Characterization of Angiotensin II Receptors

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

Angiotensin II (Ang II) is the primary mediator of the renin angiotensin system and acts primarily as a circulating hormone, but may also act as a paracrine or autocrine factor. Ang II binds specifically to G protein?coupled receptors and triggers a number of characteristic cellular responses, including vascular smooth muscle contraction and adrenal cellular aldosterone secretion. There are multiple receptor subtypes for Ang II; however, the distinction between binding sites versus receptors with their associated functional manifestations has been controversial. There are two principal human receptor subtypes, designated AT₁ and AT₂ , that are distinguished by the selective affinity of the nonpeptide antagonists losartan and PD123177. In rodents, additional isoforms of the Ang II receptor have been cloned (e.g., AT_1A and AT_1B ), although their functional distinctions are undetermined. The focus of this unit is the identification of Ang II binding sites and receptor subtypes. Radioligand competition binding assays are provided for determining the affinity of Ang II for receptors on adrenal cortical microsomes, whole adrenal membranes, or CHO cells transfected with the AT_1A receptor.

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Basic Protocol 1: Measurement of Angiotensin II Affinity Using Competition Binding Assays in Adrenal Cortical Microsomes
Alternate Protocol 1: Measurement of Angiotensin II Affinity for Specific Receptor Subtypes in Whole Adrenal Membranes
Alternate Protocol 2: Measurement of Angiotensin II Affinity for Cloned Receptors on Transfected Cells
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Measurement of Angiotensin II Affinity Using Competition Binding Assays in Adrenal Cortical Microsomes

Materials

Adult male rats (250 to 350 g)
Sucrose buffer, pH 7.4 (see recipe ), ice cold
Tris buffer 1, pH 7.2 (see recipe ), ice cold
Bio‐Rad Protein Assay Reagent (optional)
8% dimethylsulfoxide (DMSO; EM Science)
80 µM unlabeled Ang II working solution (see recipe )
Unlabeled peptides and test compounds in 8% DMSO
1.2 nM [¹²⁵ I]Ang II working solution (see recipe )
2 mM 2‐n ‐butyl‐4‐chloro‐5‐(hydroxymethyl)‐1‐{[2′‐(1H ‐tetrazol‐5‐yl) biphenyl‐4‐yl]methyl}‐imidazole (losartan; mol. wt. 461; research samples for nonhuman studies can be obtained by qualified individuals free of charge from Merck Research Laboratories)
2 mM S ‐(+)‐1‐{[4‐dimethylamino‐3‐methylphenyl]methyl}‐5‐(diphenylacetyl)‐4,5,6,7‐tetrahydro‐1H ‐imidazo[4,5,‐c]pyridine‐6‐carboxylic acid ditrifluoroacetate (PD123319; Research Biochemicals)
0.2% (v/v) polyethylenimine (PEI; available as 50% aqueous solution; Sigma)

Chilled aluminum block or equivalent cold dissection surface
Ground‐glass tissue grinder or tissue homogenizer with Teflon pestle and 0.004‐ to 0.006‐in. clearance between pestle and tube (Ten Broeck or equivalent)
Talboy model 1342 T‐line stirrer
30‐ml polycarbonate centrifuge tube with cap (Sorvall)
Sorvall RT 6000 tabletop centrifuge
Fine cheesecloth
Sorvall OTD 65B ultracentrifuge with T865 rotor
Ultrasonicator (Braun‐sonic 1510 with 3/8‐in. intermediate probe)
Plate reader (Dynatech Labs)
96‐well polypropylene microtiter plates (Costar)
934AH or GF/B glass fiber filters (Whatman)
Cell harvester (Packard Micromate 196 or Brandel M‐24 or M‐48)
80°C oven
Gamma counter (Packard TopCount or Packard Cobra II)
12 × 75–mm polystyrene test tubes

Additional reagents and equipment for Bradford assay ( appendix 3A ) with BSA standard

Alternate Protocol 1: Measurement of Angiotensin II Affinity for Specific Receptor Subtypes in Whole Adrenal Membranes

Tris buffer 2, pH 7.7 (see recipe ), ice cold
Resuspension buffer (see recipe ), ice cold
52 µM unlabeled [Sar¹ Ile⁸ ]angiotensin II acetate salt ([Sar¹ Ile⁸ ]Ang II; mol. wt. 968.2; Sigma) in 8% DMSO
1.4 nM [¹²⁵ I][Sar¹ Ile⁸ ]Ang II working solution (see recipe )
Binding assay buffer (see recipe )
0.1% (w/v) BSA (Sigma)
Tris/NaCl buffer (see recipe ), ice cold
Polytron tissue homogenizer (Brinkmann) with ST‐20 probe
37‐ml polypropylene centrifuge tubes (Sorvall)
Sorvall RC5B superspeed centrifuge and SS‐34 rotor
96‐deep‐well polypropylene microtiter plates (Beckman)

Alternate Protocol 2: Measurement of Angiotensin II Affinity for Cloned Receptors on Transfected Cells

AT_1A ‐transfected Chinese hamster ovary (CHO) cells (NEN Life Sciences)
CHO growth medium (see recipe )
0.25 nM [¹²⁵ I]Ang II working solution (see recipe )
50 µM unlabeled Ang II working solution (see recipe )
HEPES buffer (see recipe ), ice cold and room temperature
50× test compound (inhibitor) in HEPES buffer
CHAPS buffer (see recipe )
24‐well culture plates

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Figures

Figure 1.10.1 Displacement of [¹²⁵ I]Ang II by unlabeled Ang II in rat adrenal cortical microsomes plotted by KaleidaGraph curve fit (m0 = [Ang II]; m1 = IC₅₀ ). NA, not applicable.

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Figure 1.10.2 Inhibition of [¹²⁵ I][Sar¹ Ile⁸ ]Ang II binding by unlabeled Ang II and saralasin in rat whole adrenal membranes pretreated with PD123319 to block AT₂ sites. The IC₅₀ values for Ang II and saralasin are 16 nM and 0.36 nM, respectively. The IC₅₀ values were calculated by linear regression analysis of the logit transform of the parent bound data.

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Figure 1.10.3 The inhibition of [¹²⁵ I]Ang II binding in intact Chinese hamster ovary cells expressing AT_1A receptors. The IC₅₀ values are 2.3 nM (Ang II), 22 nM (losartan), >1 µM (PD123177), 1.7 nM (saralasin), 87 nM (Ang I—reflects conversion to Ang II), and 6.4 nM (Ang III). IC₅₀ calculations were performed as in Table and Figure .

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Figure 1.10.4 Displacement of [¹²⁵ I]Ang II by unlabeled Ang II in rat adrenal cortical microsomes. (A ) Increasing concentrations of [¹²⁵ I]Ang II were added to a fixed amount of adrenal cortical membrane homogenate to create a ligand displacement curve (data from Table ). Nonspecific binding was determined in the presence of 10 µM unlabeled Ang II. (B ) The Scatchard plot produces a straight line and provides evidence for interaction of Ang II with a single site. The K _d for unlabeled Ang II is 3.934 nM, and the B _max is 2.468 pmol/mg of protein. Data were analyzed using LIGAND. B, bound; F, free; T, unlabeled [Ang II] (M).

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Videos

Literature Cited

	Catt, K.J., Sandberg, K., and Balla, T. 1993. Angiotensin II receptors and signal transduction mechanisms. In Cellular and molecular biology of the renin angiotensin system (M.K. Raizada, M.I. Phillips, and C. Sumners, eds.) pp. 307‐356. CRC Press, Boca Raton, Fla.
	Chang, R.S.L., Lotti, V.J., Chen, T.B., and Faust, K.A. 1990. Two angiotensin II binding sites in rat brain revealed using 125I‐Sar1‐Ile8‐angiotensin II and selective nonpeptide antagonists. Biochem. Biophys. Res. Commun. 171:813‐817.
	Chang, R.S.L., Lotti, V.J., Chen, T.B., Omalley, S.S., Bendesky, R.J., Kling, P.J., Kivlighn, S.D., Siegl, P.K.S., Ondeyka, D., Greenlee, W.J., and Mantlo, N.B. 1995. In vitro pharmacology of an angiotensin AT(1) receptor antagonist with balanced affinity for AT(2) receptors. Eur. J. Pharmacol. 294:429‐437.
	Chiu, A.T., Herblin, W.F., Ardecky, R.J., McCall, D.E., Carini, D.J., Duncia, J.V., Pease, L.J., Wexler, R.R., Wong, P.C., Johnson, A.L., and Timmermans, P.B.M.W.M. 1989a. Identification of angiotensin II receptor subtypes. Biochem. Biophys. Res. Commun. 165:196‐203.
	Chiu, A.T., Duncia, J.V., McCall, D.E., Wong, P.C., Price, W.A., Thoolen, M.J.M.C., Carini, D.J., Johnson, A.L., and Timmermans, P.B.M.W.M. 1989b. Nonpeptide angiotensin II receptor antagonists. III. Structure‐function studies. J. Pharmacol. Exp. Ther. 250:867‐874.
	Chiu, A.T., McCall, D.E., Aldrich, P.E., and Timmermans, P.B.M.W.M. 1990. 3H]DuP 753, a highly potent and specific radioligand for the angiotensin II‐1 receptor subtype. Biochem. Biophys. Res. Commun. 172:1195‐1202.
	Chiu, A.T., Carini, D.J., Duncia, J.V., Leung, K.H., McCall, D.E., Price, W.A., Wong, P.C., Smith, R.D., Wexler, R.R., and Timmermans, P.B.M.W.M. 1991. DuP 532: A second generation of nonpeptide angiotensin II receptor antagonists. Biochem. Biophys. Res. Commun. 177:209‐217.
	Chiu, A.T., Dunscomb, J.H., McCall, D.E., Benfield, P., Baubonis, W. and Sauer, B. 1993. Characterization of angiotensin AT1A receptor isoform by its ligand binding signature. Regul. Pept. 44:141‐147.
	Christ, D.D. 1995. Human plasma protein binding of the angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) and its pharmacologically active metabolite EXP3174. J. Clin. Pharmacol. 35:515‐520.
	Cox, B.E., Word, R.A., and Rosenfeld, C.R. 1996. Angiotensin II receptor characteristics and subtype expression in uterine arteries and myometrium during pregnancy. J. Clin. Endocrinol. Metab. 81:49‐58.
	DeGasparo, M., Husain, A., Alexander, W., Catt, K.J., Chiu, A.T., Drew, M., Goodfriend, T., Harding, J.W., Inagami, T. and Timmermans, P.B.M.W.M. 1995. Proposed update of the angiotensin receptor nomenclature. Hypertension 25:924‐927.
	Feral, C., Benhaim, A., and Leymarie, P. 1996. Angiotensin II receptor type I on granulosa and thecal cells of rabbit preovulatory follicles. Biochim. Biophys. Acta 1284:221‐226.
	Fukushige, S. and Sauer, B. 1992. Genomic targeting with a positive selection 10X integration reactor allows highly reproducible gene expression in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 89:7905‐7909.
	Griendling, K.K., Lassegue, B., and Alexander, R.W. 1996. Angiotensin receptors and their therapeutic implications. Annu. Rev. Pharmacol. Toxicol. 36:281‐306.
	Grove, K.L., and Speth, R.C. 1993. Angiotensin II and nonangiotensin II displaceable binding sites for [3H]losartan in the rat liver. Biochem. Pharmacol. 46:1653‐1660.
	Johren, O., Viswanathan, M., and Saavedra, J.M. 1995. Expression of the non‐angiotensin II I‐12undefinedCGP‐42112 binding sites on activated microglia after kainic acid‐induced neurodegeneration. Brain Res. 702:153‐161.
	McQueen, J., and Semple, P.F. 1991. Angiotensin receptor assay and characterization. In Methods in neurosciences (P.M. Conn, ed.) pp. 312‐330. Academic Press, San Diego.
	Meffert, S., Stoll, M., Steckelings, U.M., Bottari, S.P., and Unger, T. 1996. The angiotensin II AT2 receptor inhibits proliferation and promotes differentiation in PC12W cells. Mol. Cell. Endocrinol. 122:59‐67.
	Nouet, S., Dodey, P., Renaut, P., Marie, J., Pruneau, D., Larguier, R., Lombard, C., and Bonnafous, J.C. 1994. Properties of 3H‐LF7‐0156, a new nonpeptide antagonist radioligand for the type 1 angiotensin II receptor. Mol. Pharmacol. 46:693‐701.
	Nozawa, Y., Miyake, H., Haruno, A., Yamada, S., Uchida, S., Ohkura, T., Kimura, R., Suzuki, H., and Hoshino, T. 1996. Down regulation of angiotensin II receptors in hypertrophied human myocardium. Clin. Exp. Pharmacol. Physiol. 23:514‐518.
	Rogg, H., DeGasparo, M., Graedel, E., Stulz, P., Burkart, F., Eberhard, M., and Erne, P. 1996. Angiotensin II receptor subtypes in human atria and evidence for alterations in patients with cardiac dysfunction. Eur. Heart J. 17:1112‐1120.
	Schambye, H.T., Hjorth, S.A., Weinstock, J., and Schwartz, T.W. 1995. Interaction between the nonpeptide angiotensin antagonist SKF108566 and histidine‐256 (HISVI‐16) of the angiotensin type 1 receptor. Mol. Pharmacol. 47:425‐431.
	Smith, R.D., and Timmermans, P.B.M.W.M. 1994. Human angiotensin receptor subtypes. Curr. Opin. Nephrol. Hypertens. 3:112‐122.
	Timmermans, P.B.M.W.M., and Smith, R.D. 1994. Angiotensin II receptor subtypes: Selective antagonists and functional correlates. Eur. Heart J. 15(Suppl D):79‐87.
	Timmermans, P.B.M.W.M., Wong, P.C., Chiu, A.T., Herblin, W.F., Benfield, P., Carini, D.J., Lee, R.J., Wexler, R.R., Saye, J.M., and Smith, R.D. 1993. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol. Rev. 45:205‐251.
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	Whitebread, S., Mele, M., Kamber, B., and DeGasparo, M. 1989. Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem. Biophys. Res. Commun. 163:284‐291.
Key References
	DeGasparo et al., 1995. See above.
	Provides recommended nomenclature and operational characteristics for Ang II receptors.
	Griendling et al., 1996. See above.
	Current overview of Ang II receptors describing receptor structure, function, and therapeutic implications.
	McQueen, and Semple, 1991. See above.
	Excellent review of Ang II receptor assay methodology.
	Timmermans et al., 1993. See above.
	Comprehensive review of history of Ang II, Ang II receptor heterogeneity, and the discovery of nonpeptide receptor antagonists.

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Characterization of Angiotensin II Receptors

Abstract

Table of Contents

Materials

Basic Protocol 1: Measurement of Angiotensin II Affinity Using Competition Binding Assays in Adrenal Cortical Microsomes

Alternate Protocol 1: Measurement of Angiotensin II Affinity for Specific Receptor Subtypes in Whole Adrenal Membranes

Alternate Protocol 2: Measurement of Angiotensin II Affinity for Cloned Receptors on Transfected Cells

Figures

Videos

Literature Cited