TY - JOUR T1 - Brain AT<sub>1</sub> Angiotensin Receptor Subtype Binding: Importance of Peptidase Inhibition for Identification of Angiotensin II as Its Endogenous Ligand JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 170 LP - 177 DO - 10.1124/jpet.109.157461 VL - 331 IS - 1 AU - Vardan T. Karamyan AU - Rama Gadepalli AU - John M. Rimoldi AU - Robert C. Speth Y1 - 2009/10/01 UR - http://jpet.aspetjournals.org/content/331/1/170.abstract N2 - The existence and localization of brain angiotensin receptors is well established. However, questions regarding the endogenous ligand for brain angiotensin type 1 (AT1) receptors necessitates re-examination of brain angiotensin receptor binding studies. To assess the ability of angiotensin II to bind to the brain AT1 receptor, radioligand binding studies of rat brain AT1 receptors were performed using both 125I-angiotensin II and 125I-sarcosine1, isoleucine8 angiotensin II. Determination of binding kinetics and competition by an AT1 receptor antagonist was carried out to reveal the identity of the membrane binding sites and to identify the bound 125I-labeled molecules. Initial analysis of 125I-angiotensin II binding to hypothalamic membranes using an established protocol revealed that a negligible amount of intact radioligand was bound to the membranes. In contrast, binding of 125I-sarcosine1, isoleucine8 angiotensin II was saturable, of high affinity, and primarily as intact radioligand. Sequential addition of four peptidase inhibitors—o-phenanthroline, puromycin, phenymethylsulfonyl fluoride, and glutamate phosphonate—to the assay buffer dramatically increased the binding of 125I-angiotensin II to rat brain membranes: more than 75% of the bound 125I was the intact radioligand, and the binding was of high affinity and saturable. Some, but not all, of the binding could be displaced by the AT1-selective antagonist losartan. This demonstrates that 125I-angiotensin II can bind to brain AT1 receptors and does not require conversion to 125I-angiotensin III to bind to brain AT1 receptors. © 2009 by The American Society for Pharmacology and Experimental Therapeutics ER -