Characterization of Tachykinin Receptors

Vladimir V. Karpitskiy1

1 Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.15
DOI:  10.1002/0471141755.ph0115s04
Online Posting Date:  May, 2001
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Abstract

The mammalian tachykinin peptides are a family of neuropeptides characterized by a common C‐terminal amino acid sequence of the form Phe‐X‐Gly‐Leu‐Met‐NH2, where X is either Phe or Val. Tachykinin peptides have various biological activities, including excitation of both peripheral and central neurons, stimulation of smooth muscle contraction, stimulation of exocrine and endocrine gland secretion, and involvement in immune and inflammatory responses. This unit presents basic methods for the characterization of tachykinin receptors by radioligand binding assay. The protocols detail methods for three major classes of tachykinin receptors and techniques for the generation of high specific activity iodinated radioligands.

     
 
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Table of Contents

  • Basic Protocol 1: Competition Binding Assay for NK1 or NK2 Receptor–Bearing Cells and Tissues
  • Alternate Protocol 1: Competition Binding Assay for NK3 Receptor–Bearing Cells and Tissues
  • Basic Protocol 2: Saturation Binding Assay for NK1 or NK2 Receptor–Bearing Cells and Tissues
  • Alternate Protocol 2: Saturation Binding Assay for NK3 Receptor–Bearing Cells and Tissues
  • Support Protocol 1: Radioiodination of the Tachykinin Peptides
  • Support Protocol 2: Preparation of Membrane from Tissue Bearing Tachykinin Receptors
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Competition Binding Assay for NK1 or NK2 Receptor–Bearing Cells and Tissues

  Materials
  • Cells or tissue membrane preparations to be assayed (see protocol 6)
  • Radioligand peptide: e.g., 2175 Ci/mmol [125I]Tyr−1‐substance P ([125I]Tyr−1‐SP for NK 1), [125I]histidyl–neurokinin A ([125I]histidyl‐NKA for NK 2), or other 125I‐labeled tachykinin peptide (NEN Life Science, Amersham, or see protocol 5)
  • Unlabeled Tyr−1‐SP, NKA, or other tachykinin peptide (Bachem)
  • Test compound (competitor)
  • 0.2% (v/v) polyethyleneimine (PEI; Sigma)
  • NK 1/NK 2 cell binding buffer (see recipe), ice cold
  • Membrane binding buffer (see recipe), ice cold
  • PBS, ice cold: 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2A), containing 120 mM NaCl
  • Tris‐buffered saline (TBS)/MnCl 2, ice cold: 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A), containing 120 mM NaCl and 3 mM MnCl 2
  • Tissue culture tubes
  • 2.5‐cm no. 32 glass filters (Schleicher & Schuell)
  • 12 × 75–mm plastic and glass tubes
  • Platform shaker at 4°C
  • Vacuum filtration manifold or other type of membrane‐harvesting apparatus
  • γ scintillation counter

Alternate Protocol 1: Competition Binding Assay for NK3 Receptor–Bearing Cells and Tissues

  • Tris‐buffered saline (TBS), ice cold: 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A), containing 120 mM NaCl
  • 66 to 69 Ci/mmol [3H]senktide (NEN Life Science) radioligand solution
  • NK 3 cell binding buffer (see recipe) or membrane binding buffer (see recipe), ice cold
  • Unlabeled senktide (Bachem)
  • TBS/BSA/Tween 20: TBS containing 2% (w/v) BSA and 0.1% (w/v) Tween 20 (Sigma)
  • TBS/0.01% (w/v) SDS
  • Liquid scintillation solution (e.g., ScintiSafe 30%; Fisher)
  • 5‐ml liquid scintillation vials
  • Liquid scintillation counter

Basic Protocol 2: Saturation Binding Assay for NK1 or NK2 Receptor–Bearing Cells and Tissues

  Materials
  • Cells or tissue membrane preparations to be assayed (see protocol 6)
  • Radioligand peptide: e.g., 2175 Ci/mmol [125I]Tyr−1‐substance P ([125I]Tyr−1‐SP for NK 1), [125I]histidyl–neurokinin A ([125I]histidyl‐NKA for NK 2), or other 125I‐labeled tachykinin peptide (NEN Life Science, Amersham, or see protocol 5)
  • Unlabeled Tyr−1‐SP, NKA, or other tachykinin peptide (Bachem)
  • 0.2% (v/v) polyethyleneimine (PEI; Sigma)
  • For cells:
  • NK 1/NK 2 cell binding buffer (see recipe), ice cold
  • PBS, ice cold: 50 mM phosphate buffer, pH 7.4 ( appendix 2A), containing 120 mM NaCl
  • For membranes:
  • Membrane binding buffer (see recipe), ice cold
  • Tris‐buffered saline (TBS)/MnCl 2, ice cold: 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A), containing 120 mM NaCl and 3 mM MnCl 2
  • Tissue culture tubes
  • 2.5‐cm no. 32 glass filters (Schleicher & Schuell)
  • 12 × 75–mm plastic and glass tubes
  • Platform shaker at 4°C
  • Vacuum filtration manifold or other type of membrane‐harvesting apparatus
  • γ scintillation counter
  • Computer program for analysis of binding data (e.g., LIGAND)

Alternate Protocol 2: Saturation Binding Assay for NK3 Receptor–Bearing Cells and Tissues

  Materials
  • Tachykinin peptide (>95% purity by HPLC; Bachem): e.g., Tyr−1‐substance P (Tyr−1‐SP), neurokinin A (NKA), Tyr−1‐NKA, neuropeptide γ (NPγ), or [MePhe7]NKB
  • 0.1% (v/v) trifluoroacetic acid (TFA)
  • 80% (v/v) acetonitrile in 0.1% TFA
  • Chloramine‐T (Sigma)
  • Sodium metabisulfite (Sigma)
  • Bovine serum albumin (BSA; Sigma)
  • 0.2 M sodium phosphate buffer, pH 7.4 ( appendix 2A) and pH 8.5
  • 1 mCi Na125I, carrier free (NENsure vial; NEN Life Science)
  • HPLC solvent A: 0.1 M phosphoric acid (HPLC grade) containing 0.1 M NaH 2PO 4, pH 2.5
  • HPLC solvent B: 100% acetonitrile (HPLC grade)
  • 2‐Mercaptoethanol (2‐ME; Sigma)
  • Sep‐Pak cartridge Classic (Waters Division of Millipore)
  • 10‐ml and 3‐ml disposable syringes with Luer‐lock tip
  • 0.5‐ml and 1.5‐ml plastic, conical microcentrifuge tubes
  • 12 × 75–mm plastic and glass tubes
  • SpeedVac evaporator (Savant)
  • Tabletop 65°C water bath
  • Programmable HPLC system utilizing separate columns (e.g., 15‐cm analytical Vydac C 4) and injectors
  • γ scintillation counter

Support Protocol 1: Radioiodination of the Tachykinin Peptides

  Materials
  • Receptor‐containing tissue
  • Tris‐buffered saline (TBS), ice cold: 50 mM Tris⋅Cl buffer, pH 7.4, containing 120 mM NaCl
  • 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A), ice cold
  • 50 mM Tris⋅Cl, pH 7.4, containing 300 mM KCl and 10 mM EDTA, ice cold
  • TBS containing 3 mM MnCl 2, ice cold
  • Polytron and Teflon/glass (Dounce) homogenizers
  • Additional reagents and equipment for determining protein concentration ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Cheng, Y.‐C. and Prusoff, W.H. 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction. Biochem. Pharm. 22:3099‐3108.
   Hanley, M.R., Sandberg, B.E.B., Lee, C.M., Iversen, L.L., Brundish, D.E., and Wade, R. 1980. Specific binding of 3H‐substance P to rat brain membranes. Nature 286:810‐812.
   Krause, J.E., Blount, P., and Sachais, B.B. 1994. Molecular biology of receptors: Structures, expression and regulatory mechanisms. In The Tachykinin Receptors (S.H. Buck, ed.) pp. 165‐218. Humana Press, Totowa, N.J.
   Krause, J.E., Staveteig, P.T., Nave‐Mentzer, J., Schmidt, S.K., Tucker, J.B., Brodbeck, R.M., Bu, J.‐Y., and Karpitskiy, V.V. 1997. Functional expression of a novel human neurokinin‐3 receptor homologue that binds [3H]senktide and [125I‐MePhe7]‐NKB, and is responsive to tachykinin peptide agonist. Proc. Natl. Acad. Sci. U.S.A. 94:310‐315.
   Lee, C.M., Iversen, L.L., Hanley, M.R., and Sandberg, B.E.B. 1982. The possible existence of multiple receptors for substance P. Naunyn‐Schmiedeberg's Arch. Pharmacol. 318:281‐287.
   Maggi, C.A., Patacchine, R., Rovero, P., and Giachetti, A. 1993. Tachykinin receptors and tachykinin receptor antagonists. J. Autonom. Pharmacol. 13:23‐93.
   Munson, P.J. and Rodbard, D. 1980. LIGAND: A versatile computerized approach for characterization of ligand binding systems. Anal. Biochem. 107:220‐239.
   Nakanishi, S. 1991. Mammalian tachykinin receptors. Annu. Rev. Neurosci. 14:123‐136.
   Otsuka, M. and Yoshioka, K. 1993. Neurotransmitter functions of mammalian tachykinins. Physiol. Rev. 73:229‐308.
Key References
  Cheng and Prusoff, 1973. See above.
  Describes the appropriate level of substrate to use in competition binding experiments such that IC50 values are equal to Ki values.
   Limbird, L.E. 1986. Cell Surface Receptors: A Short Course of Theory and Methods. Martinus Nij‐hoff Publishing, Zoetermeer, The Netherlands.
  An excellent source of basic information about theoretical and practical aspects of ligand‐receptor interactions.
  Munson and Rodbard, 1980. See above.
  Describes a computerized method to determine Kd and number of binding sites.
   Takeda, Y., Cremins, J.D., Takeda, J., and Krause, J.E. 1991. Analysis of tachykinin peptide family gene expression patterns by combined high‐performance liquid chromatography‐radioimmunoassay. Methods Neurosci. 6:119‐130.
  Describes HPLC procedures and conditions for separating closely related peptides.
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