Pharmaceutical Research

, Volume 17, Issue 12, pp 1482–1488 | Cite as

Inhibition of Angiotensin II-Induced Inositol Phosphate Production by Triacid Nonpeptide Antagonists in CHO Cells Expressing Human AT1 Receptors

  • Patrick M. L. VanderheydenEmail author
  • Ilse Verheijen
  • Frederik L. P. Fierens
  • Jean-Paul DeBacker
  • Georges Vauquelin


Purpose. The aim of the present work is to describe the inhibitory properties of LY301875 and LY303336, two polysubstituted 4-aminoimidazole AT1 receptor antagonists, on CHO cells expressing human recombinant AT1 receptors.

Methods. The binding of [3H]-angiotensin II to intact cells as well as to angiotensin II induced inositol phosphate accumulation is measured.

Results. Both antagonists inhibit specific [3H]-angiotensin II binding to AT1 receptors in these cells, with IC50 values of 5.9 and 5.2 nM, respectively. Preincubation of the cells with LY301875 results in a decline of up to 80 % of the maximal angiotensin II-stimulated inositol phosphate (IP) production. A near complete decline of the maximal response is observed for LY303336. This insurmountable inhibition is attenuated for both antagonists when losartan is included during the preincubation of the cells.

Conclusions. Functional recovery experiments, in which antagonist-preincubated cells are washed and exposed to fresh media, suggest that the insurmountable inhibition by LY301875 and LY303336 is related to their relatively slow dissociation from the AT1 receptors. As already described for losartan and the derived insurmountable AT1 antagonists candesartan, EXP3174, and irbesartan, coincubation experiments reveal that LY301875 and LY303336 interact with the AT1 receptor in a manner that is competitive with angiotensin II.

LY301875 LY303336 CHO cells AT1 receptor binding 


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  1. 1.
    M. J. Robertson. Angiotensin antagonists. In Receptor-based drug design, Marcel Dekker, New York, 1998 pp. 207–229.Google Scholar
  2. 2.
    G. H. Anderson, D. H. Streeten, and T. G. Dalakos. Pressor response to 1-sar-8-ala-angiotensin II (saralasin) in hypertensive subjects. Circ. Res. 40:243–250 (1977).Google Scholar
  3. 3.
    J. V. Duncia, D. J. Carini, A. T. Chiu, A. L. Johnson,W. A. Price, P. C. Wong, R. R. Wexler, and P. B. M. W. M. Timmermans. The discovery of Dup 753, a potent, orally active nonpeptide angiotensin II receptor antagonist. Med. Res. Rev. 12:149–191 (1992).Google Scholar
  4. 4.
    P. B. M. W. M Timmermans, P. C. Wong, A. T. Chiu, W. F. Herblin, P. Benfield, D. J. Carini, R. J. Lee, R. R. Wexler, J. M. Saye, and R. D. Smith. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol. Rev. 45:205–251 (1993).Google Scholar
  5. 5.
    J. C. Zhang, A. Van Meel, M. Pfaffendorf, and P. Van Zwieten. Different types of angiotensin II receptor antagonism induced by BIBS 222 in the rat portal vein and rabbit aorta; the influence of receptor reserve. J. Pharmacol. Exp. Ther. 269:509–514 (1993).Google Scholar
  6. 6.
    S. Mochizuki, T. Sato, K. Furata, K. Hase, Y. Ohkura, C. Fukai, K. Kosakai, S. Wakabayashi, and A. Tomiyama. Pharmacological properties of KT3-671, a novel nonpeptide angiotensin II receptor antagonist. J. Cardiovasc. Pharmacol. 25:22–29 (1995).Google Scholar
  7. 7.
    M. J. Robertson, J. C. Barnes, M. G. Drew, K. L. Clark, F. H. Marshall, A. Michel, D. Middlemiss, B. C. Ross, D. Scopes, and M. D. Dowle. Pharmacological profile of GR 117289 in vitro: A novel, potent and specific non-peptide angiotensin AT1 receptor antagonist. Br. J. Pharmacol. 107:1173–1180 (1992).Google Scholar
  8. 8.
    Y. J. Liu, N. P. Shankley, N. J. Welsh, and J. W. Black. Evidence that the apparent complexity of receptor antagonism by angiotensin II analogues is due to a reversible and syntopic action. Br. J. Pharmacol. 106:233–241 (1992).Google Scholar
  9. 9.
    L. Criscione, M. de Gasparo, P. Buhlmayer, S. Whitebread, H. P. Ramjoue, and J. Wood. Pharmacological profile of valsartan: a potent, orally active, nonpeptide antagonist of the angiotensin II AT1-receptor subtype. Br. J. Pharmacol. 110:761–771 (1993).Google Scholar
  10. 10.
    C. Cazaubon, J. Gougat, F. Bousquet, P. Guiraudou, R. Gayraud, C. Lacour, A. Roccon, G. Galindo, G. Barthelemy, B. Gautret, C. Bernhart, P. Perreaut, J.-C. Breliere, G. Le Fur, and D. Nisato. Pharmacological characterization of SR 47436, a new non-peptide AT1 subtype angiotensin II receptor antagonist. J. Pharmacol. Exp. Ther. 265:826–834 (1993).Google Scholar
  11. 11.
    M. Noda, Y. Shibouta, Y. Inada, M. Ojima, T. Wada, T. Sanada, K. Kubo, Y. Kohara, T. Naka, and K. Nishikawa. Inhibition of rabbit aortic angiotensin II (AII) receptor by CV-11974, a new neuropeptide AII antagonist. Biochem. Pharmacol. 46:311–318 (1993).Google Scholar
  12. 12.
    P. M. L. Vanderheyden, F. L. P. Fierens, J. P. De Backer, and G. Vauquelin. Reversible and syntopic interaction between angiotensin receptor antagonists on Chinese Hamster Ovary cells expressing human angiotensin II type 1 receptors. Biochem. Pharmacol. (in press).Google Scholar
  13. 13.
    F. L. P. Fierens, P. M. L. Vanderheyden, J. P. De Backer and G. Vauquelin. Insurmountable angiotensin AT1 receptor antagonists: the role of tight antagonist binding. Eur. J. Pharmacol. 372:199–206 (1999).Google Scholar
  14. 14.
    A. D. Palkowitz, M. I. Steinberg, K. J. Thrasher, J. K. Reel, K. L. Hauser, K. M. Zimmerman, S. A. Wiest, C. A. Whitesitt, R. L. Simon, W. Pfeifer, S. L. Lifer, D. B. Boyd, C. J. Barnett, T. M. Wilson, J. B. Deeter, K. Takeuchi, R. E. Riley, W. D. Miller, and W. S. Marshall. Structural evolution and pharmacology of a novel series of triacid angiotensin II receptor antagonists. J. Med. Chem. 37:4508–4521 (1994).Google Scholar
  15. 15.
    M. I. Steinberg, A. D. Palkowitz, K. J. Thrasher, J. K. Reel, K. M. Zimmerman, C. A. Whitesitt, R. L. Simon, K. L. Hauser, S. L. Lifer, W. Pfeifer, K. Takeuchi, S. A. Wiest, V. Vasudevan, K. G. Bemi, J. B. Deeter, C. J. Barnett, T. M. Wilson, W. S. Marshall, and D. B. Boyd. Chiral recognition of the angiotensin II (AT1) receptor by a highly potent phenoxyproline octanoamide. Bioorg. & Med. Chemistry Letts. 4:51–56 (1994).Google Scholar
  16. 16.
    P. M. L. Vanderheyden, F. L. P. Fierens, J. P. De Backer, N. Fraeyman, and G. Vauquelin. Distinction between surmountable and insurmountable selective AT1 receptor antagonists by use of CHO-K1 cells expressing human angiotensin II AT1 receptors. Br. J. Pharmacol. 126:1057–1065 (1999).Google Scholar
  17. 17.
    H. Furuta, D. F. Guo, and T. Inagami. Molecular cloning and sequencing of the gene encoding human angiotensin II type 1 receptor. Biochem. Biophys. Res. Commun. 183:8–13 (1992).Google Scholar
  18. 18.
    F. L. P. Fierens, P. M. L. Vanderheyden, J. P. De Backer, and G. Vauquelin. Binding of the antagonist [3H]candesartan to angiotensin II AT1 receptor-tranfected Chinese hamster ovary cells. Eur. J. Pharmacol. 367:413–422 (1999).Google Scholar
  19. 19.
    M. E. Pierson and R. J. Freer. Analysis of the active conformation of angiotensin II: A comparison of AII and non-peptide AII antagonists. Peptide Res. 5:102–105 (1992).Google Scholar
  20. 20.
    R. M. Keenan, J. Weinstock, J. A. Finkelstein, R. G. Franz, D. E. Gaitanopoulos, G. R. Girard, D. T. Hill, T. M. Morgan, J. M. Samanem, J. Hempel, D. S. Eggleston, N. Aiyar, E. Griffin, E. H. Ohlstein, E. J. Stack, E. F. Weidley, and R. J. Edwards. 1-(carboxybenzyl) imidazole-5-acrylic acids: potent and selective angiotensin II receptor antagonists. J. Med. Chem. 34:1514–1517 (1991).Google Scholar
  21. 21.
    P. B. M. W. M. Timmermans, P. C. Wong, A. T. Chiu, and W. F. Herblin. Nonpeptide Angiotensin II Receptor Antagonists. Trends Pharmacol Sci 12:55–62 (1991).Google Scholar
  22. 22.
    D. de Chaffoy de Courcelles, J. E. Leysen, P. Roevens, and H. Van Belle. The Serotonin-S2 receptor: A receptor, transducer coupling model to explain insurmountable antagonist effects. Drug Development Research 8:173–178 (1986).Google Scholar
  23. 23.
    M. J. Robertson, I. G. Dougal, D. Harper, K. C. W. Mckechnie, and P. Leff. Agonist-antagonist interactions at angiotensin receptors: application of a two-state receptor model. Trends Pharmacol. Sci. 15:364–369 (1994).Google Scholar
  24. 24.
    T. P. Kenakin. Drug antagonism. In: T. P. Kenakin (ed.), Pharmacological Analysis of Drug-Receptor Interaction, Raven Press, New York, 1987, pp. 1–30.Google Scholar
  25. 25.
    H. P. Rang. The kinetics of action of acetylcholine antagonists in smooth muscle. Proc. R. Soc. Lond. 164:488–510 (1966).Google Scholar
  26. 26.
    R. L. Panek, G. H. Lu, R. W. Overhisser, T. C. Major, J. C. Hodges, and D. G. Taylor. Functional studies but not receptor binding can distinguish surmountable from insurmountable AT1 antagonism. J. Pharm. Exp. Ther. 273:753–761 (1995).Google Scholar
  27. 27.
    Y. Shibouta, Y. Inada, M. Ojima, T. Wada, M. Noda, T. Sanada, K. Kubo, Y. Kohara, T. Naka, and K. Nishikawa. Pharmacological profile of a highly potent & long-acting angiotensin II receptor antagonist, 2-Ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1 H-benzimidazole-7-carboxylic acid (CV-11974), and its prodrug, (±)-1-(Cyclohexyloxycarbonyloxy)-ethyl 2-Ethoxy-I-[[2'-(1H-tetrazol-5-yl) biohenyl-4-yl]methyl]-1 H-benzimidazole-7-carboxylate (TCV-116). J. Pharm. Exp. Ther. 266:114–120 (1993).Google Scholar
  28. 28.
    P. Morsing, G. Adler, U. Brandt-Eliasson, L. Karp, K. Ohlson, L. Renberg, P. O. Sjöquist, and T. Abrahamson. Mechanistic differences of various AT1-receptor blockers in isolated vessels of different origin. Hypertension 33:1406–1413 (1999).Google Scholar
  29. 29.
    P. B. Timmermans. Pharmacological properties of angiotensin II receptor antagonists. Can. J. Cardiol. 15(F):26–28, (1999).Google Scholar
  30. 30.
    L. E. Limbird. Cell Surface Receptors. In: L. E. Limbird (ed.), A short course on theory and methods, Kluwer Acad. Publ., Boston, 1996 pp. 61–122.Google Scholar

Copyright information

© Plenum Publishing Corporation 2000

Authors and Affiliations

  • Patrick M. L. Vanderheyden
    • 1
    Email author
  • Ilse Verheijen
    • 2
  • Frederik L. P. Fierens
    • 2
  • Jean-Paul DeBacker
    • 2
  • Georges Vauquelin
    • 2
  1. 1.Department of Molecular and Biochemical Pharmacology, Institute of Molecular Biology and BiotechnologyFree University of Brussels (VUB)Sint-Genesius RodeBelgium
  2. 2.Department of Molecular and Biochemical Pharmacology, Institute of Molecular Biology and BiotechnologyFree University of Brussels (VUB)Sint-Genesius RodeBelgium

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