Characterization of Adrenoceptors

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

Norepinephrine and epinephrine are important neurotransmitters in both the peripheral and the central nervous systems. The actions of these neurotransmitters are mediated by adrenoceptors. Three major types of adrenoceptors have been identified on the basis of pharmacological data: ?₁ , ?₂ , and ?. Within each major type, at least three receptor subtypes have been identified. Three basic types of radioligand binding experiments are provided in this unit: (1) saturation binding from which the affinity (K _d ) of the radioligand for the adrenoceptor and the binding site density (B _max ) can be determined; (2) inhibition experiments from which the affinity (K _i ) of a competing, unlabeled compound for the receptor can be determined; and (3) kinetic assays from which the forward and reverse rate constants of the binding process can be determined. Three support protocols are provided covering the preparation of membranes containing the receptor from tissue or cells in culture, calculation of K _d and B _max from saturation experiments, and calculation of K _i from inhibition experiments.

Keywords: receptor binding; epinephrine; norepinephrine; neurotransmitter; adrenoceptor; adrenergic receptor

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Basic Protocol 1: Determination of Bmax and Kd Values by Radioligand Saturation
Basic Protocol 2: Determination of Ki Values by Radioligand Binding Inhibition
Support Protocol 1: Preparation of Receptor Membranes from Tissue or Cultured Cells
Support Protocol 2: Calculation of Kd and Bmax from Saturation Experiments
Support Protocol 3: Calculation of Ki from Inhibition Experiments
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Determination of Bmax and Kd Values by Radioligand Saturation

Materials

Membrane preparation containing the adrenoceptor of interest (see protocol 3 )
0.1 M NaOH
Assay buffer appropriate for receptor type, ice cold:
- α₁ receptors: 25 mM glycylglycine, pH 7.4 (see recipe )
- α₂ receptors (for radiolabeled antagonists): 25 mM sodium phosphate, pH 7.4 ( appendix 2A )
- α₂ receptors (for radiolabeled agonists) and β receptors: 25 mM Tris⋅Cl, pH 7.4 ( appendix 2A )
5 mM HCl
Adrenergic radioligand, at 1 µCi/µl:
- α₁ receptors: [³ H]prazosin (GE Healthcare; Perkin‐Elmer) or [¹²⁵ I]HEAT (Perkin‐Elmer)
- α₂ receptors: [³ H]RX821002 (GE Healthcare), [³ H]rauwolscine (GE Healthcare; Perkin‐Elmer), or [¹²⁵ I]iodoclonidine (Perkin‐Elmer)
- β receptors: [³ H]dihydroalprenolol (GE Healthcare; Perkin‐Elmer) or [¹²⁵ I]pindolol (Perkin‐Elmer)
10 mM (−)‐norepinephrine (see recipe ; for estimating nonspecific binding)
Wash buffer (see recipe ), ice cold
Scintillation cocktail

Tissue homogenizer: e.g., Polytron (Brinkmann) or Tissumizer (Tekmar)
12 × 75–mm borosilicate glass test tubes (dilution tubes)
12 × 75–mm polypropylene test tubes (assay tubes)
Glass fiber filters (GF/A circles and GF/B strips; Whatman)
Filtration manifold (e.g., Brandel)

Additional reagents and equipment for protein quantitation assays ( appendix 3A )

Basic Protocol 2: Determination of Ki Values by Radioligand Binding Inhibition

Materials

Membrane preparation containing the adrenoceptor of interest (see protocol 3 )
Assay buffer appropriate for receptor type, ice cold:
- α₁ receptors: 25 mM glycylglycine, pH 7.4 (see recipe )
- α₂ receptors (for radiolabeled antagonists): 25 mM sodium phosphate, pH 7.4 ( appendix 2A )
- α₂ receptors (for radiolabeled agonists) and β receptors: 25 mM Tris⋅Cl, pH 7.4 ( appendix 2A )
5 mM HCl
Adrenergic radioligand, at 1 µCi/µl:
- α₁ receptors: [³ H]prazosin (GE Healthcare; Perkin‐Elmer) or [¹²⁵ I]HEAT (Perkin‐Elmer)
- α₂ receptors: [³ H]RX821002 (GE Healthcare), [³ H]rauwolscine (GE Healthcare; Perkin‐Elmer) or [¹²⁵ I]iodoclonidine (Perkin‐Elmer)
- β receptors: [³ H]dihydroalprenolol (GE Healthcare; Perkin‐Elmer) or [¹²⁵ I]pindolol (Perkin‐Elmer)
Inhibitor (see Table 1.5.3 for suggested compounds)
Wash buffer (see recipe ), ice cold
Scintillation cocktail

Tissue homogenizer: e.g., Polytron (Brinkmann) or Tissumizer (Tekmar)
12 × 75–mm borosilicate glass test tubes (dilution tubes)
Glass fiber filters (GF/A circles and GF/B strips)
12 × 75–mm polypropylene test tubes (assay tubes)

Filtration manifold (e.g., Brandel)

Table 1.5.3 MaterialsCompounds Used in the Pharmacological Characterization of Adrenergic Receptor Subtypes

Receptor	Key compounds e
α₁	WB‐4101, 5‐methylurapidil, prazosin, oxymetazoline
α₂	Rauwolscine, BRL44408, prazosin, oxymetazoline, spiroxatrine
β	Propranolol, metoprolol, ICI118551, BRL37344

^e Abbreviations: BRL 37344, 1‐(3‐chlorophenyl)‐2‐[2‐(4‐(carboxymethoxy)phenyl)‐1‐methylethylamino]ethanol; BRL 44408, 2‐[(4,5‐dihydro‐1H ‐imidazol‐2‐ylmethyl]‐2,3‐dihydro‐1‐methyl‐1H ‐isoindole; ICI 118,551, erythro (±)1‐[(‐methylindane‐4‐yl)‐oxy]‐3‐isopropylamino‐2‐butanol; WB‐4101, 2‐(N ‐[2,6‐dimethoxyphenoxyethyl]) aminomethyl‐1,4‐benzodioxane.

Support Protocol 1: Preparation of Receptor Membranes from Tissue or Cultured Cells

Materials

Tissue of interest (see Table 1.5.4 )
Wash buffer (see recipe )

Tissue homogenizer: e.g., Polytron (Brinkmann) or Tissumizer (Tekmar)

Sorvall RC5‐B centrifuge with SS‐34 rotor, or equivalent

Table 1.5.4 MaterialsSome Tissues and Cell Lines Frequently Used in Adrenergic Receptor Binding Assays

Adrenoceptor	Tissue	Cell line
α_1A	Rabbit liver, rat submandibular gland	Transfected cells
α_1B	Rat liver, rat spleen	DDT cells, transfected cells
α_1D		Transfected cells
α_2A	Human platelets, bovine pineal (α_2D )	HT29 cells, RINm5F cells (α_2D ), transfected cells
α_2B	Neonatal rat lung, rat kidney	NG108 cells, transfected cells
α_2C	Opossum kidney, human caudate nucleus	OK cells, Y79 cells, transfected cells
β₁	Rat cerebral cortex	Transfected cells
β₂	Rat lung, rat cerebellum	Transfected cells
β₃		Transfected cells

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Figures

Figure 1.5.1 Saturation data for the binding of [³ H]rauwolscine to the α_2B adrenergic receptor. The mean bound cpm per assay tube is plotted against the amount of added radioligand (radioligand concentration). Specific binding is the difference between total binding (binding in the tubes without norepinephrine) and nonspecific binding (binding in the tubes with norepinephrine), as shown in Table . In this unit, data are given as cpm/tube. Normally, when presenting saturation data in a publication, the radioligand concentration data would be converted to a molar concentration, such as pM, and the bound radioligand to either pM or fmol/mg protein.

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Figure 1.5.2 Rosenthal plot of saturation data. The specific binding from the data in Figure (also see Table ) are transformed using the Rosenthal procedure and plotted as bound/free versus bound, which yields a linear relationship. Normally, the free concentration of radioligand data is converted to a molar concentration (e.g., pM) and the specific binding data to either pM or fmol/mg protein. If both the bound and free data are in the same units (cpm or pM, for example), then bound/free has no units.

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Figure 1.5.3 Inhibition of [³ H]rauwolscine binding by the agonist clonidine to the three α₂ ‐adrenoceptor subtypes. (A ) The data from a typical inhibition experiment are plotted as bound [³ H]rauwolscine cpm versus log[clonidine]. The curves drawn are the one‐site fit as determined by the Prism program (GraphPad). (B ) Normalized plot of the inhibition of [³ H]rauwolscine binding by the agonist clonidine. The data are taken from panel A but are presented here as percent specific binding. Because the three curves have the same maxima and minima, it is easy to visualize the relative potency of the inhibitor at the three subtypes.

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Videos

Literature Cited

	Brede, M., Philipp, M., Knaus, A., Muthig, V., and Hein, L. 2004. Alpha‐2 adrenergic receptor subtypes ‐ novel functions uncovered in gene‐targeted mouse models. Biol. Cell 96:343‐348.
	Bylund, D.B. 1986. Graphic presentation and analysis of inhibition data from ligand‐binding experiments. Anal. Biochem. 159:50‐57.
	Bylund, D.B. 1988. Subtypes of α2‐adrenoceptors: Pharmacological and molecular biological evidence converge. Trends Pharmacol. Sci. 9:356‐361.
	Bylund, D.B. 1992. Subtypes of α1‐ and α2‐adrenergic receptors. FASEB J. 6:832‐839.
	Bylund, D.B. and Murrin, L.C. 2000. Radioligand Saturation Binding Experiments over Large Concentration Ranges. Life Sci. 67:2897‐2911.
	Bylund, D.B. and Toews, M.L. 1993. Radioligand binding methods: Practical guide and tips. Am. J. Physiol. 265:L421‐429.
	Bylund, D.B., Eikenberg, D.C., Hieble, J.P., Langer, S.Z., Lefkowitz, R.J., Minneman, K.P., Molinoff, P.B., Ruffolo, R.R., Jr., and Trendelenburg, A.U. 1994. IV. International Union of Pharmacology nomenclature of adrenoceptors. Pharmacol. Rev. 46:121‐136.
	Bylund, D.B., Deupree, J.D., and Toews, M.L. 2004. Radioligand binding methods for membrane preparations and intact cells. In GPCR Signal Transduction Protocols (G.B. Willars and R.A. Challiss, eds.) pp. 1‐28. Humana Press, Totowa, N.J.
	Cheng, Y.‐C. and Prusoff, W.H. 1973. Relationship between the inhibition constant (KI and the concentration of inhibitor which causes 50 per cent inhibition (IC50 of an enzymatic reaction. Biochem. Pharmacol. 22:3099‐3108.
	Deupree, J.D., Hinton, K.A., Cerutis, D.R., and Bylund, D.B. 1996. Buffers differentially alter the binding of [3H]rauwolscine and [3H]RX821002 to the α‐2 adrenergic receptor subtypes. J. Pharmacol. Exp. Ther. 278:1215‐1227.
	Ford, A.P.D.W., Williams, T.J., Blue, D.R., and Clarke, D.E. 1994. α1‐Adrenoceptor classification: Sharpening Occam's razor. Trends Pharmacol. Sci. 15:167‐170.
	Guimaraes, S. and Moura, D. 2001. Vascular adrenoceptors: An update. Pharmacol. Rev. 53:319‐356.
	Hieble, J.P., Bondinell, W.E., and Ruffolo Jr., R.R. 1995a. Alpha‐ and beta‐adrenoceptors: From the gene to the clinic. 1. Molecular biology and adrenoceptor subclassification. J. Med. Chem. 38:3415‐3444.
	Hieble, J.P., Bylund, D.B., Clarke, D.E., Eikenburg, D.C., Langer, S.Z., Lefkowitz, R.J., Minneman, K.P., and Ruffolo, R.R., Jr. 1995b. International Union of Pharmacology. X. Recommendation for nomenclature of α1 adrenoceptors: Consensus update. Pharmacol. Rev. 47:267‐270.
	Jones, S.B., Smith, J.M., Jones, A.W., and Bylund, D.B. 1987. α1 adrenergic receptor binding in aortas from rat and dog: Comparison of [3H]prazosin and β‐iodo‐[125I]‐4‐hydroxyphenylethylaminomethyltetralone. J. Pharmacol. Exp. Ther. 241:875‐881.
	Perez, D.M. (ed.) 2006. The Adrenergic Receptor in the 21st Century. Humana Press, Totowa, N.J.
	Rosenthal, H.E. 1967. Graphical method for the determination and presentation of binding parameters in a complex system. Anal. Biochem. 20:525‐532.
	Ruffolo Jr., R.R., Bondinell, W., and Hieble, J.P. 1995. Alpha‐ and beta‐adrenoceptors: From the gene to the clinic. 2. Structure‐activity relationships and therapeutic applications. J. Med. Chem. 38:3681‐3716.
	Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660‐672.
	Strosberg, A.D. and Pietri‐Rouxel, F. 1996. Function and regulation of the β3‐adrenoceptor. Trends Pharmacol. Sci. 17:373‐381.
	Tanoue, A., Koshimizu, T.A., Shibata, K., Nasa, Y., Takeo S., and Tsujimoto, G. 2003. Insights into alpha1 adrenoceptor function in health and disease from transgenic animal studies. Trends Endocrinol. Metab. 14:107‐113.
	Zheng, M., Zhu, W., Han, Q., and Xiao, R.P. 2005. Emerging concepts and therapeutic implications of beta‐adrenergic receptor subtype signaling. Pharmacol. Ther. 108:257‐268.
Key References
	Bylund et al., 1994. See above.
	Two articles by an international committee providing a short overview of the nomenclature and the molecular and pharmacological characteristics of the three types of adrenoceptors.
	Hieble et al., 1995b. See above.
	A good introduction to the theory of receptor binding.
	Limbird, L.E. 1996. Cell Surface Receptors: A Short Course on Theory and Methods. Kluwer Academic Publishers, Boston.
	Provides a good overview of methods for receptor binding (Chapter 1), computer‐assisted analysis of binding data (Chapter 2), and receptor autoradiography (Chapter 7).
	Yamamura, H.I., Enna, S.J., and Kuhar, S.J. (eds.) 1990. Methods in Neurotransmitter Receptor Analysis. Raven Press, New York.

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Characterization of Adrenoceptors

Abstract

Table of Contents

Materials

Basic Protocol 1: Determination of Bmax and Kd Values by Radioligand Saturation

Basic Protocol 2: Determination of Ki Values by Radioligand Binding Inhibition

Support Protocol 1: Preparation of Receptor Membranes from Tissue or Cultured Cells

Figures

Videos

Literature Cited