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Immunocytochemical Localization of Endothelin Peptides, Precursors, and Endothelin-Converting Enzymes

  • Rhoda E. Kuc
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Part of the Methods in Molecular Biology™ book series (MIMB, volume 206)

Abstract

This chapter will describe the protocol used for the immunocytochemical (ICC) visualization of endothelin (ET) peptides, precursors and endothelin converting enzymes (ECEs) in both frozen cryostat tissue sections and cultured cells using polyclonal primary antisera raised in rabbits. We have raised antisera against the ET-1 carboxy-terminal heptapeptide, ET-1(15–21), which is conserved in all three ET isoforms, (thus, the antibody does not distinguish between the three mature peptides); big ET-1(31–38), big ET-2(31–37) and big ET-3(31–42) (1), the ECE-1 isoforms (2,3) and ECE-2 (4) (see Fig. 1). The specificity of these antisera have been characterized using radioimmunoassays (RIA, [5], see  Chapter 2), enzyme-linked immunosorbant assays (ELISA, see  Chapter 2) and in a comprehensive range of human and animal tissues using ICC, including comparisons with commercially available antibodies, although in most cases the staining achieved with these was less intense (1,6).
Fig. 1.

Amino acid sequences used to generate site-directed antisera in endothelin peptides and endothelin-converting enzyme (ECE) proteins.

Keywords

Mature Peptide Nonspecific Background Staining Brown Reaction Product Alcohol Bath Excess Buffer 
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.

References

  1. 1.
    Howard P. G., Plumpton C., and Davenport A. P. (1992) Anatomical localization and pharmacological activity of mature endothelins and their precursors in human vascular tissue. J. Hypertens. 10, 1379–1386.PubMedCrossRefGoogle Scholar
  2. 2.
    Davenport A. P., Kuc R. E., Plumpton C., Mockridge J. W., Barker P. J., and Huskisson N. S. (1998) Endothelin-converting enzyme in human tissues. Histochem. J. 30, 359–374.CrossRefGoogle Scholar
  3. 3.
    Mockridge J. W., Kuc R. E., Huskisson N. S., Barker P. J., and Davenport A. P. (1998) Characterization of site-directed antisera against endothelin-converting enzymes. J. Cardiovasc. Pharmacol. 31, S35–S37.PubMedCrossRefGoogle Scholar
  4. 4.
    Russell F. D. and Davenport A. P. (1999) Evidence for intracellular endothelinconverting enzyme-2 expression in cultured human vascular endothelial cells. Circ. Res. 84, 891–896.PubMedGoogle Scholar
  5. 5.
    Plumpton C., Ashby M. J., Kuc R. E., O’Reilly G., and Davenport A. P. (1996) Expression of endothelin peptides and mRNA in the human heart. Clin. Sci. 90, 37–46.PubMedGoogle Scholar
  6. 6.
    Cameron I. T., Davenport A. P., van Papendorp C., Barker P. J., Huskisson N. S., Gilmour R. S., et al. (1992) Endothelin-like immunoreactivity in human endometrium. J. Reprod. Fertil. 95, 623–628.PubMedCrossRefGoogle Scholar
  7. 7.
    Marciniak S. J., Plumpton C., Barker P. J., Huskisson N. S., and Davenport A. P. (1992) Localization of immunoreactive endothelin and proendothelin in the human lung. Pulm. Pharmacol. 5, 175–182.PubMedCrossRefGoogle Scholar
  8. 8.
    Bacon C. R. and Davenport A. P. (1996) Endothelin receptors in human coronary artery and aorta. Br. J. Pharmacol. 117, 986–992.PubMedGoogle Scholar
  9. 9.
    Cameron I. T., Plumpton C., Champeney R., van Papendorp C., Ashby M. J. and Davenport A. P. (1993) Identification of endothelin-1, endothelin-2 and endothelin-3 in human endometrium. J. Reprod. Fertil. 98, 251–255.PubMedCrossRefGoogle Scholar
  10. 10.
    Davenport A. P., Hoskins S. L., Kuc R. E., and Plumpton C. (1996) Differential distribution of endothelin peptides and receptors in human adrenal gland. Histochem. J. 28, 779–789.PubMedCrossRefGoogle Scholar
  11. 11.
    Plumpton C., Haynes W. G., Webb D. J., and Davenport A. P. (1995) Phosphoramidon inhibition of the in vivo conversion of big endothelin-1 to endothelin-1 in the human forearm. Br. J. Pharmacol. 116, 1821–1828.PubMedGoogle Scholar
  12. 12.
    Opgenorth T. J., Wu-Wong J. R., and Shiosaki K. (1992) Endothelin-converting enzymes. FASEB J. 6, 2653–2659.PubMedGoogle Scholar
  13. 13.
    Turner A. J. and Murphy L. J. (1996) Molecular pharmacology of endothelin converting enzymes. Biochem. Pharmacol. 51, 91–102.PubMedCrossRefGoogle Scholar
  14. 14.
    Xu D., Emoto N., Giaid A., Slaughter C., Kaw S., Dewit D., and Yanagisawa M. (1994) ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell 78, 473–485.PubMedCrossRefGoogle Scholar
  15. 15.
    Shimada K., Matsushita Y., Wakabayashi K., Takahashi M., Iijima Y., and Tanzawa K. (1995a) Cloning and functional expression of endothelin-converting enzyme cDNA. Biochem. Biophys. Res. Commun. 207, 807–812.PubMedCrossRefGoogle Scholar
  16. 16.
    Shimada K., Takahashi M., Ikeda M., and Tanzawa K. (1995b) Identification of two isoforms of an endothelin-converting enzyme-1. Febs. Lett. 371, 140–144.PubMedCrossRefGoogle Scholar
  17. 17.
    Emoto N. and Yanagisawa M. (1995) Endothelin converting enzyme-2 is a membrane bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum. J. Biol. Chem. 270, 15,262–15,268.PubMedCrossRefGoogle Scholar
  18. 18.
    Davenport A. P. and Kuc R. E. (2000) Cellular expression of isoforms of endothelin converting enzyme (ECE-1c, ECE-1b and ECE-1a) and Endothelinconverting enzyme-2. J. Cardiovasc. Pharmacol. 36, S12–S14.PubMedGoogle Scholar
  19. 19.
    Russell F. D. and Davenport A. P. (1999) Evidence for intracellular endothelinconverting enzyme-2 expression in cultured human vascular endothelial cells. Circ. Res. 84,8, 891–896.PubMedGoogle Scholar
  20. 20.
    Russell F. D., Coppell A. L., and Davenport A. P. (1998) In vitro enzymatic processing of radiolabeled big ET-1 in human kidney. Biochem. Pharmacol. 55, 697–701.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Rhoda E. Kuc
    • 1
  1. 1.Clinical Pharmacology UnitUniversity of CambridgeCambridgeUK

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