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).
Amino acid sequences used to generate site-directed antisera in endothelin peptides and endothelin-converting enzyme (ECE) proteins.
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References
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.
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.
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.
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.
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.
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.
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.
Bacon C. R. and Davenport A. P. (1996) Endothelin receptors in human coronary artery and aorta. Br. J. Pharmacol. 117, 986–992.
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.
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.
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.
Opgenorth T. J., Wu-Wong J. R., and Shiosaki K. (1992) Endothelin-converting enzymes. FASEB J. 6, 2653–2659.
Turner A. J. and Murphy L. J. (1996) Molecular pharmacology of endothelin converting enzymes. Biochem. Pharmacol. 51, 91–102.
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.
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.
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.
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.
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.
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.
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.
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Kuc, R.E. (2002). Immunocytochemical Localization of Endothelin Peptides, Precursors, and Endothelin-Converting Enzymes. In: Maguire, J.J., Davenport, A.P. (eds) Peptide Research Protocols. Methods in Molecular Biology™, vol 206. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-289-9:003
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DOI: https://doi.org/10.1385/1-59259-289-9:003
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