Characterization of Neuropeptide Y (NPY) Receptors

William Karbon1, Julie Marasco1, Clarence Hale1

1 Amgen, Inc., Thousand Oaks, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.11
DOI:  10.1002/0471141755.ph0111s01
Online Posting Date:  May, 2001
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Abstract

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) constitute a trio of structurally related peptides that elicit a variety of physiological responses in targets including the brain, gut, and vascular smooth muscle. These actions are mediated by a family of G proteinā€coupled receptors, each of which exhibits a distinct pharmacological fingerprint. This unit describes protocols for identifying and characterizing the pharmacological properties of two NPY receptor subtypes, Y1 and Y2. At least four other members of this receptor family have been described (Y3, Y4, Y5 and y6), and a brief description of these receptors and relevant literature citations are also provided.

     
 
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Table of Contents

  • Basic Protocol 1: Competition Analysis to Determine Y1 and Y2 Receptor Number and Affinity in Rat Brain Tissues
  • Alternate Protocol 1: Saturation Analysis to Determine Y1 and Y2 Receptor Number and Affinity in Rat Brain Tissues
  • Basic Protocol 2: Competition Analysis to Determine Y1 and Y2 Receptor Number and Affinity in Human Neuroblastoma Cell Lines
  • Alternate Protocol 2: Saturation Analysis to Determine Y1 and Y2 Receptor Number and Affinity in Human Neuroblastoma Cell Lines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Competition Analysis to Determine Y1 and Y2 Receptor Number and Affinity in Rat Brain Tissues

  Materials
  • Frozen rat cerebral cortex (Y 1) or hippocampus (Y 2), dissected fresh and immediately frozen, or purchased frozen from a commercial vendor (e.g., Pel‐Freez)
  • Homogenization buffer (see recipe), ice cold
  • Tissue binding buffer (see recipe), ice cold
  • Competing ligands: unlabeled peptide YY (PYY), reference peptides (e.g., Bachem California or Peninsula Labs; see Table 1.11.2), or other test compounds
  • 25 nM [125I]PYY (Amersham; ∼2200 Ci/mmol; store aliquots at −20°C)
  • 50 mM HEPES buffer, pH 7.4, ice cold
  • Motor‐driven, glass‐fitted Teflon homogenizer, chilled
  • High‐speed refrigerated centrifuge (e.g., Beckman model J2‐MI)
  • Motorized tissue homogenizer (e.g., Brinkmann Polytron)
  • 12 × 75–mm polypropylene tubes
  • Whatman GF/C filters
  • 0.1% (w/v) polyethyleneimine (PEI)
  • Brandel 48‐sample cell harvesting unit
NOTE: Buffers should be kept cold. Always keep tissue homogenates and pellets on ice.
Table 1.1.2   MaterialsPharmacological Characterization of [125I]PYY‐Labeled Y 1 and Y 2 Receptors in Rat Brain Membranes

Compound e K i (nM)
Y 1 (cortex) f Y 2 (cortex) g Y 2 (hippocampus)
hNPY 2.0 2.0 4.0
pNPY 1.0 2.0 3.0
hPYY 2.0 0.5 1.0
p[Leu31Pro34]NPY 2.0 >300 >300
h[Leu31Pro34]PYY 1.0 >300 >300
pNPY 2‐36 20 2.0 4.0
hNPY 3‐36 200 3.0 2.0
hPYY 3‐36 280 0.5 2.0
pNPY 13‐36 >300 10 15
hPP >300 >300 >300
BIBP 3226 8.0 >300 >300

 eh, human; p, porcine. Peptides can be purchased from Bachem California or Peninsula Labs. BIBP 3226 can be obtained from Research Biochemicals.
 fAssayed in the presence of 200 nM unlabeled pNPY 13‐36.
 gAssayed in the presence of 100 nM unlabeled p[Leu31Pro34]NPY.

Alternate Protocol 1: Saturation Analysis to Determine Y1 and Y2 Receptor Number and Affinity in Rat Brain Tissues

  Materials
  • Human neuroblastoma cell lines: SK‐N‐MC (American Type Culture Collection, ATCC, no. HTB 10), SK‐N‐BE2 (W. Karbon), or KAN‐TS (Amersham Research Biochemicals)
  • Growth medium (see recipe)
  • Competing ligands: unlabeled peptide YY (PYY), reference peptides (e.g., Bachem California or Peninsula Labs; see Table 1.11.3), or other test compounds
  • Cell binding buffer (see recipe)
  • 25 nM [125I]PYY (Amersham; ∼2200 Ci/mmol; store aliquots at −20°C)
  • Eagle's minimal essential medium (EMEM; Life Technologies), ice cold
  • 1 N NaOH
  • Humidified 95% O 2/5% CO 2 incubator
  • Fibronectin‐coated 24‐well tissue culture plates (Becton Dickinson Labware)
  • 12 × 75–mm polypropylene tubes
  • Additional reagents and equipment for counting cells (Phelan, )
    Table 1.1.3   MaterialsPharmacological Characterization of [125I]PYY‐Labeled Y 1 and Y 2 Receptors in Human Neuroblastoma Cells

    Compound h K i (nM)
    Y 1 (SK‐N‐MC) Y 2 (SK‐N‐BE2)
    hNPY 3.0 3.0
    hPYY 2.0 1.0
    p[Leu31Pro34]NPY 3.0 220
    h[Leu31Pro34]PYY 5.0 250
    pNPY 2‐36 22 5.0
    hNPY 3‐36 110 5.0
    hPYY 3‐36 80 1.0
    pNPY 13‐36 300 3.0
    hPP >300 >300
    BIBP 3226 10 >300

     hh, human; p, porcine. Peptides can be purchased from Bachem California or Peninsula Labs. BIBP 3226 can be obtained from Research Biochemicals.
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Figures

Videos

Literature Cited

Literature Cited
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