Characterization of the binding properties and physiological action of divalinal-angiotensin IV, a putative AT(4) receptor antagonist

Divalinal-Ang IV [V Psi(CH2-NH2)YV Psi(CH2-NH2)HPF] is being employed increasingly as a specific AT(4) antagonist. This use, which necessitates a comprehensive physiological and pharmacological evaluation of Divalinal-Ang IV's functional and receptor binding characteristics in order to ensure its efficacy and specificity, was the stimulus for this study using bovine adrenal membranes. [I-125]Ang IV and [I-125]Divalinal-Ang IV were shown to bind with high affinity to a similar number of binding sites, suggesting that both bound the same receptor. This notion was verified by competition curves using [I-125]Ang IV and [I-125]Divalinal-Ang IV that indicated identical rank order affinities for several angiotensin-related peptides and 100% cross-displacement by Ang IV and Divalinal-Ang IV. Furthermore, an autoradiographic comparison of [I-125]Ang IV and [I-125]Divalinal-Ang IV in 20 mu m sections of bovine adrenals revealed near identical binding distributions characterized by heavy binding in the glomerulosa layer and the medulla. Physiological studies in which test compounds were injected into the internal carotid of the rat and cerebral blood flow (CBF) was measured by laser Doppler flowmetry indicated that pretreatment with Divalinal-Ang IV, but not DuP 753 or PD123177, blocked the increased flow observed with Ang IV infusion. Conversely, DuP 753, but not Divalinal-Ang IV or PD123177, inhibited the decrease in flow witnessed with Ang II. Metabolic stability studies utilizing rat kidney homogenates as a peptidase source, demonstrated that the structural changes present in Divalinal-Ang IV greatly increased its resistance to metabolism as compared to Ang IV. Together, these studies show that Divalinal-Ang IV is a stable, efficacious and specific inhibitor of AT(4) receptors.