Brain AT1 angiotensin receptor subtype binding: importance of peptidase inhibition for identification of angiotensin II as its endogenous ligand

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-o-phenanthroline, 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)I 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 AT(1)-selective antagonist losartan. This demonstrates that (125)I-angiotensin II can bind to brain AT(1) receptors and does not require conversion to (125)I-angiotensin III to bind to brain AT(1) receptors.