Central infusion of angiotensin IV or its more stable analogues facilitates memory retention and retrieval in normal animals and reverses amnesia induced by scopolamine or by bilateral perforant pathway lesions. These peptides bind with high affinity and specificity to a novel binding site designated the angiotensin AT 4 receptor. Until now, the AT 4 receptor has eluded molecular characterization. Here we identify the AT 4 Central infusions of the hexapeptide VYIHPF (angiotensin IV, Ang IV) 1 or its more stable analogues, Nle 1 -Ang IV andNorleucinal Ang IV, facilitate memory retention and retrieval in rats in the passive avoidance and Morris water maze paradigms (1-3). In two rat models of amnesia, induced by the muscarinic antagonist, scopolamine, or bilateral perforant pathway lesion, the Ang IV analogues reversed the memory deficits detected utilizing the Morris water maze paradigm (3, 4). Enhancement of long term memory by Ang IV has also been demonstrated in species as distant as crabs (5). Angiotensin IV and its analogues enhance long term potentiation in both the dentate gyrus in vivo (6) and the CA1 region of the hippocampus in vitro (7), possibly via actions at the post-synaptic terminal. We have also shown that Ang IV enhances K ϩ -evoked acetylcholine release from rat hippocampal slices (8).The actions of Ang IV and its analogues are mediated by the angiotensin AT 4 receptor, defined by an international nomenclature committee (9) as the high affinity binding site specific for Ang IV (10). The AT 4 receptor has since been shown to bind with nanomolar affinity the decapeptide, LVVYPWTQRF (LVV-H7), isolated from sheep cerebral cortex (19).Although first identified in bovine adrenal, the receptor is widely distributed throughout the brain and peripheral organs (11). In the central nervous system, its distribution is highly conserved in guinea pig (12), macaque monkey (13), and human (14) brains. AT 4 receptors occur in high levels in the basal nucleus of Meynert, in the CA1 to CA3 regions of Ammon's horn in the hippocampus, and throughout the neocortex, areas important for cognitive processing. Despite the dramatic central effects of Ang IV and the abundance of the receptor in the central nervous system, the identity of the AT 4 receptor and the mechanism by which its ligands mediate their actions were unknown. MATERIALS AND METHODSProtein Purification-AT 4 receptors in bovine adrenal membranes (16 mg of membrane protein) were cross-linked to the photoactivatable analogue of Ang IV, [ 125 I]Nle 1 -BzPhe 6 -Gly 7 -Ang IV as described previously (15). Cross-linked membranes were solubilized in solubilization buffer (1% CHAPS, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA) with shaking at room temperature for 48 h, and insoluble material was pelleted by centrifugation at 100,000 ϫ g for 1 h at 4°C. Non-cross-linked membranes (48 mg of protein) were solubilized and centrifuged similarly, and the supernatant was combined with that from cross-linked membranes. Solubilized membrane proteins were applied to a 1-ml DEAE fas...

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT 4 receptor. However the AT 4 receptor has recently been identified as the insulin-regulated aminopeptidase (IRAP). In this study, we demonstrate that AT 4 receptor ligands, including Ang IV, branes with high affinity, which was up to 200-fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT 4 ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met-enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT 4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.

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Summary The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) regulates activation of the hypothalamic-pituitary-adrenal (HPA) axis and the adrenal gland in response to various stressors. We previously found that in response to acute psychological stress (restraint), elevated corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamic paraventricular nucleus (PVN) as well as elevated plasma corticosterone (CORT) were profoundly attenuated in PACAP-deficient mice. To determine whether HPA axis responses and stress-induced depressive-like behaviors in a chronic stress paradigm are affected by PACAP deficiency, we subjected mice to 14 days of social defeat stress. Defeat-exposed PACAP−/− mice showed a marked attenuation of stress-induced increases in serum CORT levels, cellular PVN ΔFosB immunostaining, and depressive-like behaviors (social interaction and forced swim tests) compared to wild-type control mice. The PACAP−/− mice showed reduced PVN FosB-positive cell numbers, but relatively elevated cell counts in several forebrain areas including the medial prefrontal cortex, after social stress. PACAP appears to be specific for mediating HPA activation only in psychological stress because marked elevations in plasma CORT after a systemic stressor (lipopolysaccharide administration) occurred regardless of genotype. We conclude that chronically elevated CORT is a key component of depressive effects of social defeat, and that attenuation of the CORT response at the level of the PVN, as well as extrahypothalamic forebrain regions, in PACAP-deficient mice protects from development of depressive behavior.