Characterization of Tachykinin Receptors

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 [¹²⁵ I]Tyr⁻¹ ‐substance P ([¹²⁵ I]Tyr⁻¹ ‐SP for NK₁ ), [¹²⁵ I]histidyl–neurokinin A ([¹²⁵ I]histidyl‐NKA for NK₂ ), or other ¹²⁵ I‐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)
NK₁ /NK₂ 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)/MnCl₂ , ice cold: 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A ), containing 120 mM NaCl and 3 mM MnCl₂

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 [³ H]senktide (NEN Life Science) radioligand solution
NK₃ 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 [¹²⁵ I]Tyr⁻¹ ‐substance P ([¹²⁵ I]Tyr⁻¹ ‐SP for NK₁ ), [¹²⁵ I]histidyl–neurokinin A ([¹²⁵ I]histidyl‐NKA for NK₂ ), or other ¹²⁵ I‐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:
NK₁ /NK₂ 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)/MnCl₂ , ice cold: 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A ), containing 120 mM NaCl and 3 mM MnCl₂
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 Na¹²⁵ I, carrier free (NENsure vial; NEN Life Science)
HPLC solvent A: 0.1 M phosphoric acid (HPLC grade) containing 0.1 M NaH₂ PO₄ , 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 C₄ ) 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 MnCl₂ , ice cold
Polytron and Teflon/glass (Dounce) homogenizers
Additional reagents and equipment for determining protein concentration ( appendix 3A )

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Figures

Figure 1.15.1 Results from a representative competition radioligand binding experiment using [¹²⁵ I]Tyr⁻¹ ‐NKA as the radioligand (220,000 cpm/tube or 0.045 nM) and NKB as the competitor (concentrations are indicated). CHO cells expressing cloned rat NK₂ receptor (700,000 receptors/cell) were used as the receptor source. The binding assay was performed as described (see ). (A ) Competition curve. (B ) Hill transformation plot (see step , ). Solving the equation when y = 0 gives x = log[IC₅₀ ] = −7.3495; therefore, the IC₅₀ for NKB = 44.7 nM.

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Figure 1.15.2 Example of data output from a representative saturation equilibrium radioligand binding experiment using [³ H]senktide (concentrations are indicated). CHO cells expressing cloned human NK₃ receptor were used as the receptor source. Nonspecific binding was defined with a 1000‐fold excess of unlabeled senktide. The binding assay was performed as described (see ). The inset is a Scatchard plot of the data. The values presented for K _d and B _max are the averages from four independent experiments. The values were obtained by a nonlinear least‐squares analysis using the LIGAND program. (Plotted data are from Krause et al., )

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Figure 1.15.3 Examples of HPLC separation of unlabeled and radioiodinated Tyr⁻¹ ‐SP species by reversed‐phase HPLC. (A ) HPLC separation of the radioiodinated material (see ); (B ) HPLC trace of intact (unlabeled) Tyr⁻¹ ‐SP. In both panels, the HPLC solvent gradient progression is illustrated by the horizontal line rising from left to right. Small black arrows 1 and 2 represent peaks of unlabeled SP species: peak 1 is the reduced peptide (intact form), elution time 35 min; peak 2 is the oxidized peptide (methylsulfoxide form), elution time 27 min. The other peaks represent material originating from ingredients of radioiodination reaction (e.g., BSA, 2‐ME). Small black arrows 3 and 4 represent elution times of [¹²⁵ I]Tyr⁻¹ ‐SP species: arrow 3 indicates the fraction containing oxidized [¹²⁵ I]Tyr⁻¹ ‐SP, elution time 33 min, yield 104.3 µCi/ml; arrow 4 indicates the fraction containing reduced [¹²⁵ I]Tyr⁻¹ ‐SP, elution time 40 min, yield 603.8 µCi/ml or 60% of incorporation from total 1 mCi ¹²⁵ I used in the reaction. The total amount of iodinated SP = activity collected/specific activity of ¹²⁵ I (2175 Ci/mmol) = 603.8 µCi/ml = 277.6 pmol/ml = 2.175 µCi/pmol.

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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.