Biochemical Methods and Assays

  • Paul L M. van Giersbergen
  • Stephen H. Buck
Part of the The Receptors book series (REC)


The tachykinins are a family of peptides that share the common C-terminal sequence These peptides exert their biological effects via three pharmacologically and molecular biologically well-defined receptors (see other chapters in this volume), the receptors. Substance P (SP) binds preferentially to the receptor, neurokinin A (NKA) to the receptor, and neurokinin B (NKB) to the receptor. Among other things, the number of receptor sites in tissues/cells and the affinities of compounds/peptides can be measured readily in receptor binding assays that exist for each individual tachykinin receptor. In this chapter, we will describe the development of these assays and the various factors that can influence their outcome. For reasons of clarity, the current tachykinin receptor nomenclature has been used throughout the chapter.


Divalent Cation Guanine Nucleotide Incubation Buffer Tachykinin Receptor Radioligand Concentration 
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  1. Aharony, D., Catanese, C. A., and Woodhouse, D.P. (1991) Binding of the novel ligand [4,5-31–1-Leu substance P to high-affinity receptors on guinea pig lung membranes: Modulation by GTP analogs and sulfhydryl modifying agents. J. Pharmacol. Exp. Ther. 259, 146–155.PubMedGoogle Scholar
  2. Anton, P. A., Reeve, Jr, J. R., Vidrich, A., Mayer, E., and Shanahan, F. (1991) Development of a biotinylated analog of substance P for use as a receptor probe. Lab. Invest. 64, 703–708.PubMedGoogle Scholar
  3. Bahouth, S. W., Stewart, J. M., and Musacchio, J. M. (1984) Specific binding of a125Ilabeled substance P analog to rat submaxillary gland. J. Pharmacol. Exp. Ther. 230, 116–123.PubMedGoogle Scholar
  4. Bahouth, S. W., Lazaro, D. M., Brundish, D. E., and Musacchio, J. M. (1985) Specific binding of [3H-Tyr8]physalaemin to rat submaxillary gland substance P receptor. Mol. Pharmacol. 27, 38–45.PubMedGoogle Scholar
  5. Bahouth, S. W. and Musacchio, J. M. (1985) Specific binding of [3H]substance P to the rat submaxillary gland. The effects of ions and guanine nucleotides. J. Pharmacol. Exp. Ther. 234, 326–336.PubMedGoogle Scholar
  6. Beaujouan, J. C., Torrens, Y., Herbet, A., Daguet, M.-C., Glowinski, J., and Prochiantz, A. (1982) Specific binding of an immunoreactive and biologically active125I-labeled substance P derivative to mouse mesencephalic cells in primary culture. Mol. Pharmacol. 22, 48–55.PubMedGoogle Scholar
  7. Beaujouan, J. C., Torrens, Y., Viger, A., and Glowinski, J. (1984) A new type of tachykinin binding site in the rat brain characterized by specific binding of a labeled eledoisin derivative. Mol. Pharmacol. 26, 248–254.PubMedGoogle Scholar
  8. Bergstrom, L., Beaujouan, J. C., Torrens, Y., Saffroy, M., Glowinski, J., Lavielle, S., Chassaing, G., Marquet, A., D’Orleans-Juste, P., Dion, S., and Regoli, D. (1987a) 3H-neurokinin A labels a specific tachykinin-binding site in the rat duodenal smooth muscle. Mol. Pharmacol. 32, 764–771.Google Scholar
  9. L., Torrens, Y., Saffroy, M., Beaujouan, J. C., Lavielle, S., Chassaing, G., Morgat, J. L., Glowinski, J., and Marquet, A. (1987b) [3H]Neurokinin B and 125I-Bolton Hunter eledoisin label identical tachykinin binding sites in the rat brain. J. Neurochem. 48,125–133.CrossRefGoogle Scholar
  10. Blume, A. J. (1978) Interactions of ligands with opiate receptors of brain membranes: Regulation by ions and nucleotides. Proc. Natl. Acad. Sci. USA 75, 1713–1717.PubMedCrossRefGoogle Scholar
  11. Bolton, A. E. and Hunter, W. M. (1972) A new method for labelling protein hormones with radioiodine for use in the radioimmunoassay. J. Endocrinol. 55, 30,31.Google Scholar
  12. Bolton, A. E. and Hunter, W. M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem. J. 133, 529–538.PubMedGoogle Scholar
  13. Buck, S. H. and Burcher, E. (1985) The rat submaxillary gland contains predominantly P-type tachykinin binding sites. Peptides 6, 1079–1084.PubMedCrossRefGoogle Scholar
  14. Buck, S. H., Burcher, E., Shults, C. W., Lovenberg, W., and O’Donohue, T. L. (1984) Novel pharmacology of substance K-binding sites: A third type of tachykinin receptor. Science 226, 987–989.PubMedCrossRefGoogle Scholar
  15. Buck, S. H., Helke, C. J., Burcher, E., Shults, C. W., and O’Donohue, T. L. (1986) Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in rat central nervous system. Peptides 7, 1109–1120.PubMedCrossRefGoogle Scholar
  16. Buck, S. H. and Krstenansky, J. L. (1987) The dogfish peptides scyliorhinin I and scyliorhinin II bind with differential selectivity to mammalian tachykinin receptors. Eur. J. Pharmacol. 144, 109–111.PubMedCrossRefGoogle Scholar
  17. Buck, S. H. and Shatzer, S. A. (1988) Agonist and antagonist binding to tachykinin peptide NK-2 receptors. Life Sci. 42, 2701–2708.PubMedCrossRefGoogle Scholar
  18. Burcher, E. and Buck, S. H. (1986) Multiple tachykinin binding sites in hamster, rat and guinea pig urinary bladder. Eur. J. Pharmacol. 128, 165–177.PubMedCrossRefGoogle Scholar
  19. Burcher, E., Buck, S. H., Lovenberg, W., and O’Donohue, T. L. (1986) Characterization and autoradiographic localization of multiple tachykinin binding sites in gastrointestinal tract and bladder. J. Pharmacol. Exp. Ther. 236, 819–831.PubMedGoogle Scholar
  20. Bylund, D. B. and Yamamura, H. I. (1990) Methods for receptor binding, in Methods in Neurotransmitter Receptor Analysis (Yamamura, H. I., Enna, S. J., and Kuhar, M. J., eds.), Raven, New York, pp. 37–68.Google Scholar
  21. Cascieri, M. A., Ber, E., Ming Fong, T., Sadowski, S., Bansal, A., Swain, C., Seward, E., Frances, B., Burns, D., and Strader, C. D. (1992) Characterization of the binding of a potent, selective, radioiodinated antagonist to the human neurokinin-1 receptor. Mol. Pharmacol. 42, 458–463.PubMedGoogle Scholar
  22. Cascieri, M. A. and Liang, T. (1983) Characterization of the substance P receptor in rat brain cortex membranes and the inhibition of radioligand binding by guanine nucleotides. J. Biol. Chem.. 258, 5158–5164.PubMedGoogle Scholar
  23. Cascieri, M. A. and Liang, T. (1984) Binding of [1251]Bolton hunter conjugated eledoisin to rat brain cortex membranes—Evidence for two classes of tachykinin receptors in the mammalian central nervous system. Life Sci. 35, 179–184.PubMedCrossRefGoogle Scholar
  24. Chang, R. S. L. and Snyder, S. H. (1980) Histamine H1-receptor binding sites in guinea pig brain membranes: Regulation of agonist interactions by guanine nucleotides and cations. J. Neurochem. 34, 916–922.PubMedCrossRefGoogle Scholar
  25. Childers, S. R. and Snyder, S. H. (1980) Differential regulation by guanine nucleotides of opiate agonist an antagonist receptor interactions. J. Neurochem. 34, 583–594PubMedCrossRefGoogle Scholar
  26. Conlon, J. M., Deacon, C. F., O’Toole, L., and Thim, L. (1986) Scyliorhinin I and II: Two novel tachykinins from dogfish gut. FEBS Lett. 200, 111–116.PubMedCrossRefGoogle Scholar
  27. Creese, I., Usdin, T. B., and Snyder, S. H. (1980) Dopamine receptor binding regu-lated by guanine nucleotides. Mol. Pharmacol. 16, 69–76.Google Scholar
  28. Cuatrecasas, P. and Hollenberg, M. D. (1976) Membrane receptors and hormone action. Adv. Prot. Chem. 30, 251–451.Google Scholar
  29. Ehlert, F. J., Roeske, W.R., and Yamamura, H. I. (1981) Muscarinic receptor: Regulation by guanine nucleotides, ions and N-ethylaleimide. Fed. Proc. 40, 153–159.PubMedGoogle Scholar
  30. Foster, A. C. and Tridgett, R. (1988) Comparison of the binding of radiolabelled neurokinin A and eledoisin in rat cortex synaptic membranes. Br. J. Pharmacol. 94, 602–608.PubMedCrossRefGoogle Scholar
  31. Goodman, R. R., Cooper, M. J., Gavish, M., and Snyder, S. H. (1982) Guanine nucleotide and cation regulation of the binding of [3H]cyclohexyladenosine and [3H]diethylphenylxanthine to adenosine Al receptors in brain membranes. Mol. Pharmacol. 21, 329–335.PubMedGoogle Scholar
  32. Guard, S., Watson, S. P., Maggio, J. E., Phon Too, H., and Watling, K. J. (1990) Pharmacological analysis of [311]-senktide binding to NK3 tachykinin receptors in guinea pig ileum longitudinal muscle-myenteric plexus and cerebral cortex membranes. Br. J. Pharmacol. 99, 767–773.PubMedCrossRefGoogle Scholar
  33. 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.PubMedCrossRefGoogle Scholar
  34. Henderson, A. K., Lai, J., Buck, S. H., Fujiwara, Y., Singh, G., Yamamura, m. S., Nakanishi, S., Roeske, W.R., and Yamamura, H. I. (1990) A cloned neurokinin A receptor mediates phosphatidylinositol hydrolysis in a transfected murine fibroblast. Life Sci. 47, PL-7—PL-12.CrossRefGoogle Scholar
  35. Jensen, R. T. and Gardner, J. D. (1979) Interaction of physalaemin, substance P, and eledoisin with specific membrane receptors on pancreatic acinar cells. Proc. Natl. Acad. Sci. USA 76, 5679–5683.PubMedCrossRefGoogle Scholar
  36. Laufer, R., Gilon, C., Chorev, M., and Selinger, Z. (1986) Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand. J. Biol. Chem. 261, 10,257–10,263.PubMedGoogle Scholar
  37. Lembeck, F., Mayer, N., and Schindler, G. (1977) Substance P in rat brain synaptosomes. Naunyn Schmiedeberg’s Arch. Pharmacol. 301, 17–22.CrossRefGoogle Scholar
  38. Lee, C.-M., Javitch, J. A. and Snyder, S. H. (1983) 3H-substance P binding to salivary gland membranes. Regulation by guanyl nucleotides and divalent cations. Mol. Pharmacol. 23, 563–569.PubMedGoogle Scholar
  39. Lee, C.-M., Campbell, N. J., Williams, B. J., and Iversen, L. L. (1986) Multiple tachykinin binding sites in peripheral tissues and in brain. Eur. J. Pharmacol. 130, 209–217.PubMedCrossRefGoogle Scholar
  40. Lew, R., Geraghty, D. P., Drapeau, G., Regoli, D., and Burcher, E. (1990) Binding characteristics of [125I]Bolton-Hunter [Sar9,Met(O2)“]substance P, a new selective radioligand for the NK, receptor. Eur. J. Pharmacol. 184, 97–108.PubMedCrossRefGoogle Scholar
  41. Liang, T. and Cascieri, M. A. (1980) Specific binding of an immunoreactive and biologically active 125I-labeled N(1)acylated substance P derivative to parotid cells. Biochem. Biophys. Res. Commun. 96, 1793–1799.PubMedCrossRefGoogle Scholar
  42. Liang, T. and Cascieri, M. A. (1981) Substance P receptor on parotid cell membranes. J. Neurosci. 1, 1133–1141.PubMedGoogle Scholar
  43. Luber-Narod, J., Boyd, N. D., and Leemean, S. E. (1990) Guanine nucleotides decrease the affinity of substance P binding to its receptor. Eur. J. Pharmacol. 188, 185–191.PubMedCrossRefGoogle Scholar
  44. Macdonald, S. G. and Boyd, N. D. (1989) Regulation of substance P receptor affinity by guanine nucleotide-binding proteins. J. Neurochem. 53, 264–272.PubMedCrossRefGoogle Scholar
  45. Mantyh, P. W., Gates, T., Mantyh, C. R., and Maggio, J. E. (1989) Autoradiographic localization and characterization of tachykinin receptor binding sites in the rat brain and peripheral tissues. J. Neurosci. 9, 258–279.PubMedGoogle Scholar
  46. Mantyh, P. W., Pinnock, R. D., Downes, C. P., Goedert, M., and Hunt, S. P. (1984) Correlation between inositol phospholipid hydrolysis and substance P receptors in rat CNS. Nature 309, 795–797.PubMedCrossRefGoogle Scholar
  47. Mayer, N., Lembeck, F., and Gamse, R. (1979) Substance P: Characteristics of binding to synaptic vesicles of rat brain. Naunyn Schmiedeberg’s Arch. Pharmacol. 306, 45–51.CrossRefGoogle Scholar
  48. McKnight, A. T., Maguire, J. J., Elliott, N. J., Fletcher, A. E., Foster, A. C., Tridgett, R., Williams, B. J., Longmore, J., and Iversen, L. L. (1991) Pharmacological specificity of novel, synthetic, cyclic peptides as antagonists at tachykinin receptors. Br. J. Pharmacol. 104, 355–360.PubMedCrossRefGoogle Scholar
  49. McLean, S., Ganong, A. H., Seeger, T. F., Bryce, D. K., Pratt, K. G., Reynolds, L. S., Siok, C. J., Lowe, III, J. A., and Heym, J. (1991) Activity and distribution of binding sites in brain of a nonpeptide substance P (NK1) receptor antagonist. Science 251, 437–439.PubMedCrossRefGoogle Scholar
  50. Michelot, R., Gozlan, H., Beaujouan, J. C., Besson, M. J., Torrens, Y., and Glowinski, J. (1980) Synthesis and biological activities of substance P iodinated derivatives. Biochem. Biophys. Res. Commun. 95, 491–498.PubMedCrossRefGoogle Scholar
  51. Mohini Sharma, P. and Musacchio, J. M. (1987) N-ethylmaleimide blocks the modulatory effects of divalent cations and guanine nucleotides on the brain substance P receptor. Eur. J. Pharmacol. 138, 9–19.CrossRefGoogle Scholar
  52. Mohini, P., Bahouth, S. W., Brundish, D. E., and Musacchio, J. M. (1985) Specific labeling of rat brain substance P receptor with [3H]physalaemin. J. Neurosci. 5, 2078–2085.PubMedGoogle Scholar
  53. Mussap, C. J., Geraghty, D. P., and Burcher, E. (1993) Tachykinin receptors: A radioligand binding perspective. J. Neurochem. 60, 1987–2009.PubMedCrossRefGoogle Scholar
  54. Mussap, C. J. and Burcher, E. (1990) [125I]-Bolton-Hunter scyliorhinin II: A novel, selective radioligand for the tachykinin NK3 receptor in rat brain. Peptides 11, 827–836.PubMedCrossRefGoogle Scholar
  55. Nakata, Y., Kusaka, Y., Segawa, T., Yajima, H., and Kitagawa, K. (1978) Substance P: Regional distribution and specific binding to synaptic membranes in rabbit central nervous system. Life Sci. 22, 259–268.PubMedCrossRefGoogle Scholar
  56. Park, C. H., Massari, V. J., Quirion, R., Tizabi, Y., Shults, and O’Donohue, T. L. (1984) Characteristics of 3H-substance P binding sites in rat brain membranes.Peptides 5, 833–836.Google Scholar
  57. Perrone, M. H., Diehl, R. E., and Haubrich, D. R. (1983) Binding of [3H]substance P tp putative substance P receptors in rat brain membranes. Eur. J. Pharmacol. 95, 131–133.PubMedCrossRefGoogle Scholar
  58. Petitet, F., Beaujouan, J. C., Saffroy, M., Torrens, Y., Chassaing, G., Lavielle, S., Besseyre, J., Garret, C., Carruette, A., and Glowinski, J. (1991) Further demonstration that [Pro9]-substance P is a potent and selective ligand of NK-1 tachykinin receptors. J. Neurochem. 56, 879–889.PubMedCrossRefGoogle Scholar
  59. Petitet, F., Saffroy, M., Torrens, Y., Glowinski, J., Beaujouan, J. C., Lavielle, S., Chassaing, G., and Marquet, A. (1988) 3H[Pro9]SP, a new selective ligand for NK1 binding sites. Regul. Pept. 22, 147.CrossRefGoogle Scholar
  60. Putney, J. W., Jr., Van De Walle, C. M., and Wheeler, C. S. (1980) Binding of 125Iphysalaemin to rat parotid acinar cells. J. Physiol. 301, 205–212.PubMedGoogle Scholar
  61. Rosenthal, H. E. (1967) Graphical method for the determination and presentation of binding parameters in a complex system. Anal. Biochem. 20, 525–532.PubMedCrossRefGoogle Scholar
  62. Saria, A., Mayer, N., Lembeck, F., and Pabst, M. (1980) Regional distribution and biochemical properties of’25I-Tyra-substance P binding sites in synaptic vesicles. Naunyn Schmiedeberg’s Arch. Pharmacol. 311, 151–157.CrossRefGoogle Scholar
  63. Segawa, T., Nakata, Y., Nakamura, K., Yajima, H., and Kitagawa, K. (1976) Substance P in the central nervous system of rabbits: Uptake system differs from putative transmitters. Jpn. J. Pharmacol. 26, 757–760.PubMedCrossRefGoogle Scholar
  64. Sjödin, L., Brodin, E., Nilsson, G., and Conlon, T. P. (1980) Interaction of substance P with dispersed pancreatic acinar cells from the guiea pig. Binding of radioiodinated peptide. Acta. Physiol. Scand. 109, 97–105.PubMedCrossRefGoogle Scholar
  65. Snyder, S. H. (1979) Peptide and neurotransmitter receptors in the brain: Regulation by ions and guanyl nucleotides, in Central Regulation of the Endocrine System (Fuxe, K., Hökfelt, T., and Luft, R., eds.), Plenum, New York, pp. 109–117.CrossRefGoogle Scholar
  66. Torrens, Y., Lavielle, Chassaing, G., Marquet, A., Glowinski, J., and Beaujouan, J. C. (1984) Neuromedin K, a tool to further distinguish two central tachykinin binding sites. Eur. J. Pharmacol. 102, 381,382.Google Scholar
  67. Too, H.-P. and Hanley, M. R. (1988) Solubilization and characterization of substance P-binding sites from chick brain membranes. Biochem. J. 252, 545–551.PubMedGoogle Scholar
  68. Tousignant, C., Guillemette, G., Drapeau, G., and Regoli, D. (1989) Comparison of binding assay and biological activity on a NK-1 system with new selective ago-fists. Neuropeptides 14, 275–283.PubMedCrossRefGoogle Scholar
  69. Unnerstall, J. R. (1990) Computer-assisted analysis of binding data, in Methods in NeurotransmitterReceptorAnalysis (Yamamura, H. I., Enna, S.J., and Kuhar, M. J., eds.), Raven, New York, pp. 37–68.Google Scholar
  70. U’Prichard, D. C. and Snyder, S. H. (1980) Interactions of divalent cations and guanine nucleotides at alpha2 adrenergic receptor binding sites in bovine brain membranes. J. Neurochem. 34, 385–394.PubMedCrossRefGoogle Scholar
  71. Van Giersbergen, P. L. M., Conlon, J. M., and Buck, S. H. (1991) Binding sites for tachykinin peptides in the brain and stomach of the dogfish, Scyliorhinus canicula. Peptides 12, 1161–1163.CrossRefGoogle Scholar
  72. Van Giersbergen, P. L. M., Shatzer, S. A., Henderson, A. K., Lai, J., Nakanishi, S., Yamamura, H. I., and Buck, S. H. (1991) Characterization of a tachykinin peptide NK2 receptor transfected into murine fibroblast B82 cells. Proc. Natl. Acad. Sci. USA 88, 1661–1665.PubMedCrossRefGoogle Scholar
  73. Viger, A., Beaujouan, J. C., Torrens, Y., and Glowinski, J. (1983) Specific binding of a 125I-substance P derivative to rat brain synaptosomes. J. Neurochem. 40, 1030–1039.PubMedCrossRefGoogle Scholar
  74. Watling, K. J. (1992) Nonpeptide antagonists herald new era in tachykinin research. Trends Pharmacol. Sci. 13, 266–269.PubMedCrossRefGoogle Scholar
  75. Watson, S. P. and Iversen, L. L. (1984) 3H-substance P binding to guinea pig ileum longitudinal smooth muscle membranes. Regul. Pept. 8, 273–281.PubMedCrossRefGoogle Scholar
  76. Yamada, S., Yamamura, H. I., and Roeske, W. R. (1980) The regulation of cardiac a,adrenergic receptors by guanine nucleotides and by muscarinic cholinergic ago-fists. Eur. J. Pharmacol. 63, 239–241.PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media New York 1994

Authors and Affiliations

  • Paul L M. van Giersbergen
  • Stephen H. Buck

There are no affiliations available

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