Simonin et al. 10.1073/pnas.0502090103. |
Fig. 5. Assessment of RF9 selectivity on opioid and RFamide receptors. Membranes from heterologous cells expressing the different receptors were labeled with selective radiolabeled ligands and three concentrations of RF9 (0.1, 1, and 10 mM) were tested in competition experiments. No significant competition activity was observed for RF9 on these receptors at doses up to 10 mM except on m and k, for which we observed a slight competition at this concentration. Values are mean ± SEM from two or more separate experiments performed in duplicate.
Supporting Methods
Cloning of Human Neuropeptide FF 2 (hNPFF2).
An EST encoding human NPFF2 receptor subtype (GeneBank accession no. AA449919) except the first 88 residues was purchased from Invitrogen. The 5' coding sequence of hNPFF2 was amplified by PCR from human genomic DNA. The two cDNA fragments were combined to produce the full-length hNPFF2 receptor and subcloned into the pcDNA3 expression vector using standard molecular biology methods (1).Cell Membrane Preparations and Receptor Binding Assays.
Membranes from insect cells expressing Y1 hNPY (human neuropeptide Y) receptor were prepared as described (2). Y1 hNPY receptor membranes were incubated in a final volume of 0.5 ml containing 50 mM Tris·HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% BSA, 0.05 nM [125I]YY, and the ligands to be tested as described in ref. 2. Nonspecific binding was determined in the presence of 1 mM NPY. Membranes from COS-1 cells transiently expressing m- and d-opioid receptors, and from Chinese hamster ovary (CHO) cells stably expressing k-opioid and ORL-1 receptors were prepared as described in ref. 3. m, d, k, and ORL-1 membranes were diluted in 50 mM Tris·HCl, pH 7.4/0.1% BSA and incubated with [3H]diprenorphine (0.2 nM for hMOR and hDOR and 0.4 nM for hKOR; PerkinElmer) or 0.1 nM [Leucyl-3H]nociceptin (for hORL1; Amersham Biosciences) and variable concentrations of RF9 in a total volume of 0.2 ml for 1 h at 25°C. Nonspecific binding was determined in presence of 10 mM naloxone (hMOR, hDOR, and hKOR; Sigma) or 10 mM nociceptin/orphanin FQ (hORL1, Sigma). For RFamide receptors, membranes from cells stably transfected with GPR10, GPR54, or GPR103 were purchased from Euroscreen (Brussels, Belgium). GPR10 membranes were incubated in a final volume of 0.1 ml containing 25 mM Hepes (pH 7.4), 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA, 0.03 nM [125I]PrRP-12–31 (Amersham Pharmacia Biosciences), and ligands to be tested for 30 min at 25°C. Nonspecific binding was determined in presence of 1 m M PrRP-31 (Bachem, Weil am Rhein, Germany). GPR54 membranes were incubated in a final volume of 0.1 ml containing 50 mM Tris·HCl (pH 7.4), 5 mM MgCl2, 2 mM EGTA 0.5% BSA, 0.2 nM of [125I]-Metastin (45-54)-NH2 (PerkinElmer), and competitors for 1 h et 25°C. Nonspecific binding was determined in presence of Metastin (1-54) (Phoenix, Zaltbommel, The Netherlands). GPR103 membranes were incubated in a final volume of 0.1 ml containing 50 mM Tris·HCl (pH 7.4), 10 mg/ml saponin, 0.5% BSA, 100 mg/ml bacitracin, 0.06 nM [125I]-QRFP26 (PerkinElmer), and competitors for 30 min at 25°C. Nonspecific binding was determined in presence of P518 (Bachem)Blood Pressure and Heart Rate Measurements.
Experiments were carried out on male Sprague–Dawley rats weighing 250–300 g. Animals were purchased from the University of Alberta Biological Animal Center. They were housed in a 12 h light/12 h dark cycle (lights on at 0800 h) at 21°C with free access to food and water. All protocols used in these experiments were approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta.Rats (n = 5) were anesthetized with sodium pentobarbitol (50 mg/kg i.p.; Somnotol, MTC Pharmaceuticals Hamilton, Canada) and also received 0.05 mg atropine sulfate s.c. and 500 ml/kg Ethacilin i.m. In all animals, the descending aorta was cannulated for measurement of arterial blood pressure as described in ref. 4. In the same surgical session, an in-dwelling cannula into the lateral cerebral ventricle was inserted by drilling a small hole through the skull at 1.8 mm caudal and 1.4 mm lateral from bregma. The guide cannula was set to a depth of 3.8–4.2 mm from the top of the skull, and accurate placement into the ventricle was verified by observing the outflow of the cerebrospinal fluid by withdrawing on a microsyringe attached to the cannula. A second small hole was drilled 4–5 mm caudal from the guide cannula for insertion of an anchor screw. A small amount of dental cement was applied to the screw and the guide cannula to hold it in place and allowed to harden before closure of the wound and recovery from anesthesia. Five to seven days after surgery, the arterial line was flushed with saline and connected to a pressure transducer for continuous recording of arterial blood pressure. A small internal cannula was inserted into the guide cannula and connected to a 25-ml Hamilton syringe. NPFF peptide (Bachem) was made into a stock of 1 mg/ml and frozen in 50-ml aliquots until use. RF9 was freshly prepared from a stock solution of 1 mg/ml
Measurement of Nociceptive Mechanical Threshold.
All experiments were carried out on male Sprague–Dawley rats (Iffa credo, Saint Germain sur l’Arbresle, France) weighing 250–350 g. Animals were housed and habituated to experimental procedure as described in ref. 5. Experiments were performed according to the official edict presented by the French Ministry of Agriculture (Paris, France), the recommendations of the Helsinki Declaration, and the Animals Care and Use Manual of the National Institutes of Health (1999). At the end of experiments, the rats were euthanized with pentobarbital sodium (120 mg/kg). Nociceptive mechanical thresholds in rats (n = 8) were determined by a modification of the Randall–Selitto method (6), the paw-pressure vocalization test, in which a constantly increasing pressure is applied to the hind paw until the rat squeaks. Nociceptive threshold was measured with the Basile analgesimeter (Apelex, Massy, France; stylus tip diameter, 1 mm), and a 600-g cutoff value was used for preventing tissue damage. All drugs were dissolved in normal saline (0.9%) and administered s.c. (100 ml/100 mg body weight). Control animals were injected with an equal volume of saline.1. Sambrook, J. & Russel, D. W. (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Woodbury, NY).
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4. Jhamandas, J. H. & MacTavish, D. (2003) J. Neuroendocrinol. 15, 24–32.
5. Celerier, E., Laulin, J. P., Corcuff, J. B., Le Moal, M. & Simonnet, G. (2001) J. Neurosci. 21, 4074–4080.
6. Kayser, V., Basbaum, A. I. & Guilbaud, G. (1990) Brain Res. 508, 329–332.