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Autoradiography of Enzymes, Second Messenger Systems, and Ion Channels

  • Protocol
Receptor Binding Techniques

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 306))

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Abstract

Autoradiographic detection of ligand binding to tissue sections has been used to localize, quantify, and characterize a diverse range of sites. Enzymes have been studied via selective inhibitors, ion channels using naturally occurring toxins, and second messenger systems using inositol polyphosphates. Ligand binding complements immunohistochemistry (see Chapter 8) and in situ hybridization (see Chapter 4) by permitting pharmacological characterization and quantification of active sites. Localization, affinity, and specificity of binding sites for ligands (see Chapter 5) can be correlated with functional studies performed with the same pharmacological agent. Bioactive ligands are often identified before their targets have been fully characterized, and radiolabeled ligands may become available before molecular and immunological reagents have been developed. A pharmacologically active agent may be synthesized before the endogenous ligand for its binding site has been identified, and autoradiographic methods may help elucidate the site of action of such agents.

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References

  1. Igic, R. and Behnia, R. (2003) Properties and distribution of angiotensin I converting enzyme. Curr. Pharm. Des. 9, 697–706.

    Article  PubMed  CAS  Google Scholar 

  2. Wei, L., Clauser, E., Alhenc-Gelas, F., and Corvol, P. (1992) The two homologous domains of human angiotensin I-converting enzyme interact differently with competitive inhibitors. J. Biol. Chem. 267, 13, 398–13,405.

    Google Scholar 

  3. Strittmatter, S. M. and Snyder, S. H. (1984) Angiotensin-converting enzyme in the male rat reproductive system: autoradiographic visualization with [3H]captopril. Endocrinology 115, 2332–2341.

    Article  PubMed  CAS  Google Scholar 

  4. Mendelsohn, F. A. (1984) Localization of angiotensin converting enzyme in rat forebrain and other tissues by in vitro autoradiography using 125i-labelled mk351a. Clin. Exp. Pharmacol. Physiol. 11, 431–435.

    Article  PubMed  CAS  Google Scholar 

  5. Correa, F. M., Guilhaume, S. S., and Saavedra, J. M. (1991) Comparative quantification of rat brain and pituitary angiotensin-converting enzyme with autoradiographic and enzymatic methods. Brain Res. 545, 215–222.

    Article  PubMed  CAS  Google Scholar 

  6. Sun, Y., Diaz-Arias, A. A., and Weber, K. T. (1994) Angiotensin-converting enzyme, bradykinin, and angiotensin ii receptor binding in rat skin, tendon, and heart valves: an in vitro, quantitative autoradiographic study. J. Lab. Clin. Med. 123, 372–377.

    PubMed  CAS  Google Scholar 

  7. Sun, Y., Cleutjens, J. P., Diaz-Arias, A. A., and Weber, K. T. (1994) Cardiac angiotensin converting enzyme and myocardial fibrosis in the rat. Cardiovasc. Res. 28, 1423–1432.

    Article  PubMed  CAS  Google Scholar 

  8. Sun, Y. and Weber, K. T. (1996) Angiotensin-converting enzyme and wound healing in diverse tissues of the rat. J. Lab. Clin. Med. 127, 94–101.

    Article  PubMed  CAS  Google Scholar 

  9. Walsh, D. A., Hu, D. E., Wharton, J., Catravas, J. D., Blake, D. R., and Fan, T. P (1997) Sequential development of angiotensin receptors and angiotensin I converting enzyme during angiogenesis in the rat subcutaneous sponge granuloma. Br. J. Pharmacol. 120, 1302–1311.

    Article  PubMed  CAS  Google Scholar 

  10. Zambetis-Bellesis, M., Dusting, G. J., Mendelsohn, F.A., and Richardson, K. (1991) Autoradiographic localization of angiotensin-converting enzyme and angiotensin ii binding sites in early atheroma-like lesions in rabbit arteries. Clin. Exp. Pharmacol. Physiol. 18, 337–340.

    Article  PubMed  CAS  Google Scholar 

  11. Moncada, S., Palmer, R. M., and Higgs, E. A. (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109–142.

    PubMed  CAS  Google Scholar 

  12. Michel, A. D., Phul, R. K., Stewart, T. L., and Humphrey, P. P. (1993) Characterization of the binding of [3H]-L-NG-nitro-arginine in rat brain. Br. J. Pharmacol. 109, 287–288.

    PubMed  CAS  Google Scholar 

  13. Kidd, E. J., Michel, A. D., and Humphrey, P. P. (1995) Autoradiographic distribution of [3H]L-NG-nitro-arginine binding in rat brain. Neuropharmacology 34, 63–73.

    Article  PubMed  CAS  Google Scholar 

  14. Hara, H., Waeber, C., Huang, P. L., Fujii, M., Fishman, M. C., and Moskowitz, M. A. (1996) Brain distribution of nitric oxide synthase in neuronal or endothelial nitric oxide synthase mutant mice using [3H]L-NG-nitro-arginine autoradiography. Neuroscience 75, 881–890.

    Article  PubMed  CAS  Google Scholar 

  15. Rutherford, R. A., McCarthy, A., Sullivan, M. H., Elder, M. G., Polak, J. M., and Wharton, J. (1995) Nitric oxide synthase in human placenta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies. Br. J. Pharmacol. 116, 3099–3109.

    PubMed  CAS  Google Scholar 

  16. Burazin, T. C. and Gundlach, A. L. (1995) Localization of no synthase in rat brain by [3H]L-NG-nitro-arginine autoradiography. Neuroreport 6, 1842–1844.

    Article  PubMed  CAS  Google Scholar 

  17. Jeremy, J. Y., Dashwood, M. R., Timm, M., et al. (1997) Nitric oxide synthase and adenylyl and guanylyl cyclase activity in porcine interposition vein grafts. Ann. Thorac. Surg. 63, 470–476.

    Article  PubMed  CAS  Google Scholar 

  18. Szallasi, A. and Blumberg, P. M. (1990) Specific binding of resiniferatoxin, an ultrapotent capsaicin analog, by dorsal root ganglion membranes. Brain Res. 524, 106–111.

    Article  PubMed  CAS  Google Scholar 

  19. Szallasi, A. and Blumberg, P. M. (1999) Vanilloid (capsaicin) receptors and mechanisms. Pharmacol. Rev. 51, 159–212.

    PubMed  CAS  Google Scholar 

  20. Gunthorpe, M. J., Benham, C. D., Randall, A., and Davis, J. B. (2002) The diversity in the vanilloid (TRPV) receptor family of ion channels. Trends Pharmacol. Sci. 23, 183–191.

    Article  PubMed  CAS  Google Scholar 

  21. Winter, J., Walpole, C. S., Bevan, S., and James, I. F. (1993) Characterization of resiniferatoxin binding sites on sensory neurons: co-regulation of resiniferatoxin binding and capsaicin sensitivity in adult rat dorsal root ganglia. Neuroscience 57, 747–757.

    Article  PubMed  CAS  Google Scholar 

  22. Szallasi, A., Blumberg, P. M., Nilsson, S., Hokfelt, T., and Lundberg, J. M. (1994) Visualization by [3H]Resiniferatoxin autoradiography of capsaicin-sensitive neurons in the rat, pig and man. Eur. J. Pharmacol. 264, 217–221.

    Article  PubMed  CAS  Google Scholar 

  23. Szallasi, A., Nilsson, S., Farkas-Szallasi, T., Blumberg, P. M., Hokfelt, T., and Lundberg, J. M. (1995) Vanilloid (capsaicin) receptors in the rat: distribution in the brain, regional differences in the spinal cord, axonal transport to the periphery, and depletion by systemic vanilloid treatment. Brain Res. 703, 175–183.

    Article  PubMed  CAS  Google Scholar 

  24. Gehlert, D. R. and Wamsley, J. K. (1986) In vitro autoradiographic localization of guanine nucleotide binding sites in sections of rat brain labeled with [3H]guanylyl5′-imidodiphosphate. Eur. J. Pharmacol. 129, 169–174.

    Article  PubMed  CAS  Google Scholar 

  25. Aoki, H., Onodera, H., Yamasaki, Y., Yae, T., Jian, Z., and Kogure, K. (1992) The role of GTP binding proteins in ischemic brain damage: autoradiographic and histopathological study. Brain Res. 570, 144–148.

    Article  PubMed  CAS  Google Scholar 

  26. Sim, L. J., Selley, D. E., and Childers, S. R. (1995) In vitro autoradiography of receptor-activated g proteins in rat brain by agonist-stimulated guanylyl 5′-[gamma-[35S]thio]-triphosphate binding. Proc. Natl. Acad. Sci. USA 92, 7242–7246.

    Article  PubMed  CAS  Google Scholar 

  27. Fields, T. A. and Casey, P. J. (1997) Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins. Biochem. J. 321, 561–571.

    PubMed  CAS  Google Scholar 

  28. Denhardt, D. T. (1996) Signal-transducing protein phosphorylation cascades mediated by ras/rho proteins in the mammalian cell: the potential for multiplex signalling. Biochem. J. 318, 729–747.

    PubMed  CAS  Google Scholar 

  29. Sovago, J., Dupuis, D. S., Gulyas, B., and Hall, H. (2001) An overview on functional receptor autoradiography using [35S]GTPgS. Brain Res. Brain Res. Rev. 38, 149–164.

    Article  PubMed  CAS  Google Scholar 

  30. Brandt, D. R. and Ross, E. M. (1985) GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates. J. Biol. Chem. 260, 266–272.

    PubMed  CAS  Google Scholar 

  31. Waeber, C. and Chiu, M. L. (1999) In vitro autoradiographic visualization of guanosine-5′-O-(3-[335S]thio)triphosphate binding stimulated by sphingosine 1-phosphate and lysophosphatidic acid. J. Neurochem. 73, 1212–1221.

    Article  PubMed  CAS  Google Scholar 

  32. Tanase, D., Martin, W. A., Baghdoyan, H. A., and Lydic, R. (2001) G protein activation in rat ponto-mesencephalic nuclei is enhanced by combined treatment with a mu opioid and an adenosine A1 receptor agonist. Sleep 24, 52–62.

    PubMed  CAS  Google Scholar 

  33. Shaw, J. L., Gackenheimer, S. L., and Gehlert, D. R. (2003) Functional autoradiography of neuropeptide Y Y1 and Y2 receptor subtypes in rat brain using agonist stimulated [35S]GTPgS binding. J. Chem. Neuroanat. 26, 179–193.

    Article  PubMed  CAS  Google Scholar 

  34. Hong, W. and Werling, L. (2001) Lack of effects by sigma ligands on neuropeptide Y-induced G-protein activation in rat hippocampus and cerebellum. Brain Res. 901, 208–218.

    Article  PubMed  CAS  Google Scholar 

  35. Maruo, J., Yoshida, A., Shimohira, I., Matsuno, K., Mita, S., and Ueda, H. (2000) Binding of [35S]GTPgS stimulated by (+)-pentazocine sigma receptor agonist, is abundant in the guinea pig spleen. Life Sci. 67, 599–603.

    Article  PubMed  CAS  Google Scholar 

  36. Chen, S. R., Sweigart, K. L., Lakoski, J. M., and Pan, H. L. (2002) Functional mu opioid receptors are reduced in the spinal cord dorsal horn of diabetic rats. Anesthesiology 97, 1602–1608.

    Article  PubMed  CAS  Google Scholar 

  37. Bantel, C., Childers, S. R., and Eisenach, J. C. (2002) Role of adenosine receptors in spinal G-protein activation after peripheral nerve injury. Anesthesiology 96, 1443–1449.

    Article  PubMed  CAS  Google Scholar 

  38. Walsh, D. A., Suzuki, T., Knock, G. A., Blake, D. R., Polak, J. M., and Wharton, J. (1994) AT1 receptor characteristics of angiotensin analogue binding in human synovium. Br. J. Pharmacol. 112, 435–442.

    PubMed  CAS  Google Scholar 

  39. Georgoussi, Z., Carr, C., and Milligan, G. (1993) Direct measurements of in situ interactions of rat brain opioid receptors with the guanine nucleotide-binding protein Go. Mol. Pharmacol. 44, 62–69.

    PubMed  CAS  Google Scholar 

  40. Wilcox, R. A., Primrose, W. U., Nahorski, S. R., and Challiss, R. A. (1998) New developments in the molecular pharmacology of the myo-inositol 1,4,5-trisphosphate receptor. Trends Pharmacol. Sci. 19, 467–475.

    Article  PubMed  CAS  Google Scholar 

  41. Cullen, P. J. (1998) Bridging the gap in inositol 1,3,4,5-tetrakisphosphate signalling. Biochim. Biophys. Acta. 1436, 35–47.

    PubMed  CAS  Google Scholar 

  42. Walsh, D. A., Mapp, P. I., Polak, J. M., and Blake, D. R. (1995) Autoradiographic localization and characterization of [3H]a-trinositol (1D-myo-inositol 1,2,6-trisphosphate) binding sites in human and mammalian tissues. J. Pharmacol. Exp. Ther. 273, 461–469.

    PubMed  CAS  Google Scholar 

  43. Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Inositol trisphosphate receptor localization in brain: variable stoichiometry with protein kinase C. Nature 325, 159–161.

    Article  CAS  Google Scholar 

  44. Nagata, E., Tanaka, K., Gomi, S., et al. (1994) Alteration of inositol 1,4,5-trisphosphate receptor after six-hour hemispheric ischemia in the gerbil brain. Neuroscience 61, 983–990.

    Article  PubMed  CAS  Google Scholar 

  45. Sim-Selley, L. J., Brunk, L. K., and Selley, D. E. (2001) Inhibitory effects of SR141716A on G-protein activation in rat brain. Eur J Pharmacol 414, 135–143.

    Article  PubMed  CAS  Google Scholar 

  46. Laitinen, J. T. (1999) Selective detection of adenosine A1 receptor-dependent G-protein activity in basal and stimulated conditions of rat brain [35S]guanosine 5′-(g-thio)triphosphate autoradiography. Neuroscience 90, 1265–1279.

    Article  PubMed  CAS  Google Scholar 

  47. Moore, R. J., Xiao, R., Sim-Selley, L. J., and Childers, S. R. (2000) Agonist-stimulated [35S]GTPgS binding in brain modulation by endogenous adenosine. Neuropharmacology 39, 282–289.

    Article  PubMed  CAS  Google Scholar 

  48. Sim, L. J., Selley, D. E., Xiao, R., and Childers, S. R. (1996) Differences in G-protein activation by mu-and delta-opioid, and cannabinoid, receptors in rat striatum. Eur. J. Pharmacol. 307, 97–105.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Academic Rheumatology, University of Nottingham, City Hospital, Nottingham, UK

    David A. Walsh

  2. Section on Experimental Medicine and Toxicology, Imperial College London, London, UK

    John Wharton

Authors
  1. David A. Walsh
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  2. John Wharton
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Editor information

Editors and Affiliations

  1. Clinical Pharmacology Unit, University of Cambridge, Centre for Clinical Investigation, Addenbrooke7s Hospital, Cambridge, UK

    Anthony P. Davenport

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© 2005 Humana Press Inc.

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Walsh, D.A., Wharton, J. (2005). Autoradiography of Enzymes, Second Messenger Systems, and Ion Channels. In: Davenport, A.P. (eds) Receptor Binding Techniques. Methods in Molecular Biology™, vol 306. Humana Press. https://doi.org/10.1385/1-59259-927-3:139

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  • DOI: https://doi.org/10.1385/1-59259-927-3:139

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-420-3

  • Online ISBN: 978-1-59259-927-1

  • eBook Packages: Springer Protocols

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