Characterization of Neurotensin Receptors

Patrick Kitabgi1, Didier Pélaprat1

1 Hopital St‐Antoine, Paris
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.29
DOI:  10.1002/0471141755.ph0129s24
Online Posting Date:  May, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes procedures for performing competition binding assays with neurotensin receptor subtypes 1 and 2 (NTS1 and NTS2). Binding assays using cloned receptors, brain membranes, and primary cultured mesencephalic neurons are presented. NTS1 binding assays employing either radiolabeled neurotensin or SR 48692 (a nonpeptide neurotensin antagonist) as radioligands are described. These procedures may be used to screen selective ligands at neurotensin receptor subtypes.

Keywords: neurotensin; SR 48692; neurotensin receptors; NTS1; NTS2; binding assay; brain membranes; mesencephalic neurons

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Measurement of [125I]Neurotensin Binding to Cloned Receptors in Membranes
  • Alternate Protocol 1: Measurement of [3H]SR 48692 Binding to Cloned NTS1 Receptors in Membranes
  • Alternate Protocol 2: Measurement of 125I‐Neurotensin Binding to Homogenates from Newborn Mouse Brain
  • Alternate Protocol 3: Measurement of 125I‐Neurotensin Binding to Intact Rat Embryonic Mesencephalic Neurons in Primary Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Measurement of [125I]Neurotensin Binding to Cloned Receptors in Membranes

  Materials
  • Cell lines expressing the appropriate cloned neurotensin receptor (e.g., CHO, COS, LTK) grown to confluency in 100‐mm culture dishes
  • PBS (e.g., Life Technologies)
  • Tris buffer 1 for preparing cell membranes (see recipe), 4°C
  • Protein concentration kit (Bio‐Rad protein assay kit, Bio‐Rad Laboratories)
  • Tris buffer 2 for binding assay (see recipe), room temperature and 4°C
  • 125I‐NT (2000 Ci/mmol; PE Life Sciences, Amersham)
  • Test compounds
  • Unlabeled NT (Neosystem)
  • Rubber scraper
  • 20‐ml syringe with 21‐G needle (5‐cm length, 0.8‐mm diameter)
  • 5‐ml plastic tubes
  • Cellulose acetate filters (0.2‐µm, 25 mm; Sartorius)
  • Filtration unit (1 to 36 filters, Waters Millipore)
  • Gamma counter

Alternate Protocol 1: Measurement of [3H]SR 48692 Binding to Cloned NTS1 Receptors in Membranes

  • [3H]SR 48692 (86 Ci/mmol; Amersham Pharmacia Biotech)
  • Stock solution (1 to 10 mM) of unlabeled SR 48692 (Sanofi‐Synthélabo) in dimethylsulfoxide (DMSO)
  • Water‐compatible scintillation cocktail
  • Software such as GraphPad Prism or LIGAND

Alternate Protocol 2: Measurement of 125I‐Neurotensin Binding to Homogenates from Newborn Mouse Brain

  • 7‐day‐old mice
  • Tris‐EDTA buffer 3 for homogenizing brain tissue (see recipe), 4°C
  • Tris buffer 4 for binding assay (see recipe), room temperature
  • Polytron homogenizer
  • Additional reagents and equiment for assaying protein content ( appendix 3A)

Alternate Protocol 3: Measurement of 125I‐Neurotensin Binding to Intact Rat Embryonic Mesencephalic Neurons in Primary Cultures

  • Mesencephalic primary cultures grown in 24‐well culture plates, prepared according to described protocols (Brouard et al., )
  • Serum‐free binding medium (see recipe), room temperature
  • Serum‐free rinsing medium (see recipe), 4°C
  • 0.1 N sodium hydroxide
  • Vacuum source with intermediate medium‐receiving flask with attached Pasteur pipet
  • 37°C, 5% CO 2 humidified incubator
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bachelet, CM., Scarceriaux, V., Rostene, W., and Pelaprat, D. 1997. Evidence for neurotensin autoreceptors and relationship of neurotensin and its receptors with tyrosine hydroxylase‐positive neurons in rat primary hypothalamic cultures. Eur. J. Neurosci. 9:1483‐1487.
   Brouard, A., Pelaprat, D., Dana, C., Vial, M., Lhiaubet, A.M., and Rostene, W. 1992. Mesencephalic dopaminergic neurons in primary culture express functional neurotensin receptors. J. Neurosci. 12:1409‐1415.
   Dubuc, I., Sarret, P., Labbé‐Jullié, C., Botto, J.M., Honoré, E., Bourdel, E., Martinez, J., Costentin, J., Vincent, J.P., Kitabgi, P., and Mazella, J. 1999. Identification of the receptor subtype involved in the analgesic effect of neurotensin. J. Neurosci. 19:503‐510.
   Gully, D., Canton, M., Boigegrain, R., Jeanjean, F., Molimard, J.C., Poncelet, M., Gueudet, C., Heaulme, M., Leyris, R., Brouard, A., Pelaprat, D., Labbé‐Jullié, C., Mazella, J., Soubrié, P., Maffrand, J.P., Rostène, W., Kitabgi, P., and Le Fur, G. 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.
   Gully, D., Labeeuw, B., Boigegrain, R., Oury‐Donat, F., Bachy, A., Poncelet, M., Steinberg, R., Suaud‐Chagny, M.F., Santucci, V., Vita, N., Pecceu, F., Labbé‐Jullié, C., Kitabgi, P., Soubrie, P., Le Fur, G., and Maffrand, J.P. 1997. Biochemical and pharmacological activities of SR 142948A, a new potent neurotensin receptor antagonist. J. Pharmacol. Exp. Ther. 280:802‐812.
   Kitabgi, P. 1992. Biosynthesis, maturation, release, and degradation of neurotensin and neuromedin. N. Ann. N.Y. Acad. Sci. 668:30‐42.
   Kitabgi, P. 2002. Targeting neurotensin receptors with agonists and antagonists for therapeutic purposes. Curr. Opin. Drug Discovery Dev. 5:764‐776.
   Kitabgi, P., Rostène, W., Dussaillant, M., Schotte, A., Laduron, P., and 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., Botto, J.M., Mas, M.V., Chabry, J., Mazella, J., Vincent, J.P., Gully, D., Maffrand, J.P., and Kitabgi, P. 1995. [3H]SR 48692, the first nonpeptide neurotensin antagonist radioligand; characterization of binding properties and evidence for distinct agonist and antagonist binding domains on the rat neurotensin receptor. Mol. Pharmacol. 47:1050‐1056.
   Labbé‐Jullié, C., Deschaintres, S., Gully, D., Le Fur, G., and Kitabgi, P. 1994. Effect of the nonpeptide neurotensin antagonist SR 48692 and two enantiomeric analogs SR 48527 and SR 49711 on neurotensin binding and contractile responses in guinea‐pig ileum and colon. J. Pharm. Exp. Ther. 271:267‐276.
   Lépée‐Lorgeoux, I., Betancur, C., Souazé, F., Rostène, W., Bérod, A., and Pelaprat, D. 2000. Regulation of the neurotensin NT1 receptor in the developing rat brain following chronic treatment with the antagonist SR 48692. J. Neurosci. Res. 60:362‐369.
   Mazella, J. 2001. Sortilin/neurotensin receptor 3: A new tool to investigate neurotensin signaling and trafficking? Cell. Signal. 13:1‐6.
   Richard, F., Barroso, S., Martinez, J., Labbé‐Jullié, C., and Kitabgi, P. 2001. Agonism, inverse agonism and neutral antagonism at the constitutively active human neurotensin receptor 2. Mol. Pharmacol. 60:1392‐1398.
   Scarceriaux, V., Pelaprat, D., Forgez, P., Lhiaubet, A.M., and Rostene, W. 1995. Effects of dexamethasone and forskolin on neurotensin production in rat hypothalamic cultures. Endocrinology 136:2554‐2560.
   Vincent, J.P., Mazella, J., and Kitabgi, P. 1999. Neurotensin and neurotensin receptor. Trends Pharmacol. Sci. 20:302‐309.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library