Na+ or Mg2+, the effect of GTP7S was magnified, and a reduction of 40-45% from the baseline with salt alone was observed. Na+ (100 mM) or Mg2+ (10 mM) alone had similar effects on binding of the radioligand to membrane in these studies (reduction to 63.2 ± 9.65% of control and 39.3 ± 11.9% of control, respectively; n = 3) compared to those observed for binding to intact cells (Fig. 8). Table 1 shows that the combination of Na+ and Mg2+ further enhanced the effect of GTP7S, resulting in a reduction of approximately 60% compared to the combined salts. Figure 10 illustrates the effect of increasing concentrations of GTP7S vs. GMP, both in the presence of Na (100 mM). GTP7S maximally reduced binding by 55% at 10"4 M compared to that in the group containing only salt (no GTP7S), whereas GMP had no effect. Figure 11 shows a representative Scatchard analysis of the affect of Na+ or Na + plus GTP7S on /3-[125I] endorphin binding to U937 cell membrane. Bmax values were reduced in proportion to the aforementioned bind-
Partial characterizations of a naloxone-insensitive binding site for 0-endorphin have been described on transformed immune cells, including human lymphocytes (17) and mouse EL4 thymoma cells (16, 18). To our knowledge, this is the first report that includes an analysis of the effects of fragments of /3-endorphin, NAc-/3-endorphin, cations, and GTP on /3-[125I]endorphin binding at this site. The affinity observed herein (Kd = 1.2 ± 0.5 X 10~8 M) is intermediate between those found for EL4 thymoma (Kd = 6.5 X 10"8) and transformed human lymphocytes (Kd = 3 X 10~9 M). An average of 26,000 sites/cell were present on the U937 line. This is similar to the 19,000 sites/cell on the EL4 thymoma line. Both cell lines continuously expressed the binding sites in cultures containing fetal serum. In contrast, we have reported (19) that a higher affinity (Kd = 4.1 X 10~9 M) site is present on normal murine splenocytes after 24 h in culture, although it is undetectable on fresh splenocytes obtained from viral antibody-free mice. The structural requirements for binding to this site on U937 cells is analogous to that reported for normal murine splenocytes (19). Some of the features have also been reported on the transformed cells (16, 17). Binding to U937 cells requires that /3-endorphin contain its Cterminus. Thus, the deletion of peptides /?-endorphin-(l16) and -(1-27) was completely ineffective. In contrast, /?-endorphin-(28-31) itself was able to displace /3-[125I] endorphin in a dose-dependent fashion, but with l/100th the potency of intact /3-endorphin. Elongation of the Cterminal tetrapeptide to /3-endorphin-(18-31) enhanced the potency 10-fold, whereas lengthening to /?-endorphin-(6-31) failed to further enhance the potency. Addition of the natural N-terminal peptide sequence (methionine-enkephalin) to /?-endorphin-(16-31) resulted in an analog equipotent to /3-endorphin-(l-31). These findings define two domains of ^-endorphin that are required for binding to this naloxone-insensitive binding site. The C-terminal sequence is absolutely required, and both elongation of this (to less than or equal to amino acids 18-31) and addition of the N-terminal pentapeptide are necessary for the full potency of /3-endorphin-(l-31). These findings are in agreement with functional studies showing that /3-endorphin-(l-27) was ineffective in en-
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EFFECTS OF CATIONS AND GTP ON 0-ENDORPHIN BINDING
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U937 CELLS: EFFECT OF CATIONS ON BINDING
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FIG. 8. The effects of cations on the binding of /3-[125I]endorphin to U937 cells. Increasing concentrations of monovalent (left panel; 0-500 mM) or divalent cations (right panel; 0-100 mM) were added to Tris-HCl buffer along with the radioligand (1.0 nM). The control group (0 mM cation) contained Tris-HCl buffer (50 mM) alone. Values were the mean ± SE (n = 3).
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Ca
0
Mg
6-
@ Mn
4-
i = 2-
2-
Iu 10
50
100
500
0.5
CATION [mM]
1
10
100
CATION [mM]
RAT BRAIN: EFFECT OF CATIONS ON BINDING
FIG. 9. The effects of cations on the binding of /3-[125I]endorphin to rat brain membrane. Increasing concentrations of sodium (left panel; 0-500 mM) or manganese (right panel; 0-10 mM) were added to Tris-HCl buffer along with the radioligand (1.0 nM). Values are the mean ± SE (n = 3).
T
0 SODIUM (mM] 125
TABLE 1. /3-[ I]Endorphin binding to U937 cell membranes: effects of cations and GTP % of control Cation GTP None Na+ (100 mM) Na+ (150 mM) Mg2* (10 mM) Na+ (100 mM) + Mg*+ (10 mM)
74.6 ± 54.4 ± 61.8 ± 57 ± 42.7 ±
0
4.2 5.8 5.3 12.6 10.1
GMP 98.6 ± 93.3 ± 90.3 ± 108 ± 94.5 ±
4.1 1.0 15.7 10.7 24.8
Values are the mean ± SD (n = 3). 0
G T P T S , (10" 4 M).
hancing the proliferation of murine lymphocytes (14, 30). As reported for the transformed cells (16, 17) and normal murine splenocytes (19), the /3-endorphin-binding site on U-937 cells is unaffected by either nonselec-
100
500
0
0.05
0.1
0.5
1
10
MANGANESE [mM]
tive or selective opiate receptor ligands. Thus, naloxone, morphine, the /^-selective ligand [D-Ala2,Nme,Phe4,Glyol5]-enkephalin, the natural enkephalin pentapeptides, the 6-selective ligand [D-Pen2,Pen5]-enkephalin, and the /c-selective ligand U69,593, all have failed to displace /3[125I]endorphin from this site. In contrast to this lack of efficacy of opiate ligands, JV-Ac-/3-endorphin, which is less than 1000-fold as potent as j8-endorphin in radioreceptor studies of the brain opiate receptor (25), was found to be equipotent to /3-endorphin at binding to the naloxone-resistant site on the monocyte-like U937 cell line. To validate this finding, we demonstrated that N-Ac-(3endorphin was not converted to /3-endorphin in the RRA. The same observation about the potency of JV-Ac-|8endorphin was reported for the naloxone-resistant binding site on normal murine splenocytes (19). Interestingly,
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3013
EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
Effect of Guanine Nucleotides
u.uou -
8000 n
+
GTP +Na
•
Tris-HCI
o
NaiOOmM
2.0
2.5
0.025 7000-
2 X
•
0.020 •
6000-
u.
5000-
m 0.015-
VV°
\\ "
i
4000-
0.0103000 -6
-4
loglguanlne nucleotide] (M)
FIG. 10. The effects of guanine nucleotides on the binding of /3-[126I] endorphin to U937 cell membranes. In the presence of NaCl (100 mM), increasing concentrations (10~8-10~4 M) of GMP or GTP7S were added to cell membranes along with the radioligand (1.0 nM). Each data point is the mean from quadruplicate determinations of specific binding in a single experiment. Each aliquot contained the membrane equivalent of 2 X 10e cells.
iV-Ac-jS-endorphin appears to be synthesized by normal murine splenocytes (31) and uncharacterized when /?endorphin immunoreactivity is present constitutively in secretions from normal splenic monocytes maintained in culture (32). Thus, the naloxone-insensitive binding site may be the target for iV-Ac-/3-endorphin, acting as either an agonist or antagonist. At present, two functional studies have tested iV-Ac-0-endorphin and found that it enhanced monocyte chemotaxis and lymphocyte proliferation in a naloxone-insensitive manner (30, 33). However, another study showed that /?-endorphin enhanced mitogen-stimulated calcium uptake in rat thymocytes, but failed to demonstrate a similar affect of N-Ac-fiendorphin; its potential role as an antagonist was not evaluated (15). The site of the binding sites(s) and the relative potency of /?-endorphin-(l-31), its fragment analogs, and iV-Ac/3-endorphin at displacing /?-[125I]endorphin were studied using covalent cross-linking followed by SDS-PAGE. We consistently observed three bands: 66K, 59K, and 44K. The 66K and 44K bands were equipotently and completely displaced by /3-endorphin-(l-31), JV-Ac-0-endor-
0.005 -
0.000 0.0
V \ ° \ 0.5
1.0
1.5
3.0
Bound ( H 1 0 - 1 0 M) FIG. 11. Scatchard analysis of the effects of sodium and sodium plus GTP7S on the binding of /3-[126I]endorphin to U937 cells. Cell membranes were incubated with varying concentrations of radioligand in the presence of Tris-HCI (50 mM) alone or with the addition of sodium chloride (100 mM) or sodium chloride (100 mM) plus GTP7S (lO"4 M). The binding isotherms (not shown) were subjected to Scatchard analysis using the nonlinear curve fitting routine of Ligand (22), which generated a single best fit line for each data set. Each aliquot contained 0.7 mg protein/ml. B/F, Bound to free ratio.
phin-(l-31), and ]8-endorphin-(l-5,16-31). In agreement with the rank order of potency observed in the radioreceptor competition studies, /3-endorphin-(28-31) was minimally effective at competing the 66K and 44K bands, and j8-endorphin-(l-16) had no effect. In contrast, 0endorphin-(16-31) was more effective than /?-endorphin(28-31). The 59K band showed similar structural requirements for displacement, but was only partially reduced. Schweigerer et al. (16) reported a single 76K band on murine EL4 thymoma cells, and we (19) reported bands at 66K and 57K on normal murine splenocytes.
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3014
EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
The difference between the findings on U937 cells and EL4 thymoma cells may reflect species differences and/ or a different state of the receptor which is associated with a lower affinity (Kd = 6.5 X 10~8 M) and expressed on immature immune cells. Two of the bands present on U937 cells (66K and 59K) are similar in size to those on normal murine splenocytes (66K and 57K), whereas the 44K band is unique to the U937 line. Despite these similarities, it was the 66K and 44K bands from U937 cells and the 57K band from murine splenocytes that were displaced completely. Studies were performed to determine the affect of cations on the binding of /3-[125I] endorphin to U937 cells. Early reports established that sodium and other cations inhibit the binding of opiate agonists to neuronal opiate receptors (9, 28, 29, 34). Although most cations previously tested caused a dose-dependent inhibition of agonist binding, of the monovalent cations opiate receptors were most sensitive to Na+ (9, 28). This inhibition appeared to affect either receptor affinity (28, 35) or binding capacity (9, 29). This difference may reflect the heterogeneity of opiate receptors (36), which leads to differential responses to cations. Indeed, a recent investigation showed that opiate receptors had different responses to Na+ depending on the tissue of origin, which, in turn, specified the subtype of receptor (37). The affinity of ju-receptors on 315c cells was diminished by Na+, whereas it was the Bmax of 5-receptors on NG-108-15 cells that was reduced by Na + . The present investigations show that binding of /3[125I]endorphin to rat brain membrane has a biphasic response to Na+. Low concentrations (5-10 HIM) enhanced binding, whereas higher concentrations (>50 mM) reduced binding. This differs from the monophasic response and the exquisite sensitivity to Na+ that have been shown for the binding of alkyloids, such as dihydromorphine, to rat brain; this binding was inhibited by as little as 1 mM Na+ (9), although inhibition was most pronounced with concentrations greater than 50-100 mM (9, 28). The present investigations indicate that the sensitivity to Na+-induced inhibition of binding was greater for rat brain membrane than U937 cells, which had a monophasic response to Na+. The U937 cells were equisensitive to Na+ and K+, whereas neuronal opiate receptors are known to be more sensitive to sodium. Nevertheless, some selectivity among the monovalent cations was evidenced by the minimal affect of Li+. As previously observed for neuronal opiate receptors (34, 35), binding to U937 cells was also affected by divalent cations. Manganese more potently reduced binding than calcium or magnesium, and all three were more potent than the monovalent cations. Manganese also caused a monophasic reduction of/3-[125I] endorphin binding to the brain preparation. In contrast to these observations,
Endo • 1990 Voll26«No6
most reports indicate that low concentrations ( monovalent) and appears to be coupled to a GTP-binding protein which modulates its binding.
Acknowledgments We thank Michele Burtness for her excellent technical assistance, and Linda Soumphonphakdy and Sue Klick for their secretarial support.
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EFFECTS OF CATIONS AND GTP ON /J-ENDORPHIN BINDING
References 1. Peterson PK, Sharp BM, Gekker G, Brummitt C, Keane WF 1987 Opioid-mediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity. J Immunol 138:3907 2. Sharp BM, Keane WF, Suh HJ, Gekker G, Tsukayama D, Peterson PK 1985 Opioid peptides rapidly stimulate superoxide production by human polymorphonuclear leukocytes and peritoneal macrophages. Endocrinology 117:793 3. Peterson PK, Sharp BM, Gekker G, Brummitt C, Keane WF 1987 Opioid-mediated suppression of interferon production by cultured peripheral blood mononuclear cells. J Clin Invest 80:824 4. Mathews PM, Froelich CJ, Sibbitt WL, Bankhurst AD 1983 Enhancement of natural cytotoxicity by /3-endorphin. J Immunol 130:1658 5. Mandler RN, Biddison WE, Mandler R, Serrate SA 1986 /3-Endorphin augments the cytolytic activity and interferon production of natural killer cells. J Immunol 136:934 6. Johnson HM, Smith EM, Torres BA, Blalock JE 1982 Regulation of the in vitro antibody response by neuroendocrine hormones. Proc Natl Acad Sci USA 79:4171 7. Ovadia H, Nitson P, Abramsky 0 1989 Characterization of opiate binding sites on membranes of rat lymphocytes. J Neuroimmunol 21:93 8. Carr DJJ, Kim C-H, DeCosta B, Jacobson AE, Rice KC, Blalock JE 1988 Evidence for a delta class opioid receptor on cells of the immune system. Cell Immunol 111:44 9. Pert CB, Snyder SH 1974 Opiate receptor binding of agonists and antagonists affected differentially by sodium. Mol Pharmacol 10:868 10. Blume AJ 1978 Interaction of ligands with the opiate receptors of brain membranes: regulation by ions and nucleotides. Proc Natl Acad Sci USA 75:1713 11. Gilman SC, Schwartz JM, Milner RJ, Bloom FE, Feldman JD 1982 /3-Endorphin enhances lymphocyte proliferative responses. Proc Natl Acad Sci USA 79:4226 12. McCain HW, Lamster IB, Bozzone JM, Gribic JT 1982 /3-Endorphin modulates human immune activity via non-opiate receptor mechanisms. Life Sci 31:1619 13. Puppo F, Corsini G, Mangini P, Bottaro L, Barreca T 1985 Influence of /3-endorphin on phytohemagglutinin-induced lymphocyte proliferation and on the expression of mononuclear cell surface antigens in vitro. Immunopharmacology 10:119 14. Gilmore W, Weiner LP 1988 /3-Endorphin enhances interleukin-2 (IL-2) production in murine lymphocytes. J Neuroimmunol 18:125 15. Hemmick LM, Bidlack JM 1987 0-Endorphin modulation of mitogen-stimulated calcium uptake by rat thymocytes. Life Sci 41:1971 16. Schweigerer L, Schmidt W, Teschemacher H, Wilhelm S 1985 0Endorphin: interaction with specific nonopioid binding sites on EL4 thymoma cells. Neuropeptides 6:445 17. Hazum E, Chang KJ, Cuatrecasas P 1979 Specific Nonopiate receptors for /3-endorphin. Science 205:1033 18. Schweigerer L, Schmidt W, Teschemacher H, Gramsch C 1985 /3Endorphin: surface binding and internalization in thymoma cells. Proc Natl Acad Sci USA 82:5751 19. Shahabi NA, Linner KM, Sharp BM 1990 Murine splenocytes express a naloxone-insensitive binding site for /3-endorphin. Endocrinology 126:1442 20. Ruegg UT, Cuenod S, Hiller JM, Gioannini T, Howells RD, Simon EJ 1981 Characterization and partial purification of solubilized active opiate receptors from toad brain. Proc Natl Acad Sci USA 78:4635
3015
21. Howard AD, de La Baume S, Gioannini TL, Miller JM, Simon EJ 1985 Covalent labeling of opioid receptors with radioiodinated human /3-endorphin. J Biol Chem 260:10833 22. McPherson GA 1985 Analysis of radioligand binding experiments: a collection of computer programs for IBM PC. J Pharmacol Methods 14:213 23. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 24. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 25. Akil H, Young E, Watson SJ, Coy DH 1981 Opiate binding properties of naturally occurring N- and C-terminus modified /3-endorphins. Peptides 2:289 26. Akiyama K, Gee KW, Mosberg HI, Hruby VJ 1985 Characterization of [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin binding to delta opiate receptors in the rat brain and neuroblastoma-glioma hybrid cell line (NG 108-15). Proc Natl Acad Sci USA 82:2543 27. Lahti RA, Mickelson MM, McCall JM, Von Voightlander PF 1985 [3H]U69593 A highly selective ligand for the opioid kappa receptor. Eur J Pharmacol 109:281 28. Simon EJ, Hiller JM, Groth J, Edelman I 1975 Further properties of stereospecific opiate binding sites in rat brain: on the nature of the sodium effect. J Pharmacol Exp Ther 192:531 29. Simantov R, Snowman AM, Snyder SH 1976 Temperature and ionic influences on opiate receptor binding. Mol Pharmacol 12:977 30. Gilmore W, Weiner LP 1989 The opioid specificity of beta-endorphin enhancement of murine lymphocyte proliferation. Immunopharmacology 17:19 31. Lolait SJ, Clements JA, Markwick AJ, Cheng C, McNally M, Smith AI, Funder JW 1986 Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of /3-endorphin in spleen macrophages. J Clin Invest 77:1776 32. Kavelaars A, Ballieux RE, Heijnen CJ 1989 The role of IL-1 in the corticotropin-releasing factor and arginine-vasopressin-induced secretion of immunoreactive /3-endorphin by human peripheral blood mononuclear cells. J Immunol 142:2338 33. Sacerdote P, Panerai AE 1989 Analysis of the beta endorphin structure-related activity on human monocyte chemotaxis: importance of the amino and carboxyl-terminal. Peptides 10:565 34. Pasternak GW, Snowman AM, Snyder SH 1975 Selective enhancement of [3H] opiate agonist binding by divalent cations. Mol Pharmacol 11:735 35. Blume AJ 1978 Interaction of ligands with the opiate receptors of brain membranes: regulation by ions and nucleotides. Proc Natl Acad Sci USA 75:1713 36. Goldstein A, James IF 1984 Multiple opioid receptors: criteria for identification and classification. Trends Pharmacol Sci 5:503 37. Puttfarcken P, Werling LL, Brown SR, Cote TE, Cox BM 1986 Sodium regulation of agonist binding at opioid receptors. I. Effects of sodium replacement on binding at M- and 5-type receptors in 7315c and NG108-15 cells and cell membranes. Mol Pharmacol 30:81 38. Blume AJ 1978 Opiate binding to membrane preparations of neuroblastoma X glioma hybrid cells NG108-15: effects of ions and nucleotides. Life Sci 22:1843 39. Chang K-J, Blanchard SG, Cuatrecasas P 1983 Unmasking of magnesium-dependent high-affinity binding sites for [DAla2,DLeu6] enkephalin after pretreatment of brain membranes with guanine nucleotides. Proc Natl Acad Sci USA 80:940 40. Childers SR, Snyder SH 1978 Guanine nucleotides differentiate agonist and antagonist interactions with opiate receptors. Life Sci 23:759
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Vol. 126, No. 6 Printed in U.S.A.
/?-Endorphin Binding to Naloxone-Insensitive Sites on a Human Mononuclear Cell Line (U937): Effects of Cations and Guanosine Triphosphate* NAHID A. SHAHABI, PHILLIP K. PETERSON, AND BURT SHARP Endocrine-Neuroscience Research Laboratory, Minneapolis Medical Research Foundation (N.A.S., P.K.P., B.SJ, and the Departments of Medicine, Hennepin County Medical Center and University of Minnesota (P.K.P., B.S.), Minneapolis, Minnesota 55415 phin. Increasing concentrations of mono (Na+, K+)- and divalent (Ca2+, Mg*+, Mn2+) cations reduced the binding of /S-[125I]endorphin to U937 membrane; /3-[125I]endorphin binding to rat brain membrane showed similar cation sensitivity. GTP7-sulfate (GTP7S; 10"4 M) alone reduced binding to U937 membrane by 25%. In the presence of Na+ (100 or 150 mM) or Mg2"1" (10 mM), GTP7S reduced binding by an additional 50%. Moreover, GTP7S (10"8-10~4 M) in the presence of Na+ (100 mM) reduced binding in a dose-dependent manner, whereas GMP was ineffective. In conclusion, 0-endorphin binds to sites on human U937 cells similar to those observed on normal murine splenocytes. Although naloxone insensitive, these sites exhibit properties, such as size, salt sensitivity, and coupling to a GTPbinding protein, that are similar to those observed for agonist binding to brain opiate receptors. {Endocrinology 126: 30063015,1990)
ABSTRACT. Some of the functional effects of /3-endorphin on immune cells are resistant to inhibition by naloxone. To further characterize the /3-[125I]endorphin-binding site mediating these effects and its response to cations and GTP, the human monocyte-like cell line U937 was used. Incubation of intact cells and 0-[125I]endorphin for 60 min at 4 C demonstrated a saturable, high affinity binding site [Kd = 1.2 ± 0.5 x 10~8 M (mean ± SE; n = 4] competed by equimolar /3-endorphin and iV-acetyl (Ac)/3-endorphin but not by naloxone, morphine, or selective opiate receptor agonists. Competition studies showed that /3-endorphin(6-31) and /3-endorphin-(28-31) were approximately 5- and 100fold less potent, respectively, whereas /3-endorphin-(l-16) or (1-27) was ineffective. Covalent cross-linking of |3-[125I]endorphin to intact cells and resolution by gel electrophoresis showed dominant bands at 59K and 44K and a minor band at 66K. The bands at 44K and 66K were completely displaced by increasing equivalent concentrations of /3-endorphin and iV-Ac-/3-endor-
T
HE OPIATE alkyloids and /3-endorphin affect a variety of leukocyte functions that are involved in host defense and immunity. Modulation of monocyte (1) and polymorphonuclear leukocyte superoxide generation (2), inhibition of lymphocyte 7-interferon secretion (3), enhancement of natural killer cell activity (4, 5), and opioid-induced inhibition of the antibody response to sheep erythrocytes (6) are all sensitive to reversal by the opiate receptor antagonist naloxone. These effects are probably mediated by opiate receptors such as the ^-like (7) and 5-opiate receptors (8), which have been partially characterized on murine splenocytes and human peripheral mononuclear cells. The yu-like site was sensitive to displacement by naloxone or morphine and completely resistant to /3-endorphin and leucine- or methionineenkephalin. Sodium ions inhibited the binding of [3H]
Received December 29, 1989. Address all correspondence and requests for reprints to: Dr. Burt M. Sharp, Division of Endocrinology, Metabolism, and Nutrition, Department of Medicine, Hennepin County Medical Center, 701 Park Avenue South, Minneapolis, Minnesota 55415. * This work was supported by Grant DA-04196.
naloxone, as did GTP. These effects of sodium and GTP have been well described for neural opiate receptors where these agents differentially affect the binding of agonists compared to antagonists (9, 10). The recently described 5-site on immune cells was observed after covalent cross-linkage to a 5-selective radioligand (8). A 70K band, present under reducing conditions, was completely displaced by 5-ligands, partially by naloxone, and not at all by a ^-selective ligand. In contrast to opiate receptor-mediated responses, the effects of /3-endorphin on mononuclear cell proliferative responses (11-13), interleukin-2 production (14), and calcium uptake (15) are resistant to inhibition by naloxone. The biochemical characterization of the immune cell receptor(s) involved in these naloxone-insensitive effects of /3-endorphin is incomplete. Radioligand binding studies have demonstrated the presence of naloxoneresistant binding sites for /3-endorphin on murine EL4thymoma cells and transformed human mononuclear cells (16, 17). Limited characterization of this naloxoneresistant site(s) on a thymoma cell line has demonstrated a 76K band after chemical cross-linking (18), and Scat-
3006
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EFFECTS OF CATIONS AND GTP ON 0-ENDORPHIN BINDING chard analysis showed an affinity of 65 nM (Kd). Normal murine splenocytes express a high affinity naloxoneinsensitive /3-endorphin binding site(s) (Kd = 4.1 X 10~9 M) in culture, but not on fresh whole splenic membrane or purified splenic cells taken from viral antibody-free mice (19). JV-Ac-/3-Endorphin is equipotent to /3-endorphin at this site, and the same bands, resolved on gel electrophoresis (57K and 66K), are displaced by both ligands. To further characterize this naloxone-insensitive binding site for /3-endorphin with respect to the affects of salt and guanine nucleotides, a human monocyte-like cell line (U937), was used. A descriptive biochemical profile of this site was first established from saturation, competition, and chromatographic studies to permit comparison to previous reports (16-19). Then, the effects of various cations on the binding of/3-[125I] endorphin to U937 cells were analyzed and compared to those on a rat brain preparation under the same binding conditions. Since both the classical brain opiate receptor and //-like opiate receptors on murine splenocytes appear to be coupled to GTP-binding proteins, the effect of a GTP analog, alone and in combination with cations, on /3-[125I] endorphin binding to U937 cells was determined. Our studies show that there is a naloxone-insensitive binding site for /?endorphin on U937 cells that has properties similar to those of binding sites on normal murine splenocytes (19). Moreover, this site is sensitive to cations and GTP in a way which is similar to the naloxone-responsive opiate receptors in brain and on immune cells. Materials and Methods Cell culture and preparation for radioligand binding assay The U937 cell line was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured continuously in suspension in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin, 100 tig/ml streptomycin, and 2 DIM glutamine. Cell cultures, grown as 1-1.5 X 106 cells/ml, were fed three times per week and the evening before each experiment with 20-25 ml medium. They were maintained in a humidified atmosphere of 5% CO2 in air at 37 C. Intact cells were washed three times with RPMI-1640, once with assay buffer [50 mM K2HPO4 in the presence of the protease inhibitors 50 Mg/ml bacitracin, 10 jug/ml leupeptin, 10 /xg/ml soybean trypsin inhibitor, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1 mM benzamidine, pH 7.4] and then resuspended in this buffer for use in the binding assay. Cells or cell membranes were prepared in Tris-HCl buffer (50 mM), pH 7.4, containing protease inhibitors to determine the effect of cations on radioligand binding. To study the effect of GTP in the presence or absence of cations on radioligand binding, cells were first homogenized in Tris-HCl using a Brinkmann Polytron (Westbury, NY) at a setting of 6 for 20 sec. After centrif-
3007
ugation at 38,000 x g for 20 min, the pellet was washed once and then resuspended in the same buffer. Membrane preparation for rat brain opiate receptor Male Holtzman rats, 2-300 g (Holtzman Co., Madison, WI), were housed at a constant temperature of 23 C with a 12-h light, 12-h dark cycle. Food and water were available ad libitum. Membrane from whole rat brain was prepared as described previously (20). Briefly, homogenization of brain was carried out in 20 vol Tris-HCl (50 mM) containing dipotassium EDTA (1 mM) at pH 7.4; a Brinkmann Polytron at a setting of 6 was used for 20 sec. After filtration through nylon gauze, the pellet obtained after centrifugation at 38,000 X g for 20 min was resuspended in a total of 6 vol sucrose (0.32 M) with dipotassium EDTA (1.0 mM) and then frozen at -80 C. The preparation was thawed once, just before the RRA. Radioligand binding assay The binding of/?-[125I]endorphin (2000 Ci/mmol; Amersham Corp., Arlington Heights, IL), monoiodinated at tyrosine-27, was determined by filtration assay as previously described (21). Briefly, 50-^1 aliquots of either rat brain membrane (100-200 Hg protein/aliquot) or a suspension of intact cells (0.4-1.0 X 107 cells) or their membrane equivalent was incubated in triplicate with £-[125I] endorphin in a total volume of 100 ix\K2HPO4 or Tris-HCl (for cation or membrane studies; 50 mM) buffer, pH 7.4, containing bacitracin (50 ^g/ml), leupeptin (10 ^g/ml), soybean trypsin inhibitor (10 pg/m\), PMSF (1 mM), and benzamidine (1 mM). Nonspecific binding was determined in the presence of 3 nM (final concentration) unlabeled /3-endorphin. After incubation for 60 min at 4 C, the receptor-bound radioactivity was isolated by filtration through glass fibers presoaked for at least 30 min at room temperature in a solution of K2HPO4 (50 mM), pH 7.4, containing 0.01% poly-L-lysine HBr (mol wt, 240,000) and 0.4% BSA. Thereafter, filters were washed three times with 3 ml cold K2HPO4 (50 mM), pH 7.4, buffer containing 0.01% Triton X-100. The filters were then counted in a Beckman y-counter with 75% efficiency. The competition of /3-endorphin binding by fragments of /?-endorphin (Peninsula Laboratories, Belmont, CA) and naloxone hydrochloride (DuPont Corp., Wilmington, DE) used /?-[125I] endorphin (0.05 nM). Scatchard analysis of the data was performed using the nonlinear least squares regression analysis of Ligand by McPherson (22). All studies were repeated at least three times except the dose response to GTP7 sulfate (GTPyS; Boehringer Mannheim, Indianapolis, IN: n = 1). Protein was measured using Bio-Rad reagents (23). Analysis of N-acetyl (Ac)-(i-endorphin by reverse phase HPLC To determine whether iV-Ac-/3-endorphin remained intact or was converted to 0-endorphin during the radioligand binding assay, HPLC (Gilson Corp., Middleton, WI) using a reverse phase C8 Ultrasphere column (Beckman Instruments, Inc., Palo Alto, CA) was performed. A gradient elution, starting with 0.1% trifluoroacetic acid and 3.0% acetonitrile (EM Science, Gibbstown, NJ) and reaching 60.0% acetonitrile over 40 min, was used to separate /?-endorphin (eluting at 21.5 min) from
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EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
JV-Ac-/3-endorphin (eluting at 22.5) which were detected by UV spectroscopy at 220 nm. In the absence of (3-[125I]endorphin, AT-Ac-/3-endorphin (5-10 jug) was added to the standard radioligand binding assay for 60 min at 4 C. After centrifugation to separate the cells, the supernatant was filtered (Centrex nylon, 0.2 /um, Schleicher and Schuell, Keene, NH), and approximately 100-150 ^1 were injected for analysis. Using this design, /?-endorphin alone and /3-endorphin plus N-Ac-f3-endorphin were also analyzed after incubation.
Endo• 1990 Vol 126 • No 6
40 n 4C
s
Affinity labeling and electrophoretic analysis U937 cell membrane was suspended in the K2HPO4 (50 mM) buffer used for radioligand binding with the addition of PMSF (1 mM) benzamidine (1 mM), and EDTA. Membrane (1-1.5 mg protein/ml) was incubated with |8-[125I]endorphin in the presence or absence of unlabeled ligand for 60 min at 4 C. Unbound ligand was removed by centrifugation at 20,000 x g (4 C) for 20 min after the addition of 5 vol ice-cold binding buffer. After resuspension, cross-linking was initiated with the addition of JV-hydroxysulfosuccinimide and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to yield final concentrations of 5 mM and 0.1 M, respectively. The reaction was terminated by the addition of ice-cold assay buffer, followed by centrifugation. Pellets were solubilized in Laemmli sample buffer containing sodium dodecyl sulfate (SDS) and /3-mercaptoethanol, and boiled for 5 min (24). Samples were electrophoresed by the method of Laemmli using 1.5-mm SDS-polyacrylamide (10%) slab gels (SDS-PAGE). An aliquot of each sample (40-100 Mg protein) was electrophoresed at a current of 30 mamp/slab for 3-4 h. To determine the position of marker proteins (Bio-Rad), gels were stained with 0.05% Coomassie blue in 10% acetic acid plus 50% methanol and destained in 10% acetic acid plus 10% methanol. After drying, autoradiography was performed using Kodak X-Omat AR film (Rochester, NY) with intensifying screens at -70 C. All studies were performed at least two times.
Results Intact U937 cells were used to analyze the time course of /3-[125I]endorphin binding at 4, 25, or 37 C (Fig. 1). Beginning with the first data point that was obtained after 1 min of incubation, the maximal fraction of total radioligand was bound by 5 min at 37 C, 10 min at 25 C, and 30 min at 4 C. Thereafter, binding declined most rapidly at 37 C, and a nadir was achieved by approximately 120 min at 25 and 37 C. In contrast, maximal binding was stable at 4 C for greater than 200 min. Thus, studies were performed at 4 C for 60 min. Figure 2 shows a characteristic saturation study in which U937 cells were incubated with a range of /3-[125I] endorphin concentrations from 8 pM to 35 nM. Nonspecific binding (~20-30% of total binding) was linear throughout the dosage range. Scatchard analysis using Ligand showed one site with a Kd (mean ± SE of four studies) of 1.2 ± 0.5 x 10"8 M and a binding capacity (Bmax) of 4.0 ± 0.7 X 10"14 mol /3-[125I]endorphin/l x 106 cells or 26,000 receptors/cell.
120 TIME
150
180
(min)
FIG. 1. The effects of time and temperature on /?-[126I]endorphin binding to intact U937 cells. Cells were washed and then pellets were resuspended in the radioligand binding buffer at 1.7 x 107 cells/ml, and /3-[125I]endorphin (0.1 nM) was added. Total and nonspecific binding, in triplicate, were determined for each data point. The total bound fraction (percentage) of the total count added is shown on the y-axis. For each point, the coefficient of variation was less than 15% of the mean, and specific binding was 70-80% of the total bound.
Competition studies using opiate receptor ligands and analogs of /?-endorphin-(l-31) are shown in Figs. 3 and 4. iV-Ac-j8-endorphin-(l-31), which is ineffective at binding to classical brain opiate receptors (25), was equipotent to /3-endorphin-(l-31) in the nanomolar concentration range. /3-Endorphin-(l-31) or ethylketocyclazocine displaced 7-20% of the radioligand at extremely low concentrations (femtomolar). However, a binding site with a Kd in this concentration range was not consistently demonstrable. In at least three separate experiments, morphine, naloxone, and [D-Pen2, Pen5]-enkephalin (26) or U69,593 (27) (the latter two are selective for 8- and /c-opiate receptors, respectively) failed to displace any |8-[125I] endorphin, even at concentrations as great as 10~4 M. Figure 4 shows that the C-terminal tetrapeptide i8-endorphin-(28-31) was approximately 100-fold less potent than intact /?-endorphin, and C-terminal elongation to 0-endorphin-(18-31) or 0-endorphin-(6-31) resulted in equipotent peptides that were approximately 5-fold less potent than /3-endorphin-(l-31). Addition of the natural N-terminal pentapeptide sequence to yield /?endorphin-(1-5, 16-31) restored full potency. Thus, the C-terminal of /3-endorphin-(l-31) is absolutely required for binding to this site. This was confirmed by the
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EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
3009
SCATCHARD
BINDING 3.42E-02
I.08E-10
FIG. 2. Binding isotherm and Scatchard plot of j8-[125I]endorphin binding to U937 cells. Cells were incubated for 60 min at 4 C with increasing concentrations of (3[125I]endorphin (8.0 x 10"12-8.8 x 10~8 M). The binding isotherm (left panel) shows a single site, which is confirmed by the nonlinear curve fitting analysis using the Ligand program (22), which generated a single line to fit the Scatchard data points (right panel). Kd = 9.7 X 10' 9 M; Bmax = 18,000 receptors/cell. B/F, Bound to free ratio.
/ /
L.54E-18
L89E-02 \
2.7IE-13
•76E-08
714H2
3.52E-03 2•71E-13
vs Ln (FREE)
v
3.08E-10
B/F US BOUND
100
100 80
60
40
20
20 14
13
12
11
10
8
-Log (M)
FIG. 3. Displacement of /3-[125I]endorphin binding to U937 cells by /?endorphin-(l-31), iV-Ac-/3-endorphin-(l-31), and ethylketocyclazocine (EKC). ]8-[125I]Endorphin (0.05 nM) was incubated with increasing concentrations (10~2-10~8 M) of unlabeled ligand at 4 C for 60 min.
complete failure of j8-endorphin-(l-27) or /3-endorphin(1-16) to displace the radioligand. These studies indicate that C-terminal elongation to yield /3-endorphin-(18-31) enhances potency, whereas further lengthening to yield /3-endorphin-(6-31) is unnecessary; finally, the addition of /3-endorphin-(l-5) is necessary for full potency. The integrity of iV-Ac-/3-endorphin was determined by HPLC after incubating the peptide with intact cells at 4 C for 60 min (data not shown). After incubation of NAc-/3-endorphin (5-10 i*g), analysis of a sample of supernatant showed that approximately 80-90% of the added peptide eluted at 22.5 min and none at 21.5 min (the position of /3-endorphin). The remainder of the peptide (~10-20%) appeared to be nonspecifically bound to cells, tubes, and filters, since the peak at 22.5 min was not accompanied by other novel peaks. Similarly, incubation
13
12
11
10 125
FIG. 4. Displacement of j8-[ I]endorphin binding to U937 cells by fragments of/3-endorphin-(l-31). Cells were incubated for 60 min at 4 C with /3-[125I]endorphin (0.05 nM) and increasing concentrations (10~12-10"5 M) of the unlabeled ligands.
of /3-endorphin (5-10 ng) with cells showed that approximately 80-90% of the added peptide eluted at 21.5 min. Coincubation of equivalent amounts of iV-Ac-|8-endorphin (5-10 jug) and /3-endorphin (5-10 /*g) showed equivalent peaks at 21.5 and 22.5 min. Thus, iV-Ac-/?-endorphin is not degraded to /3-endorphin during the RRA. Covalent cross-linking with carbodiimide was performed after /3-[125I]endorphin was reversibly bound to U937 membrane. The reaction product was resolved by SDS-PAGE. Figure 5 shows the effect of increasing concentrations of unlabeled /?-endorphin-(l-31) (3 x 10"12-2.5 X 10"5 M) on the bands at 66K, 59K, and 44K. The 66K and 44K bands were completely displaced by increasing concentrations, whereas the 59K band was
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EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
3010
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Endo • 1990 Vol 126 • No 6
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31 -H 22 14 FIG. 5. The effects of increasing concentrations of ]8-endorphin-(l-31) on the binding and chemical cross-linking of 0-[125I]endorphin to U937 cells. Membrane was prepared for binding assays performed at 4 C for 60 min using /3-[125I]endorphin (1.0 nM) and increasing concentrations of unlabeled j8-endorphin-(l-3l) (lane 6, 3 x 10"12 M; lane 5, 3 x 10"10 M; lane 4, 3 x 10~8 M; lane 3, 2.5 x 10"5 M). After the binding assay, chemical cross-linking and analysis using SDS-PAGE were performed, as described in Materials and Methods. Two samples were analyzed for total binding (lane 1, protein = protein of sample in lane 3; lane 2, total counts per min = total counts per min of sample in lane 3).
FIG. 6. The effects of increasing concentrations of iV-Ac-/3-endorphin(1-31) on the binding and chemical cross-linking of j8-[125I]endorphin to U937 cell membranes. The procedure was analogous to that described in Fig. 5. For competition, increasing concentrations of iV-Ac-/3-endorphin were as follows: lane 7, 3 x 10"12 M; lane 6, 3 x 10"10 M; lane 5, 3 x 10"8 M; lane 4, 3 X 10"6 M; lane 3, 2.5 X 10~6 M. Two samples were analyzed for total binding (lane 1, total counts per min = total counts per min of sample in lane 3; lane 2, protein = protein of sample in lane 3).
minimally displaced by maximum amounts of /3-endorphin-(l-31) (2.5 X 10~5 M in lane 3, compared to lane 2, which was loaded with an equivalent number of counts per min, or lane 1 which contained the protein equivalent of lane 3). Figure 6 shows a parallel experiment conducted using AT-Ac-/3-endorphin-(l-31) as the unlabeled ligand (2.5 X 10~5 M in lane 3, compared to lane 1, which was loaded with an equivalent number of counts per min, or lane 2, which contained the protein equivalent of lane 3); the results were similar. The effects of various fragments of /3-endorphin (all used at 2.5 X 10~5 M) on the appearance of the 66K, 59K, and 44K bands on SDS-PAGE were studied (Fig. 7). As expected from the aforementioned competition studies, j3-endorphin-(l-16) (lane 7) was without effect. /?-Endorphin-(28-31) (lane 6) slightly reduced the 66K and 44K bands, and /3-endorphin-(16-31) (lane 5) was more effective. 0-Endorphin-(l-5, 16-31) (lane 4) and 0-endorphin-(l-31) (lane 3) were equally effective, both completely displaced the 66K and 44K bands and partially displaced the 59K band. The coincubation of an opiate agonist and monovalent cations has been shown to reduce agonist binding to
brain homogenates (9, 28, 29). Thus, studies were performed to determine the influence of cations on the binding of /3-[125I]endorphin to intact U937 cells us. rat brain homogenates. Figure 8 shows that 100 mM Na+ or K+ reduced binding to U937 cells by approximately 35%, and inhibition to less than 95% of control occurred at a concentration of 500 mM. We also observed that 100 mM Li+ failed to affect binding, and 500 mM reduced binding by only 20% (data not shown). Figure 9 indicates that binding of /?-[125I]endorphin to rat brain membrane was more sensitive to inhibition by Na+, since concentrations of 50, 100, and 500 mM reduced binding by 23%, 63%, and 88%, respectively. U937 cells were more sensitive to inhibition by divalent than monovalent cations (Fig. 8). At 5 mM, Ca2+ or Mg2+ reduced binding by 54% and 40%, respectively; the degree of inhibition was similar at 10 mM. Mn2+ (1 or 10 mM) also inhibited binding by 35% and 70%, respectively. In contrast to the enhanced sensitivity of rat brain membrane to monovalent cations, Fig. 9 indicates that the divalent cation Mn2+ at 1 or 10 mM, which reduced binding by 30% and 75%, respectively, was equally effective on rat brain and U937 cells. To determine whether the naloxone-resistant binding
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EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
3011
ing studies. Na+ (100 mM) significantly reduced the Bmax from 393.7 ± 21.4 to 203.7 ± 7.9 fmol/mg protein (n = 3; P < 0.01), and Na+ (100 mM) plus GTP7S (10~4 M) further reduced the Bmax to 121.9 ± 6.2 fmol/mg protein (n = 3; P < 0.01). Kd values were not altered significantly by Na+ or Na+ plus GTP7S (control, Na+, and Na+ plus GTP7S: 2.0 ± 0.5 X 10~8, 1.0 ± 0.1 X 10"8, and 7.0 ± 1.4 X 10"9 M, respectively; n = 3).
Discussion
FIG. 7. The effect of fragments of /3-endorphin-(l-31) on the binding and chemical cross-linking of /S-[128I] endorphin to U937 cell membranes. The procedure was analogous to that described in Fig. 5. For competition, the concentration of all fragments was 25 jtM [lane 3, /3endorphin-(l-31); lane 4, (8-endorphin-(l-5, 16-31); lane 5, /3-endorphin-( 18-31); lane 6, /8-endorphin-(28-31); lane 7, /3-endorphin(1-16)]. As previously described (Fig. 6), two samples were analyzed (lanes 1 and 2) for total binding.
site for /?-endorphin on U937 is coupled to a guanine nucleotide-binding protein, the effect of a GTP analog on /3-[125I]endorphin binding to cell membrane was studied. Table 1 shows that GTP7S (10~4 M) reduced the binding of /?-[125I] endorphin by 25%. In the presence of either Na+ or Mg2+, the effect of GTP7S was magnified, and a reduction of 40-45% from the baseline with salt alone was observed. Na+ (100 mM) or Mg2+ (10 mM) alone had similar effects on binding of the radioligand to membrane in these studies (reduction to 63.2 ± 9.65% of control and 39.3 ± 11.9% of control, respectively; n = 3) compared to those observed for binding to intact cells (Fig. 8). Table 1 shows that the combination of Na+ and Mg2+ further enhanced the effect of GTP7S, resulting in a reduction of approximately 60% compared to the combined salts. Figure 10 illustrates the effect of increasing concentrations of GTP7S vs. GMP, both in the presence of Na (100 mM). GTP7S maximally reduced binding by 55% at 10"4 M compared to that in the group containing only salt (no GTP7S), whereas GMP had no effect. Figure 11 shows a representative Scatchard analysis of the affect of Na+ or Na + plus GTP7S on /3-[125I] endorphin binding to U937 cell membrane. Bmax values were reduced in proportion to the aforementioned bind-
Partial characterizations of a naloxone-insensitive binding site for 0-endorphin have been described on transformed immune cells, including human lymphocytes (17) and mouse EL4 thymoma cells (16, 18). To our knowledge, this is the first report that includes an analysis of the effects of fragments of /3-endorphin, NAc-/3-endorphin, cations, and GTP on /3-[125I]endorphin binding at this site. The affinity observed herein (Kd = 1.2 ± 0.5 X 10~8 M) is intermediate between those found for EL4 thymoma (Kd = 6.5 X 10"8) and transformed human lymphocytes (Kd = 3 X 10~9 M). An average of 26,000 sites/cell were present on the U937 line. This is similar to the 19,000 sites/cell on the EL4 thymoma line. Both cell lines continuously expressed the binding sites in cultures containing fetal serum. In contrast, we have reported (19) that a higher affinity (Kd = 4.1 X 10~9 M) site is present on normal murine splenocytes after 24 h in culture, although it is undetectable on fresh splenocytes obtained from viral antibody-free mice. The structural requirements for binding to this site on U937 cells is analogous to that reported for normal murine splenocytes (19). Some of the features have also been reported on the transformed cells (16, 17). Binding to U937 cells requires that /3-endorphin contain its Cterminus. Thus, the deletion of peptides /?-endorphin-(l16) and -(1-27) was completely ineffective. In contrast, /?-endorphin-(28-31) itself was able to displace /3-[125I] endorphin in a dose-dependent fashion, but with l/100th the potency of intact /3-endorphin. Elongation of the Cterminal tetrapeptide to /3-endorphin-(18-31) enhanced the potency 10-fold, whereas lengthening to /?-endorphin-(6-31) failed to further enhance the potency. Addition of the natural N-terminal peptide sequence (methionine-enkephalin) to /?-endorphin-(16-31) resulted in an analog equipotent to /3-endorphin-(l-31). These findings define two domains of ^-endorphin that are required for binding to this naloxone-insensitive binding site. The C-terminal sequence is absolutely required, and both elongation of this (to less than or equal to amino acids 18-31) and addition of the N-terminal pentapeptide are necessary for the full potency of /3-endorphin-(l-31). These findings are in agreement with functional studies showing that /3-endorphin-(l-27) was ineffective in en-
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EFFECTS OF CATIONS AND GTP ON 0-ENDORPHIN BINDING
3012
Endo • 1990 Voll26.No6
U937 CELLS: EFFECT OF CATIONS ON BINDING
Na
FIG. 8. The effects of cations on the binding of /3-[125I]endorphin to U937 cells. Increasing concentrations of monovalent (left panel; 0-500 mM) or divalent cations (right panel; 0-100 mM) were added to Tris-HCl buffer along with the radioligand (1.0 nM). The control group (0 mM cation) contained Tris-HCl buffer (50 mM) alone. Values were the mean ± SE (n = 3).
1
£4
8-
K
D
Cont.
•
CONTRX
0
Ca
0
Mg
6-
@ Mn
4-
i = 2-
2-
Iu 10
50
100
500
0.5
CATION [mM]
1
10
100
CATION [mM]
RAT BRAIN: EFFECT OF CATIONS ON BINDING
FIG. 9. The effects of cations on the binding of /3-[125I]endorphin to rat brain membrane. Increasing concentrations of sodium (left panel; 0-500 mM) or manganese (right panel; 0-10 mM) were added to Tris-HCl buffer along with the radioligand (1.0 nM). Values are the mean ± SE (n = 3).
T
0 SODIUM (mM] 125
TABLE 1. /3-[ I]Endorphin binding to U937 cell membranes: effects of cations and GTP % of control Cation GTP None Na+ (100 mM) Na+ (150 mM) Mg2* (10 mM) Na+ (100 mM) + Mg*+ (10 mM)
74.6 ± 54.4 ± 61.8 ± 57 ± 42.7 ±
0
4.2 5.8 5.3 12.6 10.1
GMP 98.6 ± 93.3 ± 90.3 ± 108 ± 94.5 ±
4.1 1.0 15.7 10.7 24.8
Values are the mean ± SD (n = 3). 0
G T P T S , (10" 4 M).
hancing the proliferation of murine lymphocytes (14, 30). As reported for the transformed cells (16, 17) and normal murine splenocytes (19), the /3-endorphin-binding site on U-937 cells is unaffected by either nonselec-
100
500
0
0.05
0.1
0.5
1
10
MANGANESE [mM]
tive or selective opiate receptor ligands. Thus, naloxone, morphine, the /^-selective ligand [D-Ala2,Nme,Phe4,Glyol5]-enkephalin, the natural enkephalin pentapeptides, the 6-selective ligand [D-Pen2,Pen5]-enkephalin, and the /c-selective ligand U69,593, all have failed to displace /3[125I]endorphin from this site. In contrast to this lack of efficacy of opiate ligands, JV-Ac-/3-endorphin, which is less than 1000-fold as potent as j8-endorphin in radioreceptor studies of the brain opiate receptor (25), was found to be equipotent to /3-endorphin at binding to the naloxone-resistant site on the monocyte-like U937 cell line. To validate this finding, we demonstrated that N-Ac-(3endorphin was not converted to /3-endorphin in the RRA. The same observation about the potency of JV-Ac-|8endorphin was reported for the naloxone-resistant binding site on normal murine splenocytes (19). Interestingly,
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3013
EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
Effect of Guanine Nucleotides
u.uou -
8000 n
+
GTP +Na
•
Tris-HCI
o
NaiOOmM
2.0
2.5
0.025 7000-
2 X
•
0.020 •
6000-
u.
5000-
m 0.015-
VV°
\\ "
i
4000-
0.0103000 -6
-4
loglguanlne nucleotide] (M)
FIG. 10. The effects of guanine nucleotides on the binding of /3-[126I] endorphin to U937 cell membranes. In the presence of NaCl (100 mM), increasing concentrations (10~8-10~4 M) of GMP or GTP7S were added to cell membranes along with the radioligand (1.0 nM). Each data point is the mean from quadruplicate determinations of specific binding in a single experiment. Each aliquot contained the membrane equivalent of 2 X 10e cells.
iV-Ac-jS-endorphin appears to be synthesized by normal murine splenocytes (31) and uncharacterized when /?endorphin immunoreactivity is present constitutively in secretions from normal splenic monocytes maintained in culture (32). Thus, the naloxone-insensitive binding site may be the target for iV-Ac-/3-endorphin, acting as either an agonist or antagonist. At present, two functional studies have tested iV-Ac-0-endorphin and found that it enhanced monocyte chemotaxis and lymphocyte proliferation in a naloxone-insensitive manner (30, 33). However, another study showed that /?-endorphin enhanced mitogen-stimulated calcium uptake in rat thymocytes, but failed to demonstrate a similar affect of N-Ac-fiendorphin; its potential role as an antagonist was not evaluated (15). The site of the binding sites(s) and the relative potency of /?-endorphin-(l-31), its fragment analogs, and iV-Ac/3-endorphin at displacing /?-[125I]endorphin were studied using covalent cross-linking followed by SDS-PAGE. We consistently observed three bands: 66K, 59K, and 44K. The 66K and 44K bands were equipotently and completely displaced by /3-endorphin-(l-31), JV-Ac-0-endor-
0.005 -
0.000 0.0
V \ ° \ 0.5
1.0
1.5
3.0
Bound ( H 1 0 - 1 0 M) FIG. 11. Scatchard analysis of the effects of sodium and sodium plus GTP7S on the binding of /3-[126I]endorphin to U937 cells. Cell membranes were incubated with varying concentrations of radioligand in the presence of Tris-HCI (50 mM) alone or with the addition of sodium chloride (100 mM) or sodium chloride (100 mM) plus GTP7S (lO"4 M). The binding isotherms (not shown) were subjected to Scatchard analysis using the nonlinear curve fitting routine of Ligand (22), which generated a single best fit line for each data set. Each aliquot contained 0.7 mg protein/ml. B/F, Bound to free ratio.
phin-(l-31), and ]8-endorphin-(l-5,16-31). In agreement with the rank order of potency observed in the radioreceptor competition studies, /3-endorphin-(28-31) was minimally effective at competing the 66K and 44K bands, and j8-endorphin-(l-16) had no effect. In contrast, 0endorphin-(16-31) was more effective than /?-endorphin(28-31). The 59K band showed similar structural requirements for displacement, but was only partially reduced. Schweigerer et al. (16) reported a single 76K band on murine EL4 thymoma cells, and we (19) reported bands at 66K and 57K on normal murine splenocytes.
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3014
EFFECTS OF CATIONS AND GTP ON /3-ENDORPHIN BINDING
The difference between the findings on U937 cells and EL4 thymoma cells may reflect species differences and/ or a different state of the receptor which is associated with a lower affinity (Kd = 6.5 X 10~8 M) and expressed on immature immune cells. Two of the bands present on U937 cells (66K and 59K) are similar in size to those on normal murine splenocytes (66K and 57K), whereas the 44K band is unique to the U937 line. Despite these similarities, it was the 66K and 44K bands from U937 cells and the 57K band from murine splenocytes that were displaced completely. Studies were performed to determine the affect of cations on the binding of /3-[125I] endorphin to U937 cells. Early reports established that sodium and other cations inhibit the binding of opiate agonists to neuronal opiate receptors (9, 28, 29, 34). Although most cations previously tested caused a dose-dependent inhibition of agonist binding, of the monovalent cations opiate receptors were most sensitive to Na+ (9, 28). This inhibition appeared to affect either receptor affinity (28, 35) or binding capacity (9, 29). This difference may reflect the heterogeneity of opiate receptors (36), which leads to differential responses to cations. Indeed, a recent investigation showed that opiate receptors had different responses to Na+ depending on the tissue of origin, which, in turn, specified the subtype of receptor (37). The affinity of ju-receptors on 315c cells was diminished by Na+, whereas it was the Bmax of 5-receptors on NG-108-15 cells that was reduced by Na + . The present investigations show that binding of /3[125I]endorphin to rat brain membrane has a biphasic response to Na+. Low concentrations (5-10 HIM) enhanced binding, whereas higher concentrations (>50 mM) reduced binding. This differs from the monophasic response and the exquisite sensitivity to Na+ that have been shown for the binding of alkyloids, such as dihydromorphine, to rat brain; this binding was inhibited by as little as 1 mM Na+ (9), although inhibition was most pronounced with concentrations greater than 50-100 mM (9, 28). The present investigations indicate that the sensitivity to Na+-induced inhibition of binding was greater for rat brain membrane than U937 cells, which had a monophasic response to Na+. The U937 cells were equisensitive to Na+ and K+, whereas neuronal opiate receptors are known to be more sensitive to sodium. Nevertheless, some selectivity among the monovalent cations was evidenced by the minimal affect of Li+. As previously observed for neuronal opiate receptors (34, 35), binding to U937 cells was also affected by divalent cations. Manganese more potently reduced binding than calcium or magnesium, and all three were more potent than the monovalent cations. Manganese also caused a monophasic reduction of/3-[125I] endorphin binding to the brain preparation. In contrast to these observations,
Endo • 1990 Voll26«No6
most reports indicate that low concentrations ( monovalent) and appears to be coupled to a GTP-binding protein which modulates its binding.
Acknowledgments We thank Michele Burtness for her excellent technical assistance, and Linda Soumphonphakdy and Sue Klick for their secretarial support.
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EFFECTS OF CATIONS AND GTP ON /J-ENDORPHIN BINDING
References 1. Peterson PK, Sharp BM, Gekker G, Brummitt C, Keane WF 1987 Opioid-mediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity. J Immunol 138:3907 2. Sharp BM, Keane WF, Suh HJ, Gekker G, Tsukayama D, Peterson PK 1985 Opioid peptides rapidly stimulate superoxide production by human polymorphonuclear leukocytes and peritoneal macrophages. Endocrinology 117:793 3. Peterson PK, Sharp BM, Gekker G, Brummitt C, Keane WF 1987 Opioid-mediated suppression of interferon production by cultured peripheral blood mononuclear cells. J Clin Invest 80:824 4. Mathews PM, Froelich CJ, Sibbitt WL, Bankhurst AD 1983 Enhancement of natural cytotoxicity by /3-endorphin. J Immunol 130:1658 5. Mandler RN, Biddison WE, Mandler R, Serrate SA 1986 /3-Endorphin augments the cytolytic activity and interferon production of natural killer cells. J Immunol 136:934 6. Johnson HM, Smith EM, Torres BA, Blalock JE 1982 Regulation of the in vitro antibody response by neuroendocrine hormones. Proc Natl Acad Sci USA 79:4171 7. Ovadia H, Nitson P, Abramsky 0 1989 Characterization of opiate binding sites on membranes of rat lymphocytes. J Neuroimmunol 21:93 8. Carr DJJ, Kim C-H, DeCosta B, Jacobson AE, Rice KC, Blalock JE 1988 Evidence for a delta class opioid receptor on cells of the immune system. Cell Immunol 111:44 9. Pert CB, Snyder SH 1974 Opiate receptor binding of agonists and antagonists affected differentially by sodium. Mol Pharmacol 10:868 10. Blume AJ 1978 Interaction of ligands with the opiate receptors of brain membranes: regulation by ions and nucleotides. Proc Natl Acad Sci USA 75:1713 11. Gilman SC, Schwartz JM, Milner RJ, Bloom FE, Feldman JD 1982 /3-Endorphin enhances lymphocyte proliferative responses. Proc Natl Acad Sci USA 79:4226 12. McCain HW, Lamster IB, Bozzone JM, Gribic JT 1982 /3-Endorphin modulates human immune activity via non-opiate receptor mechanisms. Life Sci 31:1619 13. Puppo F, Corsini G, Mangini P, Bottaro L, Barreca T 1985 Influence of /3-endorphin on phytohemagglutinin-induced lymphocyte proliferation and on the expression of mononuclear cell surface antigens in vitro. Immunopharmacology 10:119 14. Gilmore W, Weiner LP 1988 /3-Endorphin enhances interleukin-2 (IL-2) production in murine lymphocytes. J Neuroimmunol 18:125 15. Hemmick LM, Bidlack JM 1987 0-Endorphin modulation of mitogen-stimulated calcium uptake by rat thymocytes. Life Sci 41:1971 16. Schweigerer L, Schmidt W, Teschemacher H, Wilhelm S 1985 0Endorphin: interaction with specific nonopioid binding sites on EL4 thymoma cells. Neuropeptides 6:445 17. Hazum E, Chang KJ, Cuatrecasas P 1979 Specific Nonopiate receptors for /3-endorphin. Science 205:1033 18. Schweigerer L, Schmidt W, Teschemacher H, Gramsch C 1985 /3Endorphin: surface binding and internalization in thymoma cells. Proc Natl Acad Sci USA 82:5751 19. Shahabi NA, Linner KM, Sharp BM 1990 Murine splenocytes express a naloxone-insensitive binding site for /3-endorphin. Endocrinology 126:1442 20. Ruegg UT, Cuenod S, Hiller JM, Gioannini T, Howells RD, Simon EJ 1981 Characterization and partial purification of solubilized active opiate receptors from toad brain. Proc Natl Acad Sci USA 78:4635
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21. Howard AD, de La Baume S, Gioannini TL, Miller JM, Simon EJ 1985 Covalent labeling of opioid receptors with radioiodinated human /3-endorphin. J Biol Chem 260:10833 22. McPherson GA 1985 Analysis of radioligand binding experiments: a collection of computer programs for IBM PC. J Pharmacol Methods 14:213 23. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 24. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 25. Akil H, Young E, Watson SJ, Coy DH 1981 Opiate binding properties of naturally occurring N- and C-terminus modified /3-endorphins. Peptides 2:289 26. Akiyama K, Gee KW, Mosberg HI, Hruby VJ 1985 Characterization of [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin binding to delta opiate receptors in the rat brain and neuroblastoma-glioma hybrid cell line (NG 108-15). Proc Natl Acad Sci USA 82:2543 27. Lahti RA, Mickelson MM, McCall JM, Von Voightlander PF 1985 [3H]U69593 A highly selective ligand for the opioid kappa receptor. Eur J Pharmacol 109:281 28. Simon EJ, Hiller JM, Groth J, Edelman I 1975 Further properties of stereospecific opiate binding sites in rat brain: on the nature of the sodium effect. J Pharmacol Exp Ther 192:531 29. Simantov R, Snowman AM, Snyder SH 1976 Temperature and ionic influences on opiate receptor binding. Mol Pharmacol 12:977 30. Gilmore W, Weiner LP 1989 The opioid specificity of beta-endorphin enhancement of murine lymphocyte proliferation. Immunopharmacology 17:19 31. Lolait SJ, Clements JA, Markwick AJ, Cheng C, McNally M, Smith AI, Funder JW 1986 Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of /3-endorphin in spleen macrophages. J Clin Invest 77:1776 32. Kavelaars A, Ballieux RE, Heijnen CJ 1989 The role of IL-1 in the corticotropin-releasing factor and arginine-vasopressin-induced secretion of immunoreactive /3-endorphin by human peripheral blood mononuclear cells. J Immunol 142:2338 33. Sacerdote P, Panerai AE 1989 Analysis of the beta endorphin structure-related activity on human monocyte chemotaxis: importance of the amino and carboxyl-terminal. Peptides 10:565 34. Pasternak GW, Snowman AM, Snyder SH 1975 Selective enhancement of [3H] opiate agonist binding by divalent cations. Mol Pharmacol 11:735 35. Blume AJ 1978 Interaction of ligands with the opiate receptors of brain membranes: regulation by ions and nucleotides. Proc Natl Acad Sci USA 75:1713 36. Goldstein A, James IF 1984 Multiple opioid receptors: criteria for identification and classification. Trends Pharmacol Sci 5:503 37. Puttfarcken P, Werling LL, Brown SR, Cote TE, Cox BM 1986 Sodium regulation of agonist binding at opioid receptors. I. Effects of sodium replacement on binding at M- and 5-type receptors in 7315c and NG108-15 cells and cell membranes. Mol Pharmacol 30:81 38. Blume AJ 1978 Opiate binding to membrane preparations of neuroblastoma X glioma hybrid cells NG108-15: effects of ions and nucleotides. Life Sci 22:1843 39. Chang K-J, Blanchard SG, Cuatrecasas P 1983 Unmasking of magnesium-dependent high-affinity binding sites for [DAla2,DLeu6] enkephalin after pretreatment of brain membranes with guanine nucleotides. Proc Natl Acad Sci USA 80:940 40. Childers SR, Snyder SH 1978 Guanine nucleotides differentiate agonist and antagonist interactions with opiate receptors. Life Sci 23:759
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