Received December 24, 2004; Revised March 7, 2005; Accepted March 14, 2005. Author to whom all correspondence and reprint requests should be addressed: Robert C. Speth, PhD, Department of Pharmacology, School of Pharmacy, University of Mississippi, University, MS 38677. E-mail: speth@olemiss.edu Sarcosine 1 , Glycine 8 Angiotensin II Is a Functional AT 1 Angiotensin Receptor Antagonist R. C. Speth, 1,4 Kwan Hee Kim, 2 Terry S. Elton, 3,5 and Steve Simasko 1 1 Department of Veterinary and Comparative Anatomy Pharmacology and Physiology, and 2 School of Molecular Biosciences, Washington State University, Pullman, WA 99164; 3 Department of Chemistry, Brigham Young University, Provo, UT; 4 Present Address: Research Institute of Pharmaceutical Sciences and Department of Pharmacology, School of Pharmacy, University of Mississippi, University, MS 38677; and 5 Present Address, Division of Pharmacology, College of Pharmacy, Ohio State University, Columbus, OH 43210 Endocrine, vol. 26, no. 2, 83–87, March 2005 0969–711X/05/26:83–87/$30.00 © 2005 by Humana Press Inc. All rights of any nature whatsoever reserved. 83 Based on functional studies in the brain (6), SG Ang II was classified as a potent Ang II receptor antagonist based on its ability to block the dipsogenic actions of intracerebro- ventricular (ICV) Ang II. However, the inability of SG Ang II to compete for 125 I-Ang II binding sites in the bovine cere- bellum (7) raised questions about the mechanism whereby SG Ang II blocked the dipsogenic response to Ang II. Sub- sequent discovery that the predominant Ang II receptor subtype in the bovine cerebellum was the AT 2 subtype (8), and that the predominant receptor mediating the dipsogenic response to Ang II is the AT 1 subtype (9–11) led to the sug- gestion that SG Ang II was an AT 1 selective ligand (5). The studies described herein demonstrate that SG Ang II is a functional antagonist of AT 1 receptor-mediated stim- ulation of intracellular Ca 2+ mobilization and confirm the AT 1 receptor selectivity of this Ang II analog. Results The ability of SG Ang II and sarcosine 1 , isoleucine 8 Ang II (SI Ang II) to compete for 125 I-SI Ang II binding to cells stably transfected with Ang II receptors is demonstrated in Fig. 1 and Table 1. SG Ang II bound with high affinity to both AT 1A and AT 1B transfected CHO cells. In contrast, the K I of SG Ang II for AT 2 transfected cells was more than 1000-fold lower than for the AT 1 receptor transfected cells. The K I for SI Ang II was about threefold higher affinity than that for SG Ang II in AT 1A and AT 1B transfected cells and about 900-fold higher than that for SG Ang II in AT 2 transfected cells. The affinity of 125 I-SG Ang II and 125 I-SI Ang II for the Ang II receptor transfected CHO cells was similar to that of the corresponding un-iodinated peptides (Table 1). The K D for 125 I-SG Ang II binding in AT 1A and AT 1B transfected CHO cells was 4 and 2.2 nM, respectively. It was not pos- sible to determine a K D for 125 I-SG Ang II binding in the AT 2 transfected cells. The K D for 125 I-SI Ang II binding in AT 1A and AT 1B transfected CHO cells was 0.32 and 0.73 nM, respectively, while the K D of 125 I-SI Ang II for AT 2 transfected cells was 16.6 nM. The B max values for 125 I-SG Ang II and 125 I-SI Ang II were similar (Table 1). Sarcosine 1 , glycine 8 angiotensin II (SG Ang II) displayed unusual characteristics in early pharmacological stud- ies. It was a potent antagonist of the dipsogenic actions of intracerebroventricularly administered Ang II in the rat, but showed low affinity for bovine cerebellar Ang II receptors. It has recently been shown that SG Ang II binds preferentially to the AT 1 receptor. To deter- mine if SG Ang II is a functional antagonist of the AT 1 receptor-mediated calcium signaling, CHO cells stably transfected with AT 1 receptors were exposed to Ang II in the presence and absence of SG Ang II. At concen- trations of 10–100 nM, SG Ang II completely inhibited Ang II-stimulated intracellular Ca 2+ mobilization in AT 1A and AT 1B receptor-transfected cells. SG Ang II and 125 I- SG Ang II bound to AT 1A and AT 1B receptor-transfected cells with K D and K I values of 2–4 nM. In contrast, SG Ang II bound to AT 2 transfected cells with a K I value of 7.86 μM. These results demonstrate that SG Ang II is a selective and functional peptide antagonist of the AT 1 angiotensin II receptor subtype. Key Words: AT 1 receptors; calcium signaling; sarco- sine 1 , glycine 8 angiotensin II; antagonist; CHO cells; transfection. Introduction The actions of angiotensin II (Ang II) are mediated by two receptor subtypes: AT 1 and AT 2 (1). This discovery arose from the development of peptidomimetic and non- peptide antagonists selective for the two major Ang II recep- tor subtypes (2). Although some analogs of Ang II have been demonstrated to have AT 2 selectivity (3,4), it is only recently that an Ang II analog selective for the AT 1 receptor has been observed (5).