Endothelin-Converting Enzyme Activity in Vascular Smooth Muscle Preparations In Vitro

  • Janet J. Maguire
Part of the Methods in Molecular Biology™ book series (MIMB, volume 206)


The synthesis of ET-1, by the specific cleavage of its larger precursor big ET-1, by one or more endothelin-converting enzymes (ECE) has been discussed in Chapter 7. In the human vasculature ET-1, big ET-1 and ECE-1 are localized to secretory and storage granules (1) within endothelial cells (2). ET-1 is released via the constitutive and stimulated pathways, together with big ET-1 (3), and it is possible that additional conversion of this released big ET-1 occurs on the surface of the underlying smooth muscle cells. This is predicted by the observation that big ET-1, infused into the human forearm, significantly increases plasma levels of immunoreactive ET and decreases forearm blood flow in a phosphoramidon-sensitive manner. The hemodynamic response to big ET-1 occurred too quickly for appreciable amounts of phosphoramidon to have penetrated cell membranes, suggesting that the ECE responsible for conversion of infused big ET-1 is probably an ectoenzyme. As endothelial ECE has a predominantly intracellular localization (1), expression of ECE on the surface of human smooth muscle cells may account for the rapid response to exogenous big ET-1. This is consistent with reports that, in isolated vascular preparations, removal of the endothelium does not alter responses to big ET-1 (5-8) implying the presence of a nonendothelial, presumably smooth muscle enzyme. Indeed, ECE activity has been reported in cultured vascular smooth muscle cells (9,10) and one isoform of ECE-1, ECE-1b, has been localized to the smooth muscle cell plasma membrane (11).


Fumaric Acid Human Coronary Artery Bathing Medium Penetrate Cell Membrane Human Smooth Muscle Cell 
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  1. 1.
    Russell F. D., Skepper J. N., and Davenport A. P. (1998) Human endothelial cell storage granules: a novel intracellular site for isoforms of the endothelinconverting enzyme. Circ. Res. 80, 314–321.Google Scholar
  2. 2.
    Davenport A. P., Kuc R. E., Plumpton C., Mockridge J. W., Barker P. J., and Huskinsson N. S. (1998) Endothelin-converting enzyme in human tissue. Histochem. J. 30, 1–16.CrossRefGoogle Scholar
  3. 3.
    Plumpton C., Kalinka S., Martin R. C., Horton J. K., and Davenport A. P. (1994) Effects of phosphoramidon and pepstatin A on the secretion of endothelin-1 and big endothelin-1 by human umbilical vein endothelial cells: measurement by two-site enzyme-linked immunosorbent assays. Clin. Sci. 87, 245–251.PubMedGoogle Scholar
  4. 4.
    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
  5. 5.
    Fukuroda T., Noguchi K., Tsuchida S., Nishikibe M., Ikemoto F., Okada K., and Yano M. (1990) Inhibition of biological actions of big endothelin-1 by phosphoramidon. Biochem. Biophys. Res. Commun.. 172, 390–395.PubMedCrossRefGoogle Scholar
  6. 6.
    Yano M., Okada K., Takada J., Hioki Y., Matsuyama K., Fukuroda T., et al. (1991) Endothelin-converting enzyme and its in vitro and in vivo inhibition. J. Cardiovasc. Pharmacol. 17(Suppl. 7), S26–S28.PubMedCrossRefGoogle Scholar
  7. 7.
    Hisaki K., Matsumura Y., Nishiguchi S., Fujita K., Takaoka M., and Morimoto S. (1993) Endothelium-independent pressor effect of Big endothelin-1 and its inhibition by phosphoramidon in rat mesenteric artery. Eur. J. Pharmacol. 241, 75–81.PubMedCrossRefGoogle Scholar
  8. 8.
    Mombouli J.-V., Le S.Q., Wasserstrum N., and Vanhoutte P. (1993) Endothelins 1 and 3 and big endothelin-1 contract isolated human placental veins. J. Cardiovasc. Pharmacol. 22(Suppl.8), S278–S281.PubMedCrossRefGoogle Scholar
  9. 9.
    Kent A. and Keenan A. K. (1995) Evidence for signalling by big endothelin-1 via conversion to endothelin-1 in pulmonary-artery smooth-muscle cells. Life Sci. 57, 1191–1196.PubMedCrossRefGoogle Scholar
  10. 10.
    Yu J. C. M. and Davenport A. P. (1995) Secretion of endothelin-1 and endothelin-3 by human cultured vascular smooth-muscle cells. Br. J. Pharmacol. 114, 551–557.PubMedGoogle Scholar
  11. 11.
    Emoto N., Nurhantari Y., Alimsardjono H., Xie J., Yamada T., Yanagisawa M., and Matsuo M. (1999) Constitutive lysosomal targeting and degradation of bovine endothelin-converting enzyme-1a mediated by novel signals in its alternatively spliced cytoplasmic tail. J. Biol. Chem. 274, 1509–1518.PubMedCrossRefGoogle Scholar
  12. 12.
    Takahashi M., Fukuda K., Shimada K., Barnes K., Turner A.J., Ikeda M., et al. (1995) Localization of rat endothelin-converting enzyme to vascular endothelial cells and some secretory cells. Biochem. J. 311, 657–665.PubMedGoogle Scholar
  13. 13.
    Maguire J. J., Johnson C. M., Mockridge J. W., and Davenport A. P. (1997) Endothelin converting enzyme (ECE) activity in human vascular smooth muscle. Br. J. Pharmacol. 122, 1647–1654.PubMedCrossRefGoogle Scholar
  14. 14.
    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
  15. 15.
    Matsumoto H., Suzuki N., Kitada C., and Fujino M. (1994) Endothelin family peptides in human plasma and urine: their molecular form and concentration. Peptides 15, 505–510.Google Scholar
  16. 16.
    Lerman A., Edwards B. S., Hallett J. W., Heublein D. M., Sandberg S. M., and Burnett J. C. (1991) Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis. N. Engl. J. Med. 325, 997–1001.PubMedCrossRefGoogle Scholar
  17. 17.
    Zeiher A. M., Goebel H., Schächinger V., and Ihling C. (1995) Tissue endothelin-1 immunoreactivity in the active coronary atherosclerotic plaque. A clue to the mechanism of increased vasoreactivity of the culprit lesion in unstable angina. Circulation 91, 941–947.PubMedGoogle Scholar
  18. 18.
    Bacon C. R., Cary N. R. B., and Davenport A. P. (1996) Endothelin peptide and receptors in human atherosclerotic coronary artery and aorta. Circ. Res. 79, 794–801.PubMedGoogle Scholar
  19. 19.
    Minamino T., Kurihara H., Takahashi M., Shimada K., Maemura K., Oda H., et al. (1997) Endothelin-converting enzyme expression in the rat vascular injury model and human coronary atherosclerosis. Circulation 95, 221–230.PubMedGoogle Scholar
  20. 20.
    Davenport A. P. and Kuc R. E. (2000) Cellular expression of isoforms of endothelin-converting enzyme-1 (ECE-1c, ECE-1b and ECE-1a) and endothelinconverting enzyme-2. J. Cardiovasc. Pharmacol. 36(Suppl. 1), S12–S14.PubMedGoogle Scholar
  21. 21.
    Maguire J. J. and Davenport A. P. (1998) Increased response to big endothelin-1 in atherosclerotic human coronary artery: functional evidence for up-regulation of endothelin-converting enzyme activity in disease. Br. J. Pharmacol. 125, 238–240.PubMedCrossRefGoogle Scholar
  22. 22.
    Grantham J. A., Schirger J. A., Williamson E. E., Heublein D., Wennberg P. W., Kirchengast M., et al. (1998) Enhanced endothelin-converting enzyme immunoreactivity in early atherosclerosis. J. Cardiovasc. Pharmacol. 31(Suppl.1), S22–S26.PubMedCrossRefGoogle Scholar
  23. 23.
    Ahn K., Sisneros A. M., Herman S. B., Pan S. M., Hupe D., Lee C., et al. (1998) Novel selective quinazoline inhibitors of endothelin converting enzyme-1. Biochem. Biophys. Res. Commum. 243, 184–190.CrossRefGoogle Scholar
  24. 24.
    Maguire J. J., Ahn K., and Davenport A. P. (1999) Inhibition of big endothelin-1 (big ET-1) responses in endothelium-denuded human coronary artery by the selective endothelin-converting enzyme-1 (ECE-1) inhibitor PD159790. Br. J. Pharmacol. 126, U102.CrossRefGoogle Scholar
  25. 25.
    Barker S., Khan N. G., Wood E. G., and Corder R. (2001) Effect of an antisense oligodeoxynucleotide to endothelin-converting enzyme-1c (ECE-1c) on ECE-1c mRNA, ECE-1 protein and endothelin-1 synthesis in bovine pulmonary artery smooth muscle cells. Mol. Pharmacol. 59, 163–169.PubMedGoogle Scholar
  26. 26.
    Maguire J. J., Kuc R. E., and Davenport A. P. (2001) Vasoconstrictor activity of novel endothelin peptide, ET-1 (1-31), in human mammary and coronary arteries in vitro. Br. J. Pharmacol. 134, 1360–1366.PubMedCrossRefGoogle Scholar
  27. 27.
    Urata H., Kinoshita A., Misono K. S., Bumpus F. M., and Husain A. (1990) Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart. J. Biol. Chem. 265, 22,348–22,357.PubMedGoogle Scholar
  28. 28.
    Zisman L. S., Abraham W. T., Meixell G. E., Vamvakias B. N., Quaife R. A., Lowes B. D., et al. (1995) Angiotensin II formation in the intact human heart: predominance of the angiotensin-converting enzyme pathway. J. Clin. Invest. 95, 1490–1498.CrossRefGoogle Scholar
  29. 29.
    Kokkonen J. O., Saarinen J., and Kovanen P. T. (1998) Angiotensin II formation in the human heart: an ACE or nonACE-mediated pathway? Ann. Med. 30(Suppl. 1), 9–13.PubMedGoogle Scholar
  30. 30.
    Turner A. J. and Murphy L. J. (1996) Molecular pharmacology of endothelin converting enzymes. Biochem. Pharmacol. 51, 91–102.PubMedCrossRefGoogle Scholar
  31. 31.
    Yap E. Y. S., Battistini B., and McKay K. O. (2000) Contraction to big endothelin-1, big endothelin-2 and big endothelin-3, and endothelin-converting enzyme inhibition in human isolated bronchi. Br. J. Pharmacol. 129, 170–176.PubMedCrossRefGoogle Scholar
  32. 32.
    Rae G. A., Calixto J. B., and D’Orléans-Juste P. D. (1993) Conversion of big endothelin-1 in rat uterus causes contraction mediated by ETA receptors. J. Cardiovasc. Pharmacol. 22(Suppl. 8), S192–S195.PubMedCrossRefGoogle Scholar
  33. 33.
    Battistini B., Brown M., and Vane J. R. (1995) Selective proteolitic activation and degradation of ETs and Big ETs in parenchymal strips of guinea-pig lung. Biochem. Biophys. Res. Commum. 207, 675–681.CrossRefGoogle Scholar
  34. 34.
    Stork A. P. and Cocks T. M. (1994) Pharmacological reactivity of human epicardial coronary arteries: phasic and tonic responses to vasoconstrictor agents differentiated by nifedipine. Br. J. Pharmacol. 113, 1093–1098.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Janet J. Maguire
    • 1
  1. 1.Clinical Pharmacology UnitUniversity of CambridgeCambridgeUK

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