Characterization of Adrenoceptors

David B. Bylund1

1 University of Nebraska Medical Center, Omaha, Nebraska
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.5
DOI:  10.1002/0471141755.ph0105s36
Online Posting Date:  March, 2007
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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: α1, α2, and β. 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 (Kd) of the radioligand for the adrenoceptor and the binding site density (Bmax) can be determined; (2) inhibition experiments from which the affinity (Ki) 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 Kd and Bmax from saturation experiments, and calculation of Ki from inhibition experiments.

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

     
 
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Table of Contents

  • 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:
    • α 1 receptors: 25 mM glycylglycine, pH 7.4 (see recipe)
    • α 2 receptors (for radiolabeled antagonists): 25 mM sodium phosphate, pH 7.4 ( appendix 2A)
    • α 2 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:
    • α 1 receptors: [3H]prazosin (GE Healthcare; Perkin‐Elmer) or [125I]HEAT (Perkin‐Elmer)
    • α 2 receptors: [3H]RX821002 (GE Healthcare), [3H]rauwolscine (GE Healthcare; Perkin‐Elmer), or [125I]iodoclonidine (Perkin‐Elmer)
    • β receptors: [3H]dihydroalprenolol (GE Healthcare; Perkin‐Elmer) or [125I]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:
    • α 1 receptors: 25 mM glycylglycine, pH 7.4 (see recipe)
    • α 2 receptors (for radiolabeled antagonists): 25 mM sodium phosphate, pH 7.4 ( appendix 2A)
    • α 2 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:
    • α 1 receptors: [3H]prazosin (GE Healthcare; Perkin‐Elmer) or [125I]HEAT (Perkin‐Elmer)
    • α 2 receptors: [3H]RX821002 (GE Healthcare), [3H]rauwolscine (GE Healthcare; Perkin‐Elmer) or [125I]iodoclonidine (Perkin‐Elmer)
    • β receptors: [3H]dihydroalprenolol (GE Healthcare; Perkin‐Elmer) or [125I]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
    α 1 WB‐4101, 5‐methylurapidil, prazosin, oxymetazoline
    α 2 Rauwolscine, BRL44408, prazosin, oxymetazoline, spiroxatrine
    β Propranolol, metoprolol, ICI118551, BRL37344

     eAbbreviations: 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
    β 1 Rat cerebral cortex Transfected cells
    β 2 Rat lung, rat cerebellum Transfected cells
    β 3 Transfected cells

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Figures

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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