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Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand
YNPEP-01689; No of Pages 6 Neuropeptides xxx (2015) xxx–xxx
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Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand Fanni Tóth a, Jayapal Reddy Mallareddy a, Dirk Tourwé b, Andrzej W. Lipkowski c, Magdalena Bujalska-Zadrozny d, Sándor Benyhe a, Steven Ballet b, Géza Tóth a, Patrycja Kleczkowska c,d,⁎ a
Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt 62, 6726 Szeged, Hungary Research Group of Organic Chemistry, Department of Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Department of Neuropeptides, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Street, 02106 Warsaw, Poland d Department of Pharmacodynamics, Centre for Preclinical Research and Technology, Medical University of Warsaw, 1B Banacha Str., 02-106 Warsaw, Poland b c
a r t i c l e
i n f o
Article history: Received 20 August 2015 Received in revised form 25 November 2015 Accepted 7 December 2015 Available online xxxx Keywords: Neuromedin N Receptor binding assays NTS2 Rat
a b s t r a c t Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [3H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454 nM in rat brain and 425 nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8 ± 30.18 nM, while the Bmax value corresponded to 3.8 ± 0.2 pmol/mg protein in rat brain membranes. The specific binding of [3H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na+ ions was observed on the binding of [3H]NN, resulting in an IC50 of 150.6 mM. In [35S]GTPγS binding experiments the Emax value was 112.3 ± 1.4% in rat brain and 112.9 ± 2.4% in rat spinal cord membranes and EC50 values of 0.7 nM and 0.79 nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [3H]NN labels NTS2 receptors in rat brain membranes. © 2015 Published by Elsevier Ltd.
1. Introduction The neurotensin-like peptide, neuromedin N (NN), was first isolated from porcine spinal cord membranes (Minamino et al. 1984). The amino acid sequence of NN, H-Lys-Ile-Pro-Tyr-Ile-Leu-OH, is homologous to
Abbreviations: BH, Bolton–Hunter; Bmax, maximal number of the binding sites; BSA, bovine serum albumin; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; EC50, half maximal effective concentration; Emax, maximal stimulation (Efficacy); GDP, guanosine 5′-diphosphate; [35S]GTPγS, guanosine-5′-[γ-35S]-triphosphate; HOBt, 1hydroxybenzotriazole; HPLC, high performance liquid chromatography; IC50, half-maximal inhibitory concentration; Kd, equilibrium dissociation constant; kobs, pseudo-first-order rate constant; levocabastine, (3S,4R)-1-[4-cyano-4-(4-fluorophenyl)cyclohexyl]-3-methyl-4phenylpiperidine-4-carboxylic acid; SR 48692, 2-([1-(7-chloro-4-quinolinyl)-5-(2,6dimethoxyphenyl)-1 H-pyrazole-3-carbonyl]amino)admantane-2-carboxylic acid; NN, neuromedin N; NT, neurotensin; NTS1, neurotensin receptor 1; NTS2, neurotensin receptor 2; PCs, proprotein convertases; PEI, polyethyleneimine; SPPS, solid phase peptide synthesis; TBTU, N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate; TEA, triethylamine; TFA, trifluoroacetic acid. ⁎ Corresponding author at: Department of Pharmacodynamics, Centre for Preclinical Research and Technology, Medical University of Warsaw, 1B Banacha Str., 02-091 Warsaw, Poland. E-mail address: hazufi[email protected] (P. Kleczkowska).
that of neurotensin (NT) (8–13), H-Arg-Arg-Pro-Tyr-Ile-Leu-OH (Minamino et al. 1984), which is the essential part of the tridecapeptide NT for biological activity (Lambert et al. 1995). NT and NN are synthesized from a common precursor (pro-NT/NN) in mammalian brain tissues (Kislauskis et al. 1988) and intestines (Dobner et al. 1987). Both sequences are located in the C-terminal part of the proNT/NN precursor and they are flanked by Lys-Arg residues, which are the known consensus sequences for cleavage by specific endoproteases that belong to the proprotein convertase (PC) family. Processing of the precursor generates NT and NN or a large peptide ending with the NT sequence in brain, gut or adrenals (Kitabgi 2010). The biological effects mediated by NT and NN, such as naloxoneinsensitive analgesia or hypothermia, result from the interaction of these peptides with specific G protein-coupled receptors (GPCRs) (Coquerel et al. 1988; Dubuc et al. 1988). It has been reported that NN displayed antinociceptive properties after intracerebroventricular (i.c.v.) injection in rat writhing and tail-flick tests. Compared to NT, the corresponding NN-induced effects required higher doses. The fact that naloxone did not significantly reduce these antinociceptive effects indicates that both NT and NN elicit analgesia in mice through an opioid independent mechanism (Coquerel et al.
http://dx.doi.org/10.1016/j.npep.2015.12.004 0143-4179/© 2015 Published by Elsevier Ltd.
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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1988). Additionally, intracerebroventricular administration of NN elicited dose- and time-dependent hypothermia in mice, an effect that was significantly increased by bestatin (Dubuc et al. 1988). NN also has an influence on digestive organs: NN induces increases of the blood flow sin superior mesenteric artery, portal vein and pancreatic tissue, and increase of pancreatic juice volume and pancreatic protein output, and that it causes a fall in systematic arterial pressure (Sumi et al. 1987). NN degradation is 3 to 20-fold faster than that of NT, and was totally inhibited by 1,10-phenanthroline and markedly inhibited by bestatin (Checler et al. 1986; Kitabgi 2006). In general, NN is less potent in binding and functional assays than NT, which was ascribed to the higher sensitivity of NN to enzymatic degradation (Rompre and Gratton 1992). Previous reports indicate that the Zn metallo-exopeptidase aminopeptidase M is largely responsible for NN degradation in vitro and in vivo in the gastrointestinal tract (Barelli et al. 1995; Kitabgi 2006). Two types of neurotensin receptors, belonging to the seven transmembrane domain GPCRs, and previously distinguished by their affinity for neurotensin and their sensitivity to the antihistaminic drug levocabastine (Schotte and Laduron 1987), were cloned. The highaffinity, levocabastine-insensitive, neurotensin receptor NTS1 was the first to be cloned (Vita et al. 1993), followed in 1996 by the lowaffinity levocabastine-sensitive neurotensin receptor NTS2 (Chalon et al. 1996). A third and fourth neurotensin subtype receptor have subsequently been reported. NTS3 was identified as gp95/sortilin (Mazella et al. 1998; Vincent et al. 1999) and NTS4 is a mosaic protein (Jacobsen et al. 2001). NTS3 and NTS4 are non G protein-coupled single transmembrane domain sorting receptors. NN competitively inhibited specific 125I-NT (2 nM) binding to CHO/rNTS2 cells with IC50 values of 21 nM, acting as an agonist at the NTS2 site (Gendron et al. 2004). In order to perform direct ligand-binding studies with the NN hexapeptide, the tritium labeled peptide Lys-Ile-3HPro-Tyr-Ile-Leu-OH was prepared (Tóth et al. 2012). Herein we report the synthesis, radiolabeling and binding characteristics of [3H]Neuromedin N, as well as the G protein activation of the cold ligand.
Pharmaceutica (Beerse, Belgium) and SR 48692: 2-([1-(7-chloro-4quinolinyl)-5-(2,6-dimethoxyphenyl)-1 H-pyrazole-3-carbonyl]amino)admantane-2-carboxylic acid was obtained from Axon Medchem (Groningen, The Netherlands). 2.3. Peptide synthesis Synthesis of neurotensin (8–13), neuromedin N (H-Lys-Ile-Pro-TyrIle-Leu-OH), and H-Lys-Ile-ΔPro-Tyr-Ile-Leu-OH was performed manually using standard Fmoc-based solid phase peptide synthesis (SPPS) on Fmoc-Leu-Wang resin. For these syntheses a reaction vessel under nitrogen bubbling was used. The protocols for amino acid dissolution, activation and coupling time, were performed according to the manufacturer's instruction. The couplings were performed for 2 h by adding protected amino acids (3 equiv.) and TBTU (3 equiv.) in the presence of HOBt (3 equiv.) and DIPEA (6 equiv.) in DMF to the resin. The resin was washed with DMF between all reaction steps. Completion of the coupling was monitored by the Kaiser test. Removal of the Fmoc protecting group from the N-terminus of the resin-bound peptide chain was achieved by treating the peptidyl resin with a 20% piperidine in DMF solution. The support was again washed with DMF and the next coupling was performed. The final hexapeptides were cleaved from the resin by treatment with trifluoroacetic acid (99%) for 2 h followed by evaporation, precipitation in cold Et2O and HPLC purification. The purification of the peptides was performed on a Shimadzu apparatus and controlled with the software package Class-VP, 5.0 (Kyoto Japan) using a C8 column (20 × 250 mm, KROMASIL) in 0.1% TFA/water and a gradient of 3–97% acetonitrile in 0.1% TFA/water, with a flow rate of 6 ml/min and using UV detection at 280 nm (Shimadzu Diode Array Detector SPD-M10 AVP). A final purity of N99% was obtained. 2.4. Preparation of 3 H-neuromedin N (Lys-Ile-3HPro-Tyr-Ile-Leu-OH)
Inbred male Wistar rats (250–300 g body weight) were housed in the local animal house of the Biological Research Center (BRC, Szeged, Hungary). Rats were kept in groups of four, allowed free access to food and water, and maintained on a 12:12-h light/dark cycle until the time of sacrifice. Animals were treated according to the European Communities Council Directives (86/609/ECC) and the Hungarian Act for the Protection of Animals in Research (XXVIII.tv. 32.§).
In the reaction vessel 3 mg of Lys-Ile-Pro-Tyr-Ile-Leu-OH precursor was dissolved in DMF and 10 mg of PdO/BaSO4 catalyst and 1 μl of TEA were added to the precursor solution (Tóth et al. 2012). Tritium gas (370 GBq(10 Ci)) was introduced to the reaction mixture which was further stirred by a magnetic stirrer for 1 h at room temperature. The catalyst was filtered through a Whatmann GF/C glass fiber filter and washed several times with ethanol. The labile tritium was removed by repeated evaporation of an ethanol-water (1:1) mixture. The total activity was 1.75 GBq (47.3 mCi). The crude peptide was purified by HPLC using an analytical column (Atlantis) and a linear gradient of the elution system from 20% up to 40% acetonitrile in water with 1% TFA over 25 min at a flow rate of 1 ml/min and detection at 216 nm. The retention time corresponded to 10.0 min. [3 H]Neuromedin N exhibited a specific radioactivity of 0.6 TBq/mmol (16 Ci/mmol) and a purity of N98%. The radioligand was stored in a refrigerator at −80 °C.
2.2. Chemicals
2.5. Rat brain and spinal cord membrane preparation
The peptide synthesis reagents N,N-diisopropylathylamine (DIPEA), 1-hydroxybenzotriazole hydrate (HOBt), as well as N,N,N′,N′tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU), Fmoc-protected amino acids and Fmoc-Leu-Wang resin were obtained from IRIS Biotech GmbH (Marktredwitz, Germany) and Sigma Aldrich (Poznan, Poland), respectively. Guanosine-5′-[γ-35S]-triphosphate (1204 Ci/mmol) was obtained from the Isotope Institute Ltd. (Budapest, Hungary). MgCl2, NaCl, EGTA, polyethyleneimine (PEI), tris(hydroxymethyl)aminomethane (Tris), protease-free bovine serum albumin (protease-free BSA, fraction V), captopril, bestatin and phosphoramidon, guanosine 5′-diphosphate sodium salt (GDP), guanosine-5′[γ-thio]-triphosphate tetra-lithium salt (GTPγS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Levocabastine hydrochloride, (3S,4R)-1-[4-cyano-4-(4-fluorophenyl)cyclohexyl]-3-methyl-4phenylpiperidine-4 carboxylic acid, was kindly provided by Janssen
A crude membrane fraction was prepared from Wistar rat brains and spinal cords according to a method reported by Pasternak et al. with small modifications (Benyhe et al. 1997). Two- to three-month old animals were decapitated, and the brains without cerebella were rapidly removed, and washed several times with chilled 50 mM Tris–HCl buffer (pH 7.4) or in the case of the spinal cord: decapitated and the vertebral foramen was surgically opened at the lumbar level between vertebrae L4–L5. The spinal cords were rapidly removed by pneumatic pressure using a 100-ml plastic syringe with a blunt steel needle, and washed several times with chilled 50 mM Tris– HCl buffer (pH 7.4). The brains and spinal cords were then weighed and suspended in 5% vol/wt of brain or spinal cord tissue of the icecold buffer. Tissues were homogenized using a Braun teflon-glass homogenizer (1000 rpm, 10–15 strokes), and filtered through four layers of gauze to remove large aggregates. Additional buffer was added to
2. Material and methods 2.1. Animals
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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reach a final buffer volume/membrane pellet ratio of 30 (ml/g). After centrifugation with a Sorvall RC5C centrifuge (40.000 × g, at 4 °C, for 20 min), the resulting pellet was re-suspended in fresh buffer (30% vol/wt) by using a vortex. The suspension was incubated at 37 °C for 30 min to remove any endogenous opioids. Centrifugation was repeated under the same conditions as described above, and the final pellet was resuspended in five volumes of 50 mM Tris–HCl buffer (pH 7.4) containing 0.32 M sucrose to give a final protein concentration of 3–4 mg/ml. The membrane samples were stored in 5 ml aliquots at −70 °C. The protein concentration was determined by the Bradford method (Bradford 1976). Before use the membranes were thawed and re-suspended in 50 mM Tris–HCl buffer (pH 7.4) and centrifuged (40.000 × g, at 4 °C, for 20 min) to remove sucrose and used immediately in binding assays. 2.6. Receptor binding assays All binding assays were performed at 25 °C for 45 min in 50 mM Tris– HCl buffer (pH 7.4) in a final volume of 1 ml, containing 1 mg BSA and 0.2–0.4 mg/ml (rat brain or spinal cord) protein. Three peptidase inhibitors: 1 μM captopril, 1 μM bestatin and 1 μM phosphoramidon were included in the assay buffer to prevent metabolic inactivation of the peptide. Samples were made in disposable plastic reaction tubes (diameter: 0.7 cm, volume 10 ml; Sarstedt Co., Numbrecht, Germany). The time course of association was measured by incubating [3H]Neuromedin N with the membrane fractions for the indicated times. In dissociation experiments the radioligand was pre-incubated with the membranes until equilibrium achieved, and the dissociation of the receptor ligand complex was initiated by the addition of unlabeled Neuromedin N, and the dissociation of the radioligand was subsequently assessed for 45 min. Saturation binding experiments were performed with 18–517 nM [3 H]Neuromedin N in rat brain membranes. Nonspecific binding was determined in the presence of 10 μM unlabeled Neuromedin N. Reaction was terminated and bound and free radioligands were separated by rapid filtration under vacuum through Whatman GF/C (pre-soaked with the PEI for 45 min before washing) glass fiber filters by using a Brandel M24R Cell Harvester. Next, the filters were washed three times with 5 ml ice-cold Tris–HCl buffer (50 mM, pH 7.4). Dried filter disks were inserted into Ultima Gold™ scintillation fluid (Packard), then rapidly removed and placed into individual counting vials (transparent glass, Packard). Bound radioactivity was determined in a Packard Tricarb 2300TR Liquid Scintillation counter. 2.7. [35S]GTPγS binding assays Rat brain membrane and spinal cord fractions (~ 10 μg of protein/ sample) were incubated at 30 °C for 60 min in Tris–EGTA buffer (50 mM Tris–HCl, 1 mM EGTA, 3 mM MgCl2, 100 mM NaCl, pH 7.4) containing [35S]GTPγS (0.05 nM) and increasing concentrations (10−9 to 10−5 M) of the compounds tested in the presence of 30 μM GDP in a final volume of 1 ml. Total binding was measured in the absence of the test compounds, while non-specific binding was determined in the presence of 10 μM unlabeled GTPγS and subtracted from the total binding to calculate the specific binding. The reaction was started by addition of [35S]GTPγS and terminated by filtrating the samples through Whatman GF/B glass fiber filters. Filters were washed three times with ice-cold 50 mM Tris–HCl buffer (pH 7.4) using Brandel M24R Cell Harvester, then dried, and bound radioactivity was detected in Ultima Gold™ scintillation cocktail (Packard). Agonist-induced receptormediated G protein stimulation was given as a percentage of the specific [35S]GTPγS binding observed in the absence of receptor ligands (basal activity).
curves were fitted by non-linear regression using the one-site competition fitting option with no ligand depletion. Kinetic curves were fitted by non-linear regression analysis using ‘one-phase association’ option. G protein stimulation data were analyzed by the sigmoid dose–response curve fit option of Prism. Experimental data were analyzed and graphically processed by GraphPad Prism software package for research (version 4.00 for Windows, GraphPad Software, San Diego, California, USA, www. graphpad.com). 3. Results To determine appropriate conditions for equilibrium binding studies of [3H]NN to rat brain membrane preparations, the temperature dependence was first studied. The specific binding and specific/non-specific binding ratio of [3H]NN was best at room temperature, and therefore all subsequent binding experiments were conducted at 25 °C. Time-dependence of binding interaction was examined by incubating the rat brain membrane preparations at 25 °C with [3H]NN until reaching an equilibrium. Kinetic studies revealed rapid, monophasic association with pseudo-first order kinetics. The steady-state level of specific binding was achieved in 10–12 min (Fig. 1). Initial binding association was described by a half-life time of 1.833 min (95% CI 1.340 to 2.902). The pseudo-first-order rate constant (kobs) value was determined to be 0.3781 (min−1 nM−1 s−1). Saturation of [3H]NN binding to the rat brain membrane preparations was performed in the absence (total binding) and in the presence (nonspecific binding) of 10 μM unlabeled NN (Fig. 2A). In saturation binding experiments (Fig. 2A and B), performed on rat brain membranes at 25 °C binding isotherms of a representative assay, reveal a curvilinear plot for the total- and a linear plot for the nonspecific binding (Fig. 2A). Specific binding of [3H]NN, defined as the difference of the total and non-specific binding values, was saturable and described by a rectangular hyperbola (Fig. 2B). Non-linear regression analysis of the specific binding data reveals interactions with a single set of homogenous binding sites (R2 = 0.99). Consequently, a linear Scatchard plot was obtained (Fig. 2B). The equilibrium dissociation constant (Kd), which measures the affinity of a ligand for a receptor, and the maximal number of binding sites (Bmax) were calculated by non-linear regression analysis of the saturation binding data: the equilibrium dissociation constant, Kd was 264.8 ± 30.18 nM, while the maximal number of the binding sites (Bmax) was 3855 ± 215 fmol/mg protein in rat brain membranes. In displacement studies (Fig. 3A and B) the unlabelled NN displaced [3H]NN with a weak affinity both in rat brain and rat spinal cord membranes. A LogIC50 of 6.34 ± 0.06 (IC50 = 454 nM) was determined in rat brain tissue, whereas the LogIC50 was 6.37 ± 0.06 (IC50 = 425 nM) in rat spinal cord membranes. NT also displaced [3H]NN with a weak
2.8. Data analysis Radioligand binding experiments were performed in duplicate and the [35S]GTPγS binding assays were carried out in triplicate. Displacement
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Fig. 1. Kinetic studies with [3H]NN in rat brain membranes.
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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Fig. 2. Saturation analysis of [3H]NN in rat brain membranes at 25 °C.
affinity (LogIC50 of 6.06 ± 0.16 or IC50 = 866.3 nM in rat brain & LogIC50 = 6.06 ± 0.23 or IC50 = 876.7 nM in rat spinal cord membranes, respectively). Moreover, the effect of Na+ ions on specific [3H]NN binding (Fig. 4) was measured with NaCl concentrations ranging from 3 to 300 mM at 25 °C for 45 min using rat brain membrane preparations. Na+ ions had a very weak effect on the binding of [3H]NN (IC50 = 150.6 mM). In the present study, the [35S]GTPγS binding assay, reflecting interaction between ligand-activated receptors and regulatory G proteins, was used to study the agonist potency of the peptide. The stimulation of [35S]GTPγS binding to rat brain membranes and rat spinal cord membranes (Fig. 5A and Fig. 5B) was measured. NN showed agonist activities in stimulating regulatory G proteins. Maximal stimulation (Efficacy, Emax) was 112.3 ± 1.4% in rat brain and 112.9 ± 2.4% in rat spinal cord while potency values, logEC50 (EC50, nM) were 9.11 ± 0.39 (0.7 nM) in rat brain and 9.1 ± 0.46 (0.79 nM) in rat spinal cord. Stimulatory effect of NN could be maximally inhibited using the specific neurotensin receptor antagonist levocabastine (10 μM), but not by the specific opioid receptor antagonist naloxone (10 μM) or the NTS1 antagonist SR48692 (10 μM) both in rat brain and rat spinal cord membranes.
4. Discussion All the common properties (the four amino acids at the carboxy terminal are identical, same pharmacological properties, common precursor, the distribution of neuromedin N parallels that of neurotensin in rat brain) of NT and NN can be explained by the known ability of NN to behave as an agonist at the neurotensin receptors.
However some differences in the pharmacological effects of both peptides led to the suggestion of the possible existence of a NN receptor (Kalivas et al. 1986). In 1994 Gaudriault et al. (1994) attempted to identify this hypothetical receptor, by synthesizing a radiolabeled analog of NN (α- and ε-125I-BH-NN). Their results did not support the existence of a specific NN receptor in rat and mouse brain, supporting the existence of a common receptor for both peptides. In displacement studies we found that the NN and NT displaced [3H]NN with a weak affinity both in rat brain- and rat spinal cord membranes. As demonstrated, specific binding of [3H]NN was saturable, a result indicative of an interaction with a single set of homogenous binding sites in rat brain membranes. Previously, Gaudriault et al. (1994) also found that α- and ε-125I-BH-neuromedin N bound to newborn mouse brain homogenate according to a reversible and saturable process. The Scatchard representation of the binding data was linear, indicating that α- and ε-125I-BH-neuromedin N recognizes a single family of non-interacting binding site (Gaudriault et al. 1994). In addition, we found that the association of the ligand-receptor complex occurred rapidly and the steady-state level of specific binding was achieved in 10–12 min. Na+ ions had a very weak inhibitory effect on the binding of [3H]NN. Several G protein-coupled receptors are sensitive to Na+ ions, as they reduce their affinity for agonists (Ceresa and Limbird 1994). NTS1 bears the highly conserved Asp residue in the second TM domain and binding to NT is sensitive to Na+ ions and GTP analogs. In contrast, the NTS2 is characterized by the absence of the Asp residue in TM II, the corresponding position being occupied by an alanine. This substitution is responsible for the low sensitivity of the NTS2 receptor to Na+ ions. Site-directed mutagenesis experiments have shown that replacing the
Fig. 3. Displacement studies with [3H]NN in rat brain membranes (A) and in rat spinal cord (B). Effect of sodium ions on specific [3H]NN binding in rat brain membranes.
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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Previously, the functional interaction of the cloned rat NT receptor with intracellular G proteins was investigated by studying neurotensin-induced binding of the radiolabeled guanylyl nucleotide analog [35S]-GTP gamma S to membranes prepared from transfected Chinese hamster ovary (CHO) cells. The stimulation of [35S]-GTP gamma S binding by the neurotensin-related peptide neuromedin N showed an EC50 value of 21 +/− 6 nM (Hermans et al. 1997). In our studies NN showed moderate agonist activities in stimulating regulatory G proteins. We wanted to investigate whether this stimulatory effect could be blocked by different receptor specific antagonists: levocabastine is a potent and selective antagonist for the neurotensin receptor NTS2, and was the first drug used to characterize the different neurotensin subtypes (Kitabgi et al. 1987). SR48692 is a non-peptide neurotensin receptor antagonist, preferentially binding to the NTS1 receptor (Gully et al. 1993; Labbé-Jullié et al. 1998) and naloxone is an opioid receptor specific antagonist. Stimulatory effect of NN could be maximally inhibited using specific NTS2 levocabastine, but neither by the opioid receptor specific antagonist naloxone nor the NTS1 antagonist SR48692. 5. Conclusion Fig. 4. Effect of sodium ions on specific [3H]NN binding in rat brain membranes.
corresponding Ala79 by an Asp residue improves the sensitivity of the NTS2 to Na+ ions but does not restore its sensitivity to GTP (Martin et al. 1999).
Based on the results above, we conclude that in adult rat brain membranes [3H]neuromedin N preferentially labels NTS2 receptors. We base this assumption on the following observations: it has a low affinity for this receptor, levocabastine can inhibit the stimulatory effect of NN, whereas SR48692 cannot. These findings are important because NTS2-
Fig. 5. Stimulation of [35S] GTPγS in rat brain membranes (5.A.) and in rat spinal cord membranes (5.B).
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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acting NT analogs may constitute a promising avenue towards new nonopioid analgesic drugs. Conflict of interest The authors declare no conflict of interest. Association kinetics of [3H]NN binding determined at 25 °C. Membranes from rat brain were incubated with 10.75 nM [3 H]NN for various time intervals. Points represent means ± S.E.M. of at least three experiments, carried out in duplicates. Rat brain membranes were incubated for 45 min at 25 °C with increasing concentrations of [3H]NN in the absence (‘total binding’) or the presence of 10 μM NN (‘non-specific’ binding). Specific binding was calculated as the difference of the above mentioned values. Real concentrations of the radioligand were determined by counting the radioactivity of aliquot drops directly taken from the radioligand dilution series. (2.A) Isotherms for total and non-specific binding in a single experiment performed in triplicate. (2.B) Concentration dependence of [3H]NN specific binding. The corresponding Scatchard plot is given as an insert. Points represent the means ± S.E.M. of several independent experiments carried out in duplicates. Rat brain or spinal cord membranes were incubated for 45 min at 25 °C with 10–11 nM [3 H]NN in the presence of increasing concentrations of cold NN and NT. IC50 values were calculated by fitting displacement binding curves using Graphpad Prism program non-linear leastsquares algorithm. Points represent the mean ± S.E.M. of three independent experiments performed in duplicate. Experiments were conducted at 25 °C for 45 min using rat brain membrane preparations. The effect of Na+ ions on [3H]NN binding was measured with NaCl concentrations ranging from 3 to 300 mM. Values represent the mean ± S.E.M. of three independent experiments performed in duplicate. Stimulation of [35S]GTPγS binding to rat brain or rat spinal cord membranes by various concentrations of unlabeled NN and NT as a control. Possible inhibition of agonist stimulated [35S]GTPγS binding by various selective antagonists was also carried out both in rat brain and rat spinal cord: NX: Naloxone (10 μM), levocabastine (10 μM) and SR 48692 (10 μM). Stimulation is given as a percentage of the non-stimulated (basal) level. Incubations were carried out for 60 min at 30 °C. Points represent means ± S.E.M. from several independent experiments, carried out in triplicates. Acknowledgements We would like to thank Katalin Horvath for the technical assistance. This work was supported by: an OTKA CK-78566 grant from the National Scientific Research Fund, Budapest, Hungary. References Barelli, H., Woskowska, Z., Cipris, S., Fox-Threlkeld, J.E., Daniel, E.E., Vincent, J.P., Checler, F., 1995. Pharmacological role and degradation processes of neuromedin N in the gastrointestinal tract: an in vitro and in vivo study. J. Pharmacol. Exp. Ther. 275, 1300–1307. Benyhe, S., Farkas, J., Tóth, G., Wollemann, M., 1997. Met5-enkephalin-Arg6-Phe7, an endogenous neuropeptide, binds to multiple opioid and non-opioid sites in rat brain. J. Neurosci. Res. 48, 249–258. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Ceresa, B.P., Limbird, L.E., 1994. Mutation of an aspartate residue highly conserved among G-protein-coupled receptors results in nonreciprocal disruption of α2-adrenergic receptor–G-protein interactions. J. Biol. Chem. 269, 29557–29564.
Chalon, P., Vita, N., Kaghad, M., Guillemot, M., Bonnin, J., Delpech, B., Le Fur, G., Ferrara, P., Caput, D., 1996. Molecular cloning of a levocabastine-sensitive neurotensin binding site. FEBS Lett. 386, 91–94. Checler, F., Vincent, J.P., Kitabgi, P., 1986. Neuromedin N: high affinity interaction with brain neurotensin receptors and rapid inactivation by brain synaptic peptidases. Eur. J. Pharmacol. 126, 239–244. Coquerel, A., Dubuc, I., Kitabgi, P., Costentin, J., 1988. Potentiation by thiorphan and bestatin of the naloxone-insensitive analgesic effects of neurotensin and neuromedin N. Neurochem. Int. 12, 361–366. Dobner, P.R., Barber, D.L., Villa-Komaroff, L., McKiernan, C., 1987. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor. Proc. Natl. Acad. Sci. U. S. A. 84, 3516–3520. Dubuc, I., Nouel, D., Coquerel, A., Menard, J.F., Kitabgi, P., Costentin, J., 1988. Hypothermic effect of neuromedin N in mice and its potentiation by peptidase inhibitors. Eur. J. Pharmacol. 151, 117–121. Gaudriault, G., Zsürger, N., Vincent, J.P., 1994. Compared binding properties of 125Ilabeled analogues of neurotensin and neuromedin N in rat and mouse brain. J. Neurochem. 62, 361–368. Gendron, L., Perron, A., Payet, M.D., Gallo-Payet, N., Sarret, P., Beaudet, A., 2004. Lowaffinity neurotensin receptor (NTS2) signaling: internalization-dependent activation of extracellular signal-regulated kinases ½. Mol. Pharmacol. 66, 1421–1430. Gully, D., Canton, M., Boigegrain, R., Jeanjean, F., Molimard, J.C., Poncelet, M., Gueudet, C., Heaulme, M., Leyris, R., Brouard, A., 1993. Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor. Proc. Natl. Acad. Sci. U. S. A. 90, 65–69. Hermans, E., Geurts, M., Maloteaux, J.M., 1997. Agonist and antagonist modulation of [35S]-GTP gamma S binding in transfected CHO cells expressing the neurotensin receptor. Br. J. Pharmacol. 121, 1817–1823. Jacobsen, L., Madsen, P., Jacobsen, C., Nielsen, M.S., Gliemann, J., Petersen, C.M., 2001. Activation and functional characterization of the mosaic receptor SorLA/LR11. J. Biol. Chem. 276, 22788–22796. Kalivas, P.W., Richardson-Carlson, R., Duffy, P., 1986. Neuromedin N mimics the actions of neurotensin in the ventral tegmental area but not in the nucleus accumbens. J. Pharmacol. Exp. Ther. 238, 1126–1131. Kislauskis, E., Bullock, B., McNeil, S., Dobner, P.R., 1988. The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences. J. Biol. Chem. 263, 4963–4968. Kitabgi, P., 2006. Inactivation of neurotensin and neuromedin N by Zn metallopeptidases. Peptides 27, 2515–2522. Kitabgi, P., 2010. Neurotensin and neuromedin N are differentially processed from a common precursor by prohormone convertases in tissues and cell lines. Results Probl. Cell Differ. 50, 85–96. Kitabgi, P., Rostène, W., Dussaillant, M., Schotte, A., Laduron, P.M., Vincent, J.P., 1987. Two populations of neurotensin binding sites in murine brain: discrimination by the antihistamine levocabastine reveals markedly different radioautographic distribution. Eur. J. Pharmacol. 140, 285–293. Labbé-Jullié, C., Barroso, S., Nicolas-Etève, D., Reversat, J.L., Botto, J.M., Mazella, J., Bernassau, J.M., Kitabgi, P., 1998. Mutagenesis and modeling of the neurotensin receptor NTR1. Identification of residues that are critical for binding SR 48692, a nonpeptide neurotensin antagonist. J. Biol. Chem. 273, 16351–16357. Lambert, P.D., Gross, R., Nemeroff, C.B., Kilts, C.D., 1995. Anatomy and mechanisms of neurotensin–dopamine interactions in the central nervous system. Ann. N. Y. Acad. Sci. 757, 377–389. Martin, S., Botto, J.M., Vincent, J.P., Mazella, J., 1999. Pivotal role of an aspartate residue in sodium sensitivity and coupling to G proteins of neurotensin receptors. Mol. Pharmacol. 55, 210–215. Mazella, J., Zsurger, N., Navarro, V., Chabry, J., Kaghad, M., Caput, D., Ferrara, P., Vita, N., Gully, D., Maffrand, J.P., Vincent, J.P., 1998. The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor. J. Biol. Chem. 273, 26273–26276. Minamino, N., Kangawa, K., Matsuo, H., 1984. Neuromedin N: a novel neurotensin-like peptide identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 122, 542–549. Rompre, P.P., Gratton, A., 1992. A comparison of the effects of mesencephalic injections of neurotensin (1–13) and neuromedin on brain electrical self stimulation. Peptides 13, 713–719. Schotte, A., Laduron, P.M., 1987. Different postnatal ontogeny of two [3 H]neurotensin binding sites in rat brain. Brain Res. 408, 326–328. Sumi, S., Inoue, K., Kogire, M., Doi, R., Takaori, K., Yajima, H., Suzuki, T., Tobe, T., 1987. Effect of synthetic neuromedin-N, a novel neurotensin-like peptide, on exocrine pancreatic secretion and splanchnic blood flow in dogs. Neuropeptides 9, 247–255. Tóth, G., Mallareddy, J.R., Tóth, F., Lipkowski, A.W., Tourwé, D., 2012. Radiotracers, tritium labelling of neuropeptides. ARKIVOC 5, 163–174. Vincent, J.P., Mazella, J., Kitabgi, P., 1999. Neurotensin and neurotensin receptors. Trends Pharmacol. Sci. 20, 302–309. Vita, N., Laurent, P., Lefort, S., Chalon, P., Dumont, X., Kaghad, M., Gully, D., Le Fur, G., Ferrara, P., Caput, D., 1993. Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor. FEBS Lett. 317, 139–142.
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand Fanni Tóth a, Jayapal Reddy Mallareddy a, Dirk Tourwé b, Andrzej W. Lipkowski c, Magdalena Bujalska-Zadrozny d, Sándor Benyhe a, Steven Ballet b, Géza Tóth a, Patrycja Kleczkowska c,d,⁎ a
Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt 62, 6726 Szeged, Hungary Research Group of Organic Chemistry, Department of Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Department of Neuropeptides, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Street, 02106 Warsaw, Poland d Department of Pharmacodynamics, Centre for Preclinical Research and Technology, Medical University of Warsaw, 1B Banacha Str., 02-106 Warsaw, Poland b c
a r t i c l e
i n f o
Article history: Received 20 August 2015 Received in revised form 25 November 2015 Accepted 7 December 2015 Available online xxxx Keywords: Neuromedin N Receptor binding assays NTS2 Rat
a b s t r a c t Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [3H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454 nM in rat brain and 425 nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8 ± 30.18 nM, while the Bmax value corresponded to 3.8 ± 0.2 pmol/mg protein in rat brain membranes. The specific binding of [3H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na+ ions was observed on the binding of [3H]NN, resulting in an IC50 of 150.6 mM. In [35S]GTPγS binding experiments the Emax value was 112.3 ± 1.4% in rat brain and 112.9 ± 2.4% in rat spinal cord membranes and EC50 values of 0.7 nM and 0.79 nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [3H]NN labels NTS2 receptors in rat brain membranes. © 2015 Published by Elsevier Ltd.
1. Introduction The neurotensin-like peptide, neuromedin N (NN), was first isolated from porcine spinal cord membranes (Minamino et al. 1984). The amino acid sequence of NN, H-Lys-Ile-Pro-Tyr-Ile-Leu-OH, is homologous to
Abbreviations: BH, Bolton–Hunter; Bmax, maximal number of the binding sites; BSA, bovine serum albumin; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; EC50, half maximal effective concentration; Emax, maximal stimulation (Efficacy); GDP, guanosine 5′-diphosphate; [35S]GTPγS, guanosine-5′-[γ-35S]-triphosphate; HOBt, 1hydroxybenzotriazole; HPLC, high performance liquid chromatography; IC50, half-maximal inhibitory concentration; Kd, equilibrium dissociation constant; kobs, pseudo-first-order rate constant; levocabastine, (3S,4R)-1-[4-cyano-4-(4-fluorophenyl)cyclohexyl]-3-methyl-4phenylpiperidine-4-carboxylic acid; SR 48692, 2-([1-(7-chloro-4-quinolinyl)-5-(2,6dimethoxyphenyl)-1 H-pyrazole-3-carbonyl]amino)admantane-2-carboxylic acid; NN, neuromedin N; NT, neurotensin; NTS1, neurotensin receptor 1; NTS2, neurotensin receptor 2; PCs, proprotein convertases; PEI, polyethyleneimine; SPPS, solid phase peptide synthesis; TBTU, N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate; TEA, triethylamine; TFA, trifluoroacetic acid. ⁎ Corresponding author at: Department of Pharmacodynamics, Centre for Preclinical Research and Technology, Medical University of Warsaw, 1B Banacha Str., 02-091 Warsaw, Poland. E-mail address: hazufi[email protected] (P. Kleczkowska).
that of neurotensin (NT) (8–13), H-Arg-Arg-Pro-Tyr-Ile-Leu-OH (Minamino et al. 1984), which is the essential part of the tridecapeptide NT for biological activity (Lambert et al. 1995). NT and NN are synthesized from a common precursor (pro-NT/NN) in mammalian brain tissues (Kislauskis et al. 1988) and intestines (Dobner et al. 1987). Both sequences are located in the C-terminal part of the proNT/NN precursor and they are flanked by Lys-Arg residues, which are the known consensus sequences for cleavage by specific endoproteases that belong to the proprotein convertase (PC) family. Processing of the precursor generates NT and NN or a large peptide ending with the NT sequence in brain, gut or adrenals (Kitabgi 2010). The biological effects mediated by NT and NN, such as naloxoneinsensitive analgesia or hypothermia, result from the interaction of these peptides with specific G protein-coupled receptors (GPCRs) (Coquerel et al. 1988; Dubuc et al. 1988). It has been reported that NN displayed antinociceptive properties after intracerebroventricular (i.c.v.) injection in rat writhing and tail-flick tests. Compared to NT, the corresponding NN-induced effects required higher doses. The fact that naloxone did not significantly reduce these antinociceptive effects indicates that both NT and NN elicit analgesia in mice through an opioid independent mechanism (Coquerel et al.
http://dx.doi.org/10.1016/j.npep.2015.12.004 0143-4179/© 2015 Published by Elsevier Ltd.
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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1988). Additionally, intracerebroventricular administration of NN elicited dose- and time-dependent hypothermia in mice, an effect that was significantly increased by bestatin (Dubuc et al. 1988). NN also has an influence on digestive organs: NN induces increases of the blood flow sin superior mesenteric artery, portal vein and pancreatic tissue, and increase of pancreatic juice volume and pancreatic protein output, and that it causes a fall in systematic arterial pressure (Sumi et al. 1987). NN degradation is 3 to 20-fold faster than that of NT, and was totally inhibited by 1,10-phenanthroline and markedly inhibited by bestatin (Checler et al. 1986; Kitabgi 2006). In general, NN is less potent in binding and functional assays than NT, which was ascribed to the higher sensitivity of NN to enzymatic degradation (Rompre and Gratton 1992). Previous reports indicate that the Zn metallo-exopeptidase aminopeptidase M is largely responsible for NN degradation in vitro and in vivo in the gastrointestinal tract (Barelli et al. 1995; Kitabgi 2006). Two types of neurotensin receptors, belonging to the seven transmembrane domain GPCRs, and previously distinguished by their affinity for neurotensin and their sensitivity to the antihistaminic drug levocabastine (Schotte and Laduron 1987), were cloned. The highaffinity, levocabastine-insensitive, neurotensin receptor NTS1 was the first to be cloned (Vita et al. 1993), followed in 1996 by the lowaffinity levocabastine-sensitive neurotensin receptor NTS2 (Chalon et al. 1996). A third and fourth neurotensin subtype receptor have subsequently been reported. NTS3 was identified as gp95/sortilin (Mazella et al. 1998; Vincent et al. 1999) and NTS4 is a mosaic protein (Jacobsen et al. 2001). NTS3 and NTS4 are non G protein-coupled single transmembrane domain sorting receptors. NN competitively inhibited specific 125I-NT (2 nM) binding to CHO/rNTS2 cells with IC50 values of 21 nM, acting as an agonist at the NTS2 site (Gendron et al. 2004). In order to perform direct ligand-binding studies with the NN hexapeptide, the tritium labeled peptide Lys-Ile-3HPro-Tyr-Ile-Leu-OH was prepared (Tóth et al. 2012). Herein we report the synthesis, radiolabeling and binding characteristics of [3H]Neuromedin N, as well as the G protein activation of the cold ligand.
Pharmaceutica (Beerse, Belgium) and SR 48692: 2-([1-(7-chloro-4quinolinyl)-5-(2,6-dimethoxyphenyl)-1 H-pyrazole-3-carbonyl]amino)admantane-2-carboxylic acid was obtained from Axon Medchem (Groningen, The Netherlands). 2.3. Peptide synthesis Synthesis of neurotensin (8–13), neuromedin N (H-Lys-Ile-Pro-TyrIle-Leu-OH), and H-Lys-Ile-ΔPro-Tyr-Ile-Leu-OH was performed manually using standard Fmoc-based solid phase peptide synthesis (SPPS) on Fmoc-Leu-Wang resin. For these syntheses a reaction vessel under nitrogen bubbling was used. The protocols for amino acid dissolution, activation and coupling time, were performed according to the manufacturer's instruction. The couplings were performed for 2 h by adding protected amino acids (3 equiv.) and TBTU (3 equiv.) in the presence of HOBt (3 equiv.) and DIPEA (6 equiv.) in DMF to the resin. The resin was washed with DMF between all reaction steps. Completion of the coupling was monitored by the Kaiser test. Removal of the Fmoc protecting group from the N-terminus of the resin-bound peptide chain was achieved by treating the peptidyl resin with a 20% piperidine in DMF solution. The support was again washed with DMF and the next coupling was performed. The final hexapeptides were cleaved from the resin by treatment with trifluoroacetic acid (99%) for 2 h followed by evaporation, precipitation in cold Et2O and HPLC purification. The purification of the peptides was performed on a Shimadzu apparatus and controlled with the software package Class-VP, 5.0 (Kyoto Japan) using a C8 column (20 × 250 mm, KROMASIL) in 0.1% TFA/water and a gradient of 3–97% acetonitrile in 0.1% TFA/water, with a flow rate of 6 ml/min and using UV detection at 280 nm (Shimadzu Diode Array Detector SPD-M10 AVP). A final purity of N99% was obtained. 2.4. Preparation of 3 H-neuromedin N (Lys-Ile-3HPro-Tyr-Ile-Leu-OH)
Inbred male Wistar rats (250–300 g body weight) were housed in the local animal house of the Biological Research Center (BRC, Szeged, Hungary). Rats were kept in groups of four, allowed free access to food and water, and maintained on a 12:12-h light/dark cycle until the time of sacrifice. Animals were treated according to the European Communities Council Directives (86/609/ECC) and the Hungarian Act for the Protection of Animals in Research (XXVIII.tv. 32.§).
In the reaction vessel 3 mg of Lys-Ile-Pro-Tyr-Ile-Leu-OH precursor was dissolved in DMF and 10 mg of PdO/BaSO4 catalyst and 1 μl of TEA were added to the precursor solution (Tóth et al. 2012). Tritium gas (370 GBq(10 Ci)) was introduced to the reaction mixture which was further stirred by a magnetic stirrer for 1 h at room temperature. The catalyst was filtered through a Whatmann GF/C glass fiber filter and washed several times with ethanol. The labile tritium was removed by repeated evaporation of an ethanol-water (1:1) mixture. The total activity was 1.75 GBq (47.3 mCi). The crude peptide was purified by HPLC using an analytical column (Atlantis) and a linear gradient of the elution system from 20% up to 40% acetonitrile in water with 1% TFA over 25 min at a flow rate of 1 ml/min and detection at 216 nm. The retention time corresponded to 10.0 min. [3 H]Neuromedin N exhibited a specific radioactivity of 0.6 TBq/mmol (16 Ci/mmol) and a purity of N98%. The radioligand was stored in a refrigerator at −80 °C.
2.2. Chemicals
2.5. Rat brain and spinal cord membrane preparation
The peptide synthesis reagents N,N-diisopropylathylamine (DIPEA), 1-hydroxybenzotriazole hydrate (HOBt), as well as N,N,N′,N′tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU), Fmoc-protected amino acids and Fmoc-Leu-Wang resin were obtained from IRIS Biotech GmbH (Marktredwitz, Germany) and Sigma Aldrich (Poznan, Poland), respectively. Guanosine-5′-[γ-35S]-triphosphate (1204 Ci/mmol) was obtained from the Isotope Institute Ltd. (Budapest, Hungary). MgCl2, NaCl, EGTA, polyethyleneimine (PEI), tris(hydroxymethyl)aminomethane (Tris), protease-free bovine serum albumin (protease-free BSA, fraction V), captopril, bestatin and phosphoramidon, guanosine 5′-diphosphate sodium salt (GDP), guanosine-5′[γ-thio]-triphosphate tetra-lithium salt (GTPγS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Levocabastine hydrochloride, (3S,4R)-1-[4-cyano-4-(4-fluorophenyl)cyclohexyl]-3-methyl-4phenylpiperidine-4 carboxylic acid, was kindly provided by Janssen
A crude membrane fraction was prepared from Wistar rat brains and spinal cords according to a method reported by Pasternak et al. with small modifications (Benyhe et al. 1997). Two- to three-month old animals were decapitated, and the brains without cerebella were rapidly removed, and washed several times with chilled 50 mM Tris–HCl buffer (pH 7.4) or in the case of the spinal cord: decapitated and the vertebral foramen was surgically opened at the lumbar level between vertebrae L4–L5. The spinal cords were rapidly removed by pneumatic pressure using a 100-ml plastic syringe with a blunt steel needle, and washed several times with chilled 50 mM Tris– HCl buffer (pH 7.4). The brains and spinal cords were then weighed and suspended in 5% vol/wt of brain or spinal cord tissue of the icecold buffer. Tissues were homogenized using a Braun teflon-glass homogenizer (1000 rpm, 10–15 strokes), and filtered through four layers of gauze to remove large aggregates. Additional buffer was added to
2. Material and methods 2.1. Animals
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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reach a final buffer volume/membrane pellet ratio of 30 (ml/g). After centrifugation with a Sorvall RC5C centrifuge (40.000 × g, at 4 °C, for 20 min), the resulting pellet was re-suspended in fresh buffer (30% vol/wt) by using a vortex. The suspension was incubated at 37 °C for 30 min to remove any endogenous opioids. Centrifugation was repeated under the same conditions as described above, and the final pellet was resuspended in five volumes of 50 mM Tris–HCl buffer (pH 7.4) containing 0.32 M sucrose to give a final protein concentration of 3–4 mg/ml. The membrane samples were stored in 5 ml aliquots at −70 °C. The protein concentration was determined by the Bradford method (Bradford 1976). Before use the membranes were thawed and re-suspended in 50 mM Tris–HCl buffer (pH 7.4) and centrifuged (40.000 × g, at 4 °C, for 20 min) to remove sucrose and used immediately in binding assays. 2.6. Receptor binding assays All binding assays were performed at 25 °C for 45 min in 50 mM Tris– HCl buffer (pH 7.4) in a final volume of 1 ml, containing 1 mg BSA and 0.2–0.4 mg/ml (rat brain or spinal cord) protein. Three peptidase inhibitors: 1 μM captopril, 1 μM bestatin and 1 μM phosphoramidon were included in the assay buffer to prevent metabolic inactivation of the peptide. Samples were made in disposable plastic reaction tubes (diameter: 0.7 cm, volume 10 ml; Sarstedt Co., Numbrecht, Germany). The time course of association was measured by incubating [3H]Neuromedin N with the membrane fractions for the indicated times. In dissociation experiments the radioligand was pre-incubated with the membranes until equilibrium achieved, and the dissociation of the receptor ligand complex was initiated by the addition of unlabeled Neuromedin N, and the dissociation of the radioligand was subsequently assessed for 45 min. Saturation binding experiments were performed with 18–517 nM [3 H]Neuromedin N in rat brain membranes. Nonspecific binding was determined in the presence of 10 μM unlabeled Neuromedin N. Reaction was terminated and bound and free radioligands were separated by rapid filtration under vacuum through Whatman GF/C (pre-soaked with the PEI for 45 min before washing) glass fiber filters by using a Brandel M24R Cell Harvester. Next, the filters were washed three times with 5 ml ice-cold Tris–HCl buffer (50 mM, pH 7.4). Dried filter disks were inserted into Ultima Gold™ scintillation fluid (Packard), then rapidly removed and placed into individual counting vials (transparent glass, Packard). Bound radioactivity was determined in a Packard Tricarb 2300TR Liquid Scintillation counter. 2.7. [35S]GTPγS binding assays Rat brain membrane and spinal cord fractions (~ 10 μg of protein/ sample) were incubated at 30 °C for 60 min in Tris–EGTA buffer (50 mM Tris–HCl, 1 mM EGTA, 3 mM MgCl2, 100 mM NaCl, pH 7.4) containing [35S]GTPγS (0.05 nM) and increasing concentrations (10−9 to 10−5 M) of the compounds tested in the presence of 30 μM GDP in a final volume of 1 ml. Total binding was measured in the absence of the test compounds, while non-specific binding was determined in the presence of 10 μM unlabeled GTPγS and subtracted from the total binding to calculate the specific binding. The reaction was started by addition of [35S]GTPγS and terminated by filtrating the samples through Whatman GF/B glass fiber filters. Filters were washed three times with ice-cold 50 mM Tris–HCl buffer (pH 7.4) using Brandel M24R Cell Harvester, then dried, and bound radioactivity was detected in Ultima Gold™ scintillation cocktail (Packard). Agonist-induced receptormediated G protein stimulation was given as a percentage of the specific [35S]GTPγS binding observed in the absence of receptor ligands (basal activity).
curves were fitted by non-linear regression using the one-site competition fitting option with no ligand depletion. Kinetic curves were fitted by non-linear regression analysis using ‘one-phase association’ option. G protein stimulation data were analyzed by the sigmoid dose–response curve fit option of Prism. Experimental data were analyzed and graphically processed by GraphPad Prism software package for research (version 4.00 for Windows, GraphPad Software, San Diego, California, USA, www. graphpad.com). 3. Results To determine appropriate conditions for equilibrium binding studies of [3H]NN to rat brain membrane preparations, the temperature dependence was first studied. The specific binding and specific/non-specific binding ratio of [3H]NN was best at room temperature, and therefore all subsequent binding experiments were conducted at 25 °C. Time-dependence of binding interaction was examined by incubating the rat brain membrane preparations at 25 °C with [3H]NN until reaching an equilibrium. Kinetic studies revealed rapid, monophasic association with pseudo-first order kinetics. The steady-state level of specific binding was achieved in 10–12 min (Fig. 1). Initial binding association was described by a half-life time of 1.833 min (95% CI 1.340 to 2.902). The pseudo-first-order rate constant (kobs) value was determined to be 0.3781 (min−1 nM−1 s−1). Saturation of [3H]NN binding to the rat brain membrane preparations was performed in the absence (total binding) and in the presence (nonspecific binding) of 10 μM unlabeled NN (Fig. 2A). In saturation binding experiments (Fig. 2A and B), performed on rat brain membranes at 25 °C binding isotherms of a representative assay, reveal a curvilinear plot for the total- and a linear plot for the nonspecific binding (Fig. 2A). Specific binding of [3H]NN, defined as the difference of the total and non-specific binding values, was saturable and described by a rectangular hyperbola (Fig. 2B). Non-linear regression analysis of the specific binding data reveals interactions with a single set of homogenous binding sites (R2 = 0.99). Consequently, a linear Scatchard plot was obtained (Fig. 2B). The equilibrium dissociation constant (Kd), which measures the affinity of a ligand for a receptor, and the maximal number of binding sites (Bmax) were calculated by non-linear regression analysis of the saturation binding data: the equilibrium dissociation constant, Kd was 264.8 ± 30.18 nM, while the maximal number of the binding sites (Bmax) was 3855 ± 215 fmol/mg protein in rat brain membranes. In displacement studies (Fig. 3A and B) the unlabelled NN displaced [3H]NN with a weak affinity both in rat brain and rat spinal cord membranes. A LogIC50 of 6.34 ± 0.06 (IC50 = 454 nM) was determined in rat brain tissue, whereas the LogIC50 was 6.37 ± 0.06 (IC50 = 425 nM) in rat spinal cord membranes. NT also displaced [3H]NN with a weak
2.8. Data analysis Radioligand binding experiments were performed in duplicate and the [35S]GTPγS binding assays were carried out in triplicate. Displacement
3
Fig. 1. Kinetic studies with [3H]NN in rat brain membranes.
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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Fig. 2. Saturation analysis of [3H]NN in rat brain membranes at 25 °C.
affinity (LogIC50 of 6.06 ± 0.16 or IC50 = 866.3 nM in rat brain & LogIC50 = 6.06 ± 0.23 or IC50 = 876.7 nM in rat spinal cord membranes, respectively). Moreover, the effect of Na+ ions on specific [3H]NN binding (Fig. 4) was measured with NaCl concentrations ranging from 3 to 300 mM at 25 °C for 45 min using rat brain membrane preparations. Na+ ions had a very weak effect on the binding of [3H]NN (IC50 = 150.6 mM). In the present study, the [35S]GTPγS binding assay, reflecting interaction between ligand-activated receptors and regulatory G proteins, was used to study the agonist potency of the peptide. The stimulation of [35S]GTPγS binding to rat brain membranes and rat spinal cord membranes (Fig. 5A and Fig. 5B) was measured. NN showed agonist activities in stimulating regulatory G proteins. Maximal stimulation (Efficacy, Emax) was 112.3 ± 1.4% in rat brain and 112.9 ± 2.4% in rat spinal cord while potency values, logEC50 (EC50, nM) were 9.11 ± 0.39 (0.7 nM) in rat brain and 9.1 ± 0.46 (0.79 nM) in rat spinal cord. Stimulatory effect of NN could be maximally inhibited using the specific neurotensin receptor antagonist levocabastine (10 μM), but not by the specific opioid receptor antagonist naloxone (10 μM) or the NTS1 antagonist SR48692 (10 μM) both in rat brain and rat spinal cord membranes.
4. Discussion All the common properties (the four amino acids at the carboxy terminal are identical, same pharmacological properties, common precursor, the distribution of neuromedin N parallels that of neurotensin in rat brain) of NT and NN can be explained by the known ability of NN to behave as an agonist at the neurotensin receptors.
However some differences in the pharmacological effects of both peptides led to the suggestion of the possible existence of a NN receptor (Kalivas et al. 1986). In 1994 Gaudriault et al. (1994) attempted to identify this hypothetical receptor, by synthesizing a radiolabeled analog of NN (α- and ε-125I-BH-NN). Their results did not support the existence of a specific NN receptor in rat and mouse brain, supporting the existence of a common receptor for both peptides. In displacement studies we found that the NN and NT displaced [3H]NN with a weak affinity both in rat brain- and rat spinal cord membranes. As demonstrated, specific binding of [3H]NN was saturable, a result indicative of an interaction with a single set of homogenous binding sites in rat brain membranes. Previously, Gaudriault et al. (1994) also found that α- and ε-125I-BH-neuromedin N bound to newborn mouse brain homogenate according to a reversible and saturable process. The Scatchard representation of the binding data was linear, indicating that α- and ε-125I-BH-neuromedin N recognizes a single family of non-interacting binding site (Gaudriault et al. 1994). In addition, we found that the association of the ligand-receptor complex occurred rapidly and the steady-state level of specific binding was achieved in 10–12 min. Na+ ions had a very weak inhibitory effect on the binding of [3H]NN. Several G protein-coupled receptors are sensitive to Na+ ions, as they reduce their affinity for agonists (Ceresa and Limbird 1994). NTS1 bears the highly conserved Asp residue in the second TM domain and binding to NT is sensitive to Na+ ions and GTP analogs. In contrast, the NTS2 is characterized by the absence of the Asp residue in TM II, the corresponding position being occupied by an alanine. This substitution is responsible for the low sensitivity of the NTS2 receptor to Na+ ions. Site-directed mutagenesis experiments have shown that replacing the
Fig. 3. Displacement studies with [3H]NN in rat brain membranes (A) and in rat spinal cord (B). Effect of sodium ions on specific [3H]NN binding in rat brain membranes.
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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Previously, the functional interaction of the cloned rat NT receptor with intracellular G proteins was investigated by studying neurotensin-induced binding of the radiolabeled guanylyl nucleotide analog [35S]-GTP gamma S to membranes prepared from transfected Chinese hamster ovary (CHO) cells. The stimulation of [35S]-GTP gamma S binding by the neurotensin-related peptide neuromedin N showed an EC50 value of 21 +/− 6 nM (Hermans et al. 1997). In our studies NN showed moderate agonist activities in stimulating regulatory G proteins. We wanted to investigate whether this stimulatory effect could be blocked by different receptor specific antagonists: levocabastine is a potent and selective antagonist for the neurotensin receptor NTS2, and was the first drug used to characterize the different neurotensin subtypes (Kitabgi et al. 1987). SR48692 is a non-peptide neurotensin receptor antagonist, preferentially binding to the NTS1 receptor (Gully et al. 1993; Labbé-Jullié et al. 1998) and naloxone is an opioid receptor specific antagonist. Stimulatory effect of NN could be maximally inhibited using specific NTS2 levocabastine, but neither by the opioid receptor specific antagonist naloxone nor the NTS1 antagonist SR48692. 5. Conclusion Fig. 4. Effect of sodium ions on specific [3H]NN binding in rat brain membranes.
corresponding Ala79 by an Asp residue improves the sensitivity of the NTS2 to Na+ ions but does not restore its sensitivity to GTP (Martin et al. 1999).
Based on the results above, we conclude that in adult rat brain membranes [3H]neuromedin N preferentially labels NTS2 receptors. We base this assumption on the following observations: it has a low affinity for this receptor, levocabastine can inhibit the stimulatory effect of NN, whereas SR48692 cannot. These findings are important because NTS2-
Fig. 5. Stimulation of [35S] GTPγS in rat brain membranes (5.A.) and in rat spinal cord membranes (5.B).
Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004
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acting NT analogs may constitute a promising avenue towards new nonopioid analgesic drugs. Conflict of interest The authors declare no conflict of interest. Association kinetics of [3H]NN binding determined at 25 °C. Membranes from rat brain were incubated with 10.75 nM [3 H]NN for various time intervals. Points represent means ± S.E.M. of at least three experiments, carried out in duplicates. Rat brain membranes were incubated for 45 min at 25 °C with increasing concentrations of [3H]NN in the absence (‘total binding’) or the presence of 10 μM NN (‘non-specific’ binding). Specific binding was calculated as the difference of the above mentioned values. Real concentrations of the radioligand were determined by counting the radioactivity of aliquot drops directly taken from the radioligand dilution series. (2.A) Isotherms for total and non-specific binding in a single experiment performed in triplicate. (2.B) Concentration dependence of [3H]NN specific binding. The corresponding Scatchard plot is given as an insert. Points represent the means ± S.E.M. of several independent experiments carried out in duplicates. Rat brain or spinal cord membranes were incubated for 45 min at 25 °C with 10–11 nM [3 H]NN in the presence of increasing concentrations of cold NN and NT. IC50 values were calculated by fitting displacement binding curves using Graphpad Prism program non-linear leastsquares algorithm. Points represent the mean ± S.E.M. of three independent experiments performed in duplicate. Experiments were conducted at 25 °C for 45 min using rat brain membrane preparations. The effect of Na+ ions on [3H]NN binding was measured with NaCl concentrations ranging from 3 to 300 mM. Values represent the mean ± S.E.M. of three independent experiments performed in duplicate. Stimulation of [35S]GTPγS binding to rat brain or rat spinal cord membranes by various concentrations of unlabeled NN and NT as a control. Possible inhibition of agonist stimulated [35S]GTPγS binding by various selective antagonists was also carried out both in rat brain and rat spinal cord: NX: Naloxone (10 μM), levocabastine (10 μM) and SR 48692 (10 μM). Stimulation is given as a percentage of the non-stimulated (basal) level. Incubations were carried out for 60 min at 30 °C. Points represent means ± S.E.M. from several independent experiments, carried out in triplicates. Acknowledgements We would like to thank Katalin Horvath for the technical assistance. This work was supported by: an OTKA CK-78566 grant from the National Scientific Research Fund, Budapest, Hungary. References Barelli, H., Woskowska, Z., Cipris, S., Fox-Threlkeld, J.E., Daniel, E.E., Vincent, J.P., Checler, F., 1995. Pharmacological role and degradation processes of neuromedin N in the gastrointestinal tract: an in vitro and in vivo study. J. Pharmacol. Exp. Ther. 275, 1300–1307. Benyhe, S., Farkas, J., Tóth, G., Wollemann, M., 1997. Met5-enkephalin-Arg6-Phe7, an endogenous neuropeptide, binds to multiple opioid and non-opioid sites in rat brain. J. Neurosci. Res. 48, 249–258. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Ceresa, B.P., Limbird, L.E., 1994. Mutation of an aspartate residue highly conserved among G-protein-coupled receptors results in nonreciprocal disruption of α2-adrenergic receptor–G-protein interactions. J. Biol. Chem. 269, 29557–29564.
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Please cite this article as: Tóth, F., et al., Synthesis and binding characteristics of [3H]neuromedin N, a NTS2 receptor ligand, Neuropeptides (2015), http://dx.doi.org/10.1016/j.npep.2015.12.004