Angiotensin II Receptor Assays

Pancras C. Wong1

1 DuPont Merck Pharmaceutical Company, Wilmington, Delaware
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.9
DOI:  10.1002/0471141755.ph0409s00
Online Posting Date:  May, 2001
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This unit describes the use of an isolated rabbit thoracic aorta preparation for determining the nature, potency, and selectivity of antagonists to the agonism of angiotensin II at AT1 receptors by measuring their effects on the tension of the tissue.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1:

  • New Zealand White male rabbit, 2 to 4 kg (10 to 40 weeks old)
  • Pentobarbitol sodium (Nembutal, Abbott Labs or equivalent)
  • Krebs‐Henseleit solution (see recipe; pH 7.4 to 7.6 at 37°C), room temperature and 37°C
  • 3.4 M KCl solution (253 mg/ml in distilled water)
  • 10 mg/ml human angiotensin II (Sigma) in distilled water (may be stored at least 2 weeks at −20°C)
  • Antagonist of interest (e.g., losartan; Merck Research Labs)
  • Carbogen gas (95% O 2/5% CO 2; tank of compressed gas with regulator suitable for releasing gas at low pressure; i.e., 3 to 4 lb/in.2)
  • Petri dish
  • Surgical instruments: forceps, scalpels, and scissors, sterile
  • Fine surgical silk thread (4‐0 G)
  • Heated circulating water bath at 37°C
  • Heated 20‐ml organ baths maintained at 37°C, bubbled with carbogen
  • Isometric transducers (Grass FT.03 or equivalent)
  • Physiological recorder (Grass model 7E polygraph or equivalent)
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Literature Cited

Literature Cited
   Cazaubon, C., Gougat, J., Bousquet, F., Guiraudou, P., Gayraud, R., Lacour, C., Roccon, A., Galindo, G., Barthelemy, G., Gautret, B., Bernhart, C., Perreaut, P., Breliere, J.‐C., Le Fur, G. and Nisato, D. 1993. Pharmacological characterization of SR 47436, a new nonpeptide AT1 subtype angiotensin II receptor antagonist. J. Pharmacol. Exp. Ther. 265:826‐834.
   Chiu, A.T., McCall, D.E., Price, W.A., Wong, P.C., Carini, D.J., Duncia, J.V., Johnson, A.L., Wexler, R.R., Yoo, S.E. and Timmermans, P.B.M.W.M. 1990. Nonpeptide angiotensin II receptor antagonists. VII. Cellular and biochemical pharmacology of DuP 753, an orally active antihypertensive agent. J. Pharmacol. Exp. Ther. 252:711‐718.
   Criscione, L., de Gasparo, M., Bühlmayer, P., Whitebread, S., Ramjoué, H.R. and Wood, J. 1993. Pharmacological profile of valsartan: A potent, orally active, nonpeptide antagonist of the angiotensin II AT1‐receptor subtype. Br. J. Pharmacol. 11:761‐771.
   Edwards, R.M., Aiyar, N., Ohlstein, E.H., Weidley, E.F., Griffin, E., Ezekiel, M., Keenan, R., Ruffolo, R.R. and Weinstock, J. 1992. Pharmacological characterization of nonpeptide angiotensin II receptor antagonist, SK&F 108566. J. Pharmacol. Exp. Ther. 260:175‐181.
   Kenakin, T.P. (ed.) 1987. Isolated‐tissue response systems. In Pharmacologic Analysis of Drug‐Receptor Interactions. pp. 52‐84. Raven Press, New York.
   Leung, E., Rapp, J.M., Walsh, K.M., Zeitung, K.D. and Eglen, R.M. 1993. Characterization of angiotensin II receptors in smooth muscle preparations of the guinea pig in vitro. J. Pharmacol. Exp. Ther. 267:1521‐1528.
   Merlos, M., Casas, A. and Castaner, J. 1997. Tasosartan. Drugs Future 22:850‐855.
   Peach, M.J. 1977. Renin‐angiotensin system: Biochemistry and mechanism of action. Physiol. Rev. 57:313‐370.
   Rhaleb, N.E., Rouissi, N., Nantel, F., D'Orleans‐Juste, P., and Regoli, D. 1991. DuP 753 is a specific antagonist for the angiotensin receptor. Hypertension 17:480‐484.
   Shibouta, Y., Inada, Y., Ojima, M., Wada, T., Noda, M., Sanada, T., Kubo, K., Kohara, Y., Naka, T. and Nishikawa, K. 1993. Pharmacological profile of a highly potent and 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‐1‐[2é(1H‐tetrazol‐5‐yl)biphenyl‐4‐yl] methyl‐1 H ‐benzimidazole‐7‐ carboxylate (TCV‐116). J. Pharmacol. Exp. Ther. 266:114‐120.
   Tallarida, R.J. and Jacob, L.S. 1979. Isolated preparations: Dose‐response data. In The Dose‐Response Relation in Pharmacology. (T.P. Kenakin ed.) pp. 137‐141. Springer‐Verlag, New York.
   Wienen, W., Haue, Van Meel, J.C., Narr, B., Ries, U. and Entzeroth, M. 1993. Pharmacological characterization of the novel nonpeptide angiotensin II receptor antagonist, BIBR 277. Br. J. Pharmacol. 11:245‐252.
   Wong, P.C. and Hajj‐ali, A.F. 1994. Functional correlates of angiotensin receptor stimulation. In Medicinal Chemistry of the Renin‐Angiotensin System (P.B.M.W.M. Timmermans and R.R. Wexler, eds.) pp. 313‐346. Elsevier, Amsterdam.
   Wong, P.C. and Timmermans, P.B.M.W.M. 1991. Nonpeptide angiotensin II receptor antagonists: Insurmountable angiotensin II antagonism of EXP3892 is reversed by the surmountable antagonist DuP 753. J. Pharmacol. Exp. Ther. 258:49‐57.
   Wong, P.C. and P.B.M.W.M. Timmermans. 1995. Physiological effects of a new class of highly specific angiotensin II receptor antagonists. In Hypertension: Pathophysiology, Diagnosis and Management, (J.H. Laragh and B.M. Brenner, eds.) pp. 3079‐3098. Raven Press, New York.
   Wong, P.C., Hart, S.D., Zaspel, A.M., Chiu, A.T., Ardecky, R.J., Smith, R.D. and Timmermans, P.B.M.W.M. 1990a. Functional studies of nonpeptide angiotensin II receptor subtype‐specific ligands: DuP 753 (AII‐1) and PD123177 (AII‐2). J. Pharmacol. Exp. Ther. 255:584‐592.
   Wong, P.C., Price, W.A., Chiu, A.T., Carini, D.J., Duncia, J.V., Wexler, R.R., Johnson, A.L. and Timmermans, P.B.M.W.M. 1990b. Nonpeptide angiotensin II receptor antagonists: Studies with EXP9270 and DuP 753. Hypertension 15:823‐834.
   Wong, P.C., Price, W.A., Chiu, A.T., Duncia, J.V., Carini, D.J., Wexler, R.R., Johnson, A.L. and Timmermans, P.B.M.W.M. 1990c. Nonpeptide angiotensin II receptor antagonists. XI. Pharmacology of EXP3174, an active metabolite of DuP 753: An orally‐active antihypertensive agent. J. Pharmacol. Exp. Ther. 255:211‐217.
Key Reference
   Kenakin, T.P. ed. 1987. Pharmacologic Analysis of Drug‐Receptor Interaction. Raven Press, New York.
  Review of the background, experimental, and mathematical information on drug‐receptor interaction using an isolated‐tissue response system such as rabbit aorta.
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