Characterization of Neuropeptide Y (NPY) Receptors

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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 protein?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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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 (Y₁ ) or hippocampus (Y₂ ), 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 [¹²⁵ I]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 [¹²⁵ I]PYY‐Labeled Y₁ and Y₂ Receptors in Rat Brain Membranes

Compound e	K _i (nM)
Y₁ (cortex) f	Y₂ (cortex) g	Y₂ (hippocampus)
hNPY	2.0	2.0	4.0
pNPY	1.0	2.0	3.0
hPYY	2.0	0.5	1.0
p[Leu³¹ Pro³⁴ ]NPY	2.0	>300	>300
h[Leu³¹ Pro³⁴ ]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

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

^f Assayed in the presence of 200 nM unlabeled pNPY_13‐36 .

^g Assayed in the presence of 100 nM unlabeled p[Leu³¹ Pro³⁴ ]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 [¹²⁵ I]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% O₂ /5% CO₂ 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 [¹²⁵ I]PYY‐Labeled Y₁ and Y₂ Receptors in Human Neuroblastoma Cells

Compound h	K _i (nM)
Y₁ (SK‐N‐MC)	Y₂ (SK‐N‐BE2)
hNPY	3.0	3.0
hPYY	2.0	1.0
p[Leu³¹ Pro³⁴ ]NPY	3.0	220
h[Leu³¹ Pro³⁴ ]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

^h h, 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

Figure 1.11.1 Specific [¹²⁵ I]PYY binding to Y₁ (A) and Y₂ (B ) receptor subtypes in rat brain cerebral cortical membranes, and (C ) to Y₂ receptors in hippocampal membranes. The binding assay was performed as described in the text (see ). Each point represents the mean of at least three independent determinations.

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Literature Cited

Literature Cited
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