Brain AT₁Angiotensin Receptor Subtype Binding: Importance of Peptidase Inhibition for Identification of Angiotensin II as Its Endogenous Ligand

Abstract

The existence and localization of brain angiotensin receptors is well established. However, questions regarding the endogenous ligand for brain angiotensin type 1 (AT 1 ) receptors necessitates re-examination of brain angiotensin receptor binding studies. To assess the ability of angiotensin II to bind to the brain AT 1 receptor, radioligand binding studies of rat brain AT 1 receptors were performed using both 125 I-angiotensin II and 125 I-sarcosine 1 , isoleucine 8 angiotensin II. Determination of binding kinetics and competition by an AT 1 receptor antagonist was carried out to reveal the identity of the membrane binding sites and to identify the bound 125 I-labeled molecules. Initial analysis of 125 I-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 125 I-sarcosine 1 , isoleucine 8 angiotensin II was saturable, of high affinity, and primarily as intact radioligand. Sequential addition of four peptidase inhibitors-ophenanthroline, puromycin, phenymethylsulfonyl fluoride, and glutamate phosphonate-to the assay buffer dramatically increased the binding of 125 I-angiotensin II to rat brain membranes: more than 75% of the bound 125

Distinct Molecular Effects of Angiotensin II and Angiotensin III in Rat Astrocytes

“…In addition, there were no differences in Ang II, its aminopeptidase-resistant analogs, and Ang III initial peak pressor responses, findings that do not support a requisite formation of Ang III to elicit a response. It has also been established that 125 I-Ang II can bind to brain AT 1 receptors, an effect that does not require conversion to 125 I-Ang III [28]. …”

“…AMA was selected since it is an APA inhibitor that is used by many investigators to study Ang II metabolism [26]. GluP was selected since it has recently received much attention as a direct APA inhibitor with a better selectivity for APA [28]. This enzyme was selected as a target for inhibition since it is the major enzyme involved in Ang II conversion to Ang III [26, 29].…”

Brain angiotensin peptides regulate sympathetic tone and blood pressure

“…The same group indeed subsequently showed that Ang II is an agonist for the rat brain AT 1 receptor and does not require prior conversion to Ang III [138].…”

Anteroposterior distribution of AT1 angiotensin receptors in caudal brainstem cardiovascular regulatory centers of the rat

“…Five of the slides from each set were then incubated in AM5 buffer containing a concentration of 125 I-SI Ang II ranging from 80 pM to 1900 pM in the presence of 10 μM PD123319 (a selective non-peptide AT 2 receptor antagonist) for 120 minutes. Under these conditions, all specific 125 I-SI Ang II binding is at the AT 1 receptor (Karamyan et al, 2009). The sixth slide from each set was incubated with 1900 pM 125 I-SI Ang II in the presence of an AT 1 receptor saturating concentration (3 μM) of Ang II for 120 minutes to determine non-specific binding of 125 I-SI Ang II.…”