Impact of PACAP and PAC1 receptor deficiency on the neurochemical and behavioral effects of acute and chronic restraint stress in male C57BL/6 mice.

Acute restraint stress (ARS) for 3 h causes corticosterone (CORT) elevation in venous blood, which is accompanied by Fos up-regulation in the paraventricular nucleus (PVN) of male C57BL/6 mice. CORT elevation by ARS is attenuated in PACAP-deficient mice, but unaffected in PAC1-deficient mice. Correspondingly, Fos up-regulation by ARS is greatly attenuated in PACAP-deficient mice, but much less so in PAC1-deficient animals. We noted that both PACAP- and PAC1-deficiency greatly attenuate CORT elevation after ARS when CORT measurements are performed on trunk blood following euthanasia by abrupt cervical separation: this latter observation is of critical importance in assessing the role of PACAP neurotransmission in ARS, based on previous reports in which serum CORT was sampled from trunk blood. Seven days of chronic restraint stress (CRS) induces non-habituating CORT elevation, and weight loss consequent to hypophagia, in wild-type male C57BL/6 mice. Both CORT elevation and weight loss following 7-day CRS are severely blunted in PACAP-deficient mice, but only slightly in PAC1-deficient mice. However, longer periods of daily restraint (14-21 days) resulted in sustained weight loss and elevated CORT in wild-type mice, and these effects of long-term chronic stress were attenuated or abolished in both PACAP- and PAC1-deficient mice. We conclude that while a PACAP receptor in addition to PAC1 may mediate some of the PACAP-dependent central effects of ARS and short-term (<7 days) CRS on the hypothalamo-pituitary-adrenal (HPA) axis, the PAC1 receptor plays a prominent role in mediating PACAP-dependent HPA axis activation, and hypophagia, during long-term (>7 days) CRS.

Over-expression of RCAN1 causes Down syndrome-like hippocampal deficits that alter learning and memory.

People with Down syndrome (DS) exhibit abnormal brain structure. Alterations affecting neurotransmission and signalling pathways that govern brain function are also evident. A large number of genes are simultaneously expressed at abnormal levels in DS; therefore, it is a challenge to determine which gene(s) contribute to specific abnormalities, and then identify the key molecular pathways involved. We generated RCAN1-TG mice to study the consequences of RCAN1 over-expression and investigate the contribution of RCAN1 to the brain phenotype of DS. RCAN1-TG mice exhibit structural brain abnormalities in those areas affected in DS. The volume and number of neurons within the hippocampus is reduced and this correlates with a defect in adult neurogenesis. The density of dendritic spines on RCAN1-TG hippocampal pyramidal neurons is also reduced. Deficits in hippocampal-dependent learning and short- and long-term memory are accompanied by a failure to maintain long-term potentiation (LTP) in hippocampal slices. In response to LTP induction, we observed diminished calcium transients and decreased phosphorylation of CaMKII and ERK1/2-proteins that are essential for the maintenance of LTP and formation of memory. Our data strongly suggest that RCAN1 plays an important role in normal brain development and function and its up-regulation likely contributes to the neural deficits associated with DS.

Neuropeptides, growth factors, and cytokines: a cohort of informational molecules whose expression is up-regulated by the stress-associated slow transmitter PACAP in chromaffin cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a co-transmitter with acetylcholine at the adrenomedullary synapse, mediating sustained hormone secretion and regulation of cellular plasticity in response to stress at the level of gene transcription. Here we have extended our investigation of PACAP-regulated neuroendocrine cell-specific genes from PC12 cells to PC12 cells expressing physiological levels of the PAC1hop receptor found on chromaffin cells in vivo. PACAP induces in these PC12_bPAC1hop cells an additional cohort of genes, compared to PC12 cells, enriched in informational molecules including cytokines, neuropeptides, and growth factors. Using two newly developed microarray platforms for expressed bovine transcripts, we further examined PACAP-induced genes in bovine chromaffin cells during a period of exposure (6 h) corresponding to a period of prolonged metabolic or psychogenic stress in vivo during which PACAP is released from the splanchnic nerve onto chromaffin cells. As in PC12_bPAC1hop cells, PACAP induced in bovine chromaffin cells a cohort of genes encoding secretory proteins, identified by tiling for cellular localization using Ingenuity Pathway Analysis, which were highly enriched in informational molecules (secreted proteins acting at extracellular receptors). These included cytokines, growth factors and hormones, as well as converting enzymes, or protease inhibitors modulating converting enzyme function. Several neuropeptide prohormone transcripts not previously shown to be PACAP-regulated in chromaffin cells, such as thyrotropin-releasing hormone, and tachykinin precursor 1, were identified. Identification of this cohort of informational molecule-encoding transcripts suggests a wider, more integrative role for PACAP as a co-transmitter specific to stress transduction in the adrenal medulla.

Food hypersensitivity-induced chronic gastrointestinal inflammation in a non-human primate model of diet-induced obesity.

Experimental non-human primate models of obesity are induced through the introduction of atypically calorically rich diets. Studies in captive-bred macaques show the development of obesity and diabetes with similar complications to humans including eye and kidney diseases, nerve damage associated with pain and blood vessel damage. Diets differ in outcomes and here we document inflammation of the gastrointestinal tract that can be exacerbated through these dietary interventions. Following baseline physiological evaluation of body composition, Southern pigtail macaques were given a high-fat diet (HFD) for three months. This HFD consisted of lard, grains (including gluten), dairy and fructose that was otherwise omitted from a standard macaque diet (Chow). Physiological parameters were then reassessed before animals were reverted back to standard Chow for a further three months (remission). Consumption of the HFD resulted in food-mediated hypersensitivity marked by chronic weight loss, alopecia, malabsorption, protein-losing enteropathy and gross diffuse intestinal villi atrophy and lamina propria hypertrophy. Physiological changes were more highly pronounced in female macaques suggesting sex-specific differences but could be fully reversed through change of diet. Care should be taken in choosing non-human primate HFD diets for creating experimental models of obesity because they can induce severe food-driven chronic inflammation of the gastrointestinal tract that can eventuate to diet-induced chronic wasting and mortality.

AT4 receptor is insulin-regulated membrane aminopeptidase: potential mechanisms of memory enhancement.

Although angiotensin IV (Ang IV) was thought initially to be an inactive product of Ang II degradation, it was subsequently shown that the hexapeptide markedly enhances learning and memory in normal rodents and reverses the memory deficits seen in animal models of amnesia. These central nervous system effects of Ang IV are mediated by binding to a specific site, known as the AT(4) receptor, which is found in appreciable levels throughout the brain and is concentrated particularly in regions involved in cognition. This field of research was redefined by the identification of the AT(4) receptor as the transmembrane enzyme, insulin-regulated membrane aminopeptidase (IRAP). Here, we explore the potential mechanisms by which Ang IV binding to IRAP leads to the facilitation of learning and memory.

Oxytocinase/insulin-regulated aminopeptidase is distributed throughout the sheep, female reproductive tract and is regulated by oestrogen in the uterus.

Using [(125)I]Angiotensin IV (Ang IV) for the autoradiographic localisation of oxytocinase/insulin-regulated aminopeptidase (IRAP), we demonstrate for the first time that IRAP is distributed throughout the female reproductive tract. The highest concentration of IRAP was detected in the outer myometrial layer of the uterus with lower levels in the inner myometrial layer and in the luminal epithelium. High levels of the enzyme was also detected in the inner mucosal lining of the ampulla segment of the fallopian tubes with lower levels in the interstitial and isthmus. In the ovary, a high level of IRAP was found in the corpus albicans with lower levels throughout the ovarian cortex and the surrounding connective tissue. In the uterine body of ovariectomised (OVX) ewes, oestrogen treatment resulted in a significant decrease (P<0.05) in the level of IRAP in the outer myometrium. These findings indicate an important role for IRAP in reproductive physiology in regulating the action of peptide hormones.

Pituitary adenylate cyclase-activating polypeptide (PACAP): a master regulator in central and peripheral stress responses.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is a master regulator of central and peripheral stress responses required to restore and maintain homeostasis. PACAP modulates the hypothalamic-pituitary-adrenal (HPA) axis in response to acute psychogenic but not systemic stressors, through activation of corticotropin-releasing hormone (CRH) release to drive adrenal corticosterone (CORT) output. During direct high-frequency stimulation of the splanchnic nerve that is designed to mimic stress, PACAP regulates adrenomedullary catecholamine secretion. In addition to transmission, PACAP simultaneously facilitates the biosynthesis of adrenomedullary catecholamines through stimulus-secretion-synthesis coupling. During periods of chronic psychogenic stress, PACAP-mediated CORT elevation fails to desensitize and contributes to the development of maladaptive behaviors such as anxiety and depression. Based on these findings, PACAP regulates not only adaptive responses to stress but also maladaptive responses to sustained psychological stress. PACAP receptor antagonists could have therapeutic relevance in preventing hyperactivity of the HPA axis and offering protection against chronic stress-associated anxiety and depression.

PACAP-deficient mice show attenuated corticosterone secretion and fail to develop depressive behavior during chronic social defeat stress.

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.

Rapgef2 connects GPCR-mediated cAMP signals to ERK activation in neuronal and endocrine cells.

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR)-mediated increases in the second messenger cyclic adenosine monophosphate (cAMP) activate the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK), and in neuroendocrine cells, this pathway leads to cAMP-dependent neuritogenesis mediated through Rap1 and B-Raf. We found that the Rap guanine nucleotide exchange factor Rapgef2 was enriched from primary bovine neuroendocrine cells by cAMP-agarose affinity chromatography and that it was specifically eluted by cAMP. With loss-of-function experiments in the rat neuronal cell line Neuroscreen-1 (NS-1) and gain-of-function experiments in human embryonic kidney 293T cells, we demonstrated that Rapgef2 connected GPCR-dependent activation of adenylate cyclase and increased cAMP concentration with the activation of ERK in neurons and endocrine cells. Furthermore, knockdown of Rapgef2 blocked cAMP- and ERK-dependent neuritogenesis. Our data are consistent with a pathway involving the cAMP-mediated activation of Rapgef2, which then stimulates Rap1, leading to increases in B-Raf, MEK, and ERK activity.

Discrete signal transduction pathway utilization by a neuropeptide (PACAP) and a cytokine (TNF-alpha) first messenger in chromaffin cells, inferred from coupled transcriptome-promoter analysis of regulated gene cohorts.

Cultured bovine adrenal chromaffin cells (BCCs) are employed to study first messenger-specific signaling by cytokines and neurotransmitters occurring in the adrenal medulla following immune-related stress responses. Here, we show that the cytokine TNF-alpha, and the neuropeptide transmitter PACAP, acting through the TNFR2 and PAC1 receptors, activate distinct signaling pathways, with correspondingly distinct transcriptomic signatures in chromaffin cells. We have carried out a comprehensive integrated transcriptome analysis of TNF-alpha and PACAP gene regulation in BCCs using two microarray platforms to maximize transcript identification. Microarray data were validated using qRT-PCR. More than 90% of the transcripts up-regulated either by TNF-alpha or PACAP were specific to a single first messenger. The final list of transcripts induced by each first messenger was subjected to multiple algorithms to identify promoter/enhancer response elements for trans-acting factors whose activation could account for gene expression by either TNF-alpha or PACAP. Distinct groups of transcription factors potentially controlling the expression of TNF-alpha or PACAP-responsive genes were found: most of the genes up-regulated by TNF-alpha contained transcription factor binding sites for members of the Rel transcription factor family, suggesting TNF-alpha-TNFR2 signaling occurs mainly through the NF-KB signaling pathway. Surprisingly, EGR1 was predicted to be the primary transcription factor controlling PACAP-modulated genes, suggesting PACAP signaling to the nucleus occurs predominantly through ERK, rather than CREB activation. Comparison of TNFR2-dependent versus TNFR1-dependent gene induction, and EGR1-mediated transcriptional activation, may provide a pharmacological avenue to the unique pathways activated by the first messengers TNF-alpha and PACAP in neuronal and endocrine cells.

PAC1hop, null and hip receptors mediate differential signaling through cyclic AMP and calcium leading to splice variant-specific gene induction in neural cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G protein-coupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. Heterologous expression of the three major splice variants of the rat PAC1 receptor, PAC1hop, null and hip, in neural NG108-15 cells conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca(2+)](i) occurred only in PAC1hop-, and to a lesser extent in PAC1null-expressing cells. Induction of vasoactive intestinal polypeptide (VIP) and stanniocalcin 1 (STC1), two genes potentially involved in PACAP's homeostatic responses, was examined as a function of the expressed PAC1 variant. VIP induction was greatest in PAC1hop-expressing cells, suggesting that a maximal transcriptional response requires combinatorial signaling through both cAMP and Ca(2+). STC1 induction was similar for all three receptor splice variants and was mimicked by the adenylate cyclase activator forskolin, indicating that cAMP elevation is sufficient to induce STC1. The degree of activation of two different second messenger pathways appears to determine the transcriptional response, suggesting that cellular responses to stressors are fine-tuned through differential receptor isoform expression. Signaling to the VIP gene proceeded through cAMP and protein kinase A (PKA) in these cells, independently of the MAP kinase ERK1/2. STC1 gene induction by PACAP was dependent on cAMP and ERK1/2, independently of PKA. Differential gene induction via different cAMP dependent signaling pathways potentially provides further targets for the design of treatments for stress-associated disorders.

ATP derived from astrocytes modulates memory in the chick.

Memory consolidation in a discriminative bead pecking task is modulated by endogenous adenosine triphosphate (ATP) acting at purinergic receptors in the hippocampus. Consolidation, from short- to intermediate- to long-term memory during two distinct periods following training, was blocked by the non-selective P2 purinergic receptor antagonist PPADS (pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) tetrasodium salt hydrate and the specific P2Y1 receptor antagonist MRS2179. Direct injections of the ATP agonists (ATPγS and ADPßS) potentiated memory consolidation and the effect of ADPßS was blocked by MRS2179, suggesting an important role of ATP on memory consolidation via the P2Y1 receptor in the chick hippocampus. Incubation of astrocytes with ATPγS and ADPßS resulted in the increase of intracellular calcium ([Ca2+]i), the latter being blocked by MRS2179 suggesting a specific role for P2Y1 receptors in the calcium response. This response was prevented by blocking astrocytic oxidative metabolism with fluoroacetate. We argue that the source of the ATP acting on neuronal P2Y1 receptors is most likely to be astrocytes. Thrombin selectively increases [Ca2+]i in astrocytes but not in neurones. The main findings of the present study are: (a) astrocytic [Ca2+]i plays an important role in the consolidation of short-term to long-term memory; and (b) ATP released from chick astrocytes during learning modulates neuronal activity through astrocytic P2Y1 receptors.

PAC1hop receptor activation facilitates catecholamine secretion selectively through 2-APB-sensitive Ca(2+) channels in PC12 cells.

PACAP is a critical regulator of long-term catecholamine secretion from the adrenal medulla in vivo, however the receptor or pathways for Ca(2+) entry triggering acute and sustained secretion have not been adequately characterized. We have previously cloned the bovine adrenal chromaffin cell PAC1 receptor that contains the molecular determinants required for PACAP-induced Ca(2+) elevation and is responsible for imparting extracellular Ca(2+) influx-dependent secretory competence in PC12 cells. Here, we use this cell model to gain mechanistic insights into PAC1hop-dependent Ca(2+) pathways responsible for catecholamine secretion. PACAP-modulated extracellular Ca(2+) entry in PC12 cells could be partially blocked with nimodipine, an inhibitor of L-type VGCCs and partially blocked by 2-APB, an inhibitor and modulator of various transient receptor potential (TRP) channels. Despite the co-existence of these two modes of Ca(2+) entry, sustained catecholamine secretion in PC12 cells was exclusively modulated by 2-APB-sensitive Ca(2+) channels. While IP3 generation occurred after PACAP exposure, most PACAP-induced Ca(2+) mobilization involved release from ryanodine-gated cytosolic stores. 2-APB-sensitive Ca(2+) influx, and subsequent catecholamine secretion was however not functionally related to intracellular Ca(2+) mobilization and store depletion. The reconstituted PAC1hop-expessing PC12 cell model therefore recapitulates both PACAP-induced Ca(2+) release from ER stores and extracellular Ca(2+) entry that restores PACAP-induced secretory competence in neuroendocrine cells. We demonstrate here that although bPAC1hop receptor occupancy induces Ca(2+) entry through two independent sources, VGCCs and 2-APB-sensitive channels, only the latter contributes importantly to sustained vesicular catecholamine release that is a fundamental characteristic of this neuropeptide system. These results emphasize the importance of establishing functional linkages between Ca(2+) signaling pathways initiated by pleotrophic signaling molecules such as PACAP, and physiologically important downstream events, such as secretion, triggered by them.

Discovery of pituitary adenylate cyclase-activating polypeptide-regulated genes through microarray analyses in cell culture and in vivo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an evolutionarily well conserved neuropeptide with multiple functions in the nervous, endocrine, and immune systems. PACAP provides neuroprotection from ischemia and toxin exposure, is anti-inflammatory in gastric inflammatory disease and sepsis, controls proliferative signaling pathways involved in neural cell transformation, and modulates glucohomeostasis. PACAP-based, disease-targeted therapeutics might thus be both effective and benign, enhancing homeostatic responses to behavioral, metabolic, oncogenic, and inflammatory stressors. PACAP signal transduction employs synergistic regulation of calcium and cyclic adenosine monophosphate (cAMP), and noncanonical activation of both calcium- and cAMP-dependent processes. Pharmacological activation of PACAP signaling should consequently have highly specific effects even in vivo. Here, a combined cellular biochemical, pharmacologic, transcriptomic, and bioinformatic approach to understanding PACAP signal transduction by identifying PACAP target genes with oligonucleotide- and cDNA-based microarray is described. Calcium- and cAMP-dependent PACAP signaling pathways for regulation of genes encoding proteins required for neuritogenesis, changes in cell morphology, and cell survival have been traced in PC12 cells. Pharmacological experiments have linked gene expression to cell physiological responses in this system, in which gene silencing can also be employed to confirm the functional significance of induction of specific transcripts. Differential transcriptional responses to metabolic, ischemic, and other stressors in wild type compared to PACAP-deficient mice establish in principle which PACAP-responsive transcripts in culture are PACAP-dependent in vivo. Bioinformatic approaches aid in creating a pipeline for identifying neuropeptide-regulated genes, validating their cellular functions, and defining their expression in the context of neuropeptide signaling physiology, required for discovery of new targets for drug action.

The hop cassette of the PAC1 receptor confers coupling to Ca2+ elevation required for pituitary adenylate cyclase-activating polypeptide-evoked neurosecretion.

We have identified the single PAC1 receptor variant responsible for Ca2+ mobilization from intracellular stores and influx through voltage-gated Ca2+ channels in bovine chromaffin cells and the domain of this receptor variant that confers coupling to [Ca2+]i elevation. This receptor (bPAC1hop) contains a 28-amino acid "hop" insertion in the third intracellular loop, with a full-length 171-amino acid N terminus. Expression of the bPAC1hop receptor in NG108-15 cells, which lack endogenous PAC1 receptors, reconstituted high affinity PACAP binding and PACAP-dependent elevation of both cAMP and intracellular Ca2+ concentrations ([Ca2+]i). Removal of the hop domain and expression of this receptor (bPAC1null) in NG108-15 cells reconstituted high affinity PACAP binding and PACAP-dependent cAMP generation but without a corresponding [Ca2+]i elevation. PC12-G cells express sufficient levels of PAC1 receptors to provide PACAP-saturable coupling to adenylate cyclase and to drive PACAP-dependent differentiation but do not express PAC1 receptors at levels found in postmitotic neuronal and endocrine cells and do not support PACAP-mediated neurosecretion. Expression of bPAC1hop, but not bPAC1(null), at levels comparable with those of bPAC1hop in bovine chromaffin cells resulted in acquisition by PC12-G cells of PACAP-dependent [Ca2+]i increase and extracellular Ca2+ influx. In addition, PC12-G cells expressing bPAC1hop acquired the ability to release [3H]norepinephrine in a Ca2+ influx-dependent manner in response to PACAP. Expression of PACAP receptors in neuroendocrine rather than nonneuroendocrine cells reveals key differences between PAC1hop and PAC1null coupling, indicating an important and previously unrecognized role of the hop cassette in PAC1-mediated Ca2+ signaling in neuroendocrine cells.

Angiotensin II receptor (type 1 and 2) expression peaks when placental growth is maximal in sheep.

In sheep, placental size is maximal by midgestation, but blood flow continues to increase until term. No nerves are present and ANG II is thought to be a major regulator of vascular tone. We hypothesized that angiotensin type 2 receptors (AT(2)) would predominate over type 1 (AT(1)) until late in gestation and be primarily expressed in the vasculature. Real-time PCR, hybridization histochemistry, and ligand-binding studies were performed on placentae and fetal membranes at 27, 45, 66 +/- 1, 100 +/- 4, 130, and 140 days of gestation (term approximately 150 days) to determine quantitative changes and localization. The maximum level of AT(1) expression occurred in the 45-day placenta and was located predominantly in the maternal stromal cells. AT(1) receptors were expressed in the endothelial cells of the chorion in the first half of pregnancy, where later in gestation, both AT(1) and AT(2) receptors were predominant in blood vessels. These results suggest that ANG II, via the AT(1) receptor, may have hitherto unsuspected important roles in the growth/function on the ovine placenta during the maximal growth phase.

Angiotensin AT4 ligands are potent, competitive inhibitors of insulin regulated aminopeptidase (IRAP).

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT4 receptor. However the AT4 receptor has recently been identified as the insulin-regulated aminopeptidase (IRAP). In this study, we demonstrate that AT4 receptor ligands, including Ang IV, Nle1-Ang IV, divalinal-Ang IV, and the structurally unrelated LVV-hemorphin-7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leu-beta-naphthylamide by recombinant human IRAP. Both Ang IV and divalinal-Ang IV display competitive kinetics, indicating that AT4 ligands mediate their effects by binding to the catalytic site of IRAP. The AT4 ligands also displaced [125I]-Nle1-Ang IV or [125I]-divalinal1-Ang IV from IRAP-HEK293T membranes 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 AT4 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 AT4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.

Structure-activity study of LVV-hemorphin-7: angiotensin AT4 receptor ligand and inhibitor of insulin-regulated aminopeptidase.

The decapeptide LVV-hemorphin-7 binds with high affinity to the angiotensin IV (Ang IV) receptor (AT(4) receptor), eliciting a number of physiological effects, including cellular proliferation and memory enhancement. We have recently shown that the AT(4) receptor is identical to insulin-regulated aminopeptidase (IRAP) and that both LVV-hemorphin-7 and Ang IV inhibit the catalytic activity of IRAP. In the current study, a series of alanine-substituted and N- or C-terminally modified analogs of LVV-hemorphin-7 were evaluated for their abilities to compete for (125)I-Ang IV binding in sheep adrenal and cerebellar membranes. Selected analogs were also analyzed for binding to recombinant human IRAP and inhibition of IRAP aminopeptidase activity. C-Terminal deletions of LVV-hemorphin-7 resulted in modest changes in affinity for IRAP, whereas deletion of the first three N-terminal residues abolished binding. Monosubstitutions of Tyr(4) and Trp(6) with alanine resulted in a 10-fold reduction in affinity. Competition binding studies using recombinant human IRAP demonstrated the same rank order of affinity as obtained for the ovine tissues. All LVV-hemorphin-7 analogs tested, except for Leu-Val-Val-Tyr, inhibit the cleavage of the synthetic substrate, leucine beta-naphthylamide, by IRAP, with K(i) values between 56 and 620 nM. We find that the Val(3) residue is crucial for LVV-hemorphin-7 binding to IRAP, whereas the C-terminal domain seems to play a minor role. The current study highlights the minimal residues necessary for binding and inhibition of IRAP and provides a basis to design peptidomimetic analogs for experimental and potentially clinical use.