Autoradiography of Enzymes, Second Messenger Systems, and Ion Channels

  • David A. Walsh
  • John Wharton
Part of the Methods in Molecular Biology™ book series (MIMB, volume 306)


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.


Nitric Oxide Synthases Vanilloid Receptor Wire Loop Inositol Polyphosphate Paper Clip 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Igic, R. and Behnia, R. (2003) Properties and distribution of angiotensin I converting enzyme. Curr. Pharm. Des. 9, 697–706.PubMedCrossRefGoogle Scholar
  2. 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. 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.PubMedCrossRefGoogle Scholar
  4. 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.PubMedCrossRefGoogle Scholar
  5. 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.PubMedCrossRefGoogle Scholar
  6. 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.PubMedGoogle Scholar
  7. 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.PubMedCrossRefGoogle Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 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.PubMedCrossRefGoogle Scholar
  10. 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.PubMedCrossRefGoogle Scholar
  11. 11.
    Moncada, S., Palmer, R. M., and Higgs, E. A. (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109–142.PubMedGoogle Scholar
  12. 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.PubMedGoogle Scholar
  13. 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.PubMedCrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 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.PubMedGoogle Scholar
  16. 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.PubMedCrossRefGoogle Scholar
  17. 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.PubMedCrossRefGoogle Scholar
  18. 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.PubMedCrossRefGoogle Scholar
  19. 19.
    Szallasi, A. and Blumberg, P. M. (1999) Vanilloid (capsaicin) receptors and mechanisms. Pharmacol. Rev. 51, 159–212.PubMedGoogle Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. 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.PubMedCrossRefGoogle Scholar
  22. 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.PubMedCrossRefGoogle Scholar
  23. 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.PubMedCrossRefGoogle Scholar
  24. 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.PubMedCrossRefGoogle Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. 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.PubMedCrossRefGoogle Scholar
  27. 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.PubMedGoogle Scholar
  28. 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.PubMedGoogle Scholar
  29. 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.PubMedCrossRefGoogle Scholar
  30. 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.PubMedGoogle Scholar
  31. 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.PubMedCrossRefGoogle Scholar
  32. 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.PubMedGoogle Scholar
  33. 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.PubMedCrossRefGoogle Scholar
  34. 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.PubMedCrossRefGoogle Scholar
  35. 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.PubMedCrossRefGoogle Scholar
  36. 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.PubMedCrossRefGoogle Scholar
  37. 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.PubMedCrossRefGoogle Scholar
  38. 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.PubMedGoogle Scholar
  39. 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.PubMedGoogle Scholar
  40. 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.PubMedCrossRefGoogle Scholar
  41. 41.
    Cullen, P. J. (1998) Bridging the gap in inositol 1,3,4,5-tetrakisphosphate signalling. Biochim. Biophys. Acta. 1436, 35–47.PubMedGoogle Scholar
  42. 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.PubMedGoogle Scholar
  43. 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.CrossRefGoogle Scholar
  44. 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.PubMedCrossRefGoogle Scholar
  45. 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.PubMedCrossRefGoogle Scholar
  46. 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.PubMedCrossRefGoogle Scholar
  47. 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.PubMedCrossRefGoogle Scholar
  48. 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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • David A. Walsh
    • 1
  • John Wharton
    • 2
  1. 1.Academic RheumatologyUniversity of Nottingham, City HospitalNottinghamUK
  2. 2.Section on Experimental Medicine and ToxicologyImperial College LondonLondonUK

Personalised recommendations