Characterization of Neurotensin Receptors

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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

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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

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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
¹²⁵ I‐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

[³ H]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% CO₂ humidified incubator

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Figures

Figure 1.29.1 Competitive inhibition of ¹²⁵ I‐NT binding by unlabeled NT and SR 48692 in membranes from NTS1‐expressing CHO cells. The data are derived from Table and were fitted using LIGAND software. K _i values are 0.17 nM and 11.2 nM for NT and SR 48692, respectively.

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Figure 1.29.2 Competitive inhibition of ¹²⁵ I‐NT binding by unlabeled NT, SR 48692, and levocabastine in membranes from NTS2‐expressing COS cells. The data are derived from Table and were fitted using LIGAND software. K _i values are 1.44 nM, 420 nM, and 170 nM for NT, SR 48692, and levocabastine, respectively.

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Figure 1.29.3 Competitive inhibition of [³ H]SR 48692 binding by unlabeled NT and SR 48692 in membranes from NTS1‐expressing CHO cells. The data are derived from Table and were fitted using LIGAND software. The K _i value is 5.6 nM for SR 48692. Note the complex inhibition curve obtained with unlabeled NT (see Commentary for discussion).

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Figure 1.29.4 Competitive inhibition of ¹²⁵ I‐NT binding by unlabeled NT and SR 48692 in primary cultures of mesencephalic cells. The data are derived from Table and were fitted using Prism (GraphPad) software. K _i values are 0.31 nM and 2.1 nM for NT and SR 48692, respectively.

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Literature Cited

Literature Cited
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