A possible correlation between oxytocin-induced and angiotensin IV-induced anti-hyperalgesia at the spinal level in rats

In our previous study, we showed that intrathecal (i.t.) administration of angiotensin IV (Ang IV), an insulin-regulated aminopeptidase (IRAP) inhibitor, attenuated inflammatory hyperalgesia in rats. Using the plantar test in rats with carrageenan-induced paw inflammation, we investigated the possible mechanism(s) of this effect. Because i.t. oxytocin was reported to produce a dose-dependent anti-hyperalgesia in rats with inflammation, we speculate that there is a possible correlation between oxytocin-induced and Ang IV-induced anti-hyperalgesia. Using i.t. co-administered atosiban (oxytocin receptor antagonist), the anti-hyperalgesia by Ang IV was completely abolished. This indicated that oxytocin could be the major IRAP substrate responsible for the anti-hyperalgesia by Ang IV. When Ang IV was co-administered with a low dose of oxytocin, there was a significant enhancing effect of Ang IV on oxytocin-induced anti-hyperalgesia. In recent reports, electrical stimulation on the paraventricular hypothalamic nucleus (PVN) was proved to increase oxytocin release at the spinal cord. Our results also showed that Ang IV could prolong the anti-hyperalgesia induced by PVN stimulation. This suggests a possible protective effect of Ang IV on endogenous oxytocin degradation/dysfunctioning. Moreover, we examined the local effect of intraplantarly injected Ang IV in the same model. Our results showed no effect of local Ang IV on hyperalgesia and paw edema, indicating that Ang IV may not regulate the peripheral inflammatory process. Overall, our study suggests that Ang IV may act through the inhibition of the activity of IRAP to reduce the degradation of oxytocin at the spinal cord, thereby leading to anti-hyperalgesia in rats with inflammation.     

Effects of ligands or substrate of insulin-regulated aminopeptidase (IRAP) on trophoblast invasion.

Insulin-regulated aminopeptidase (IRAP) activity increases during placentation and in the invasive tumor cell of trophoblast suggesting a role for this peptidase in the invasiveness of normal and malignant trophoblast. To investigate this hypothesis, we studied the effects of substrate (OT) and inhibitors (angiotensin peptides and LVV-H7) of IRAP on the first trimester trophoblast proliferation and invasion. Addition of these peptides in the culture medium of trophoblastic cells significantly decreased metalloproteinase-9 activity and cellular invasiveness while no effect was observed on cell proliferation. The peptide IRAP inhibitors could exert their effect on cytotrophoblastic cell invasiveness by inhibition of its enzymatic activity, and thus increasing half life of the known placental peptide substrate of IRAP, OT.     

Involvement of Spinal Serotonin Receptors in Electroacupuncture Anti-Hyperalgesia in an Inflammatory Pain Rat Model

We previously showed that electroacupuncture (EA) activates medulla-spinal serotonin-containing neurons. The present study investigated the effects of intrathecal 5,7-dihydroxytryptamine creatinine sulfate, a selective neurotoxin for serotonergic terminals, the 5-hydroxytryptamine 1A receptor (5-HT1AR) antagonist NAN-190 hydrobromide and the 5-HT2C receptor (5-HT2CR) antagonist SB-242,084 on EA anti-hyperalgesia. EA was given twice at acupoint GB30 after complete Freund’s adjuvant (CFA) injection into hind paw. CFA-induced hyperalgesia was measured by assessing hind paw withdrawal latency (PWL) to a noxious thermal stimulus 30 min post-EA. Serotonin depletion and the 5-HT1AR antagonist blocked EA anti-hyperalgesia; the 5-HT2CR antagonist did not. Immunohistochemical staining showed that spinal 5-HT1AR was expressed and that 5-HT2CR was absent in naive and CFA-injected animals 2.5 h post-CFA. These results show a correlation between EA anti-hyperalgesia and receptor expression. Collectively, the data show that EA activates supraspinal serotonin neurons to release 5-HT, which acts on spinal 5-HT1AR to inhibit hyperalgesia.     

Synergistic analgesic effects between neuronostatin and morphine at the supraspinal level

Neuronostatin, a 13-amino acid peptide, is encoded in the somatostatin pro-hormone. I.c.v. administration of neuronostatin produces a significant antinociceptive effect in the mouse tail-flick test, which is mediated by endogenous opioid receptor. However, the direct functional interaction between morphine and neuronostatin has not been characterized. In the present study, effect of neuronostatin on morphine analgesia was investigated in the tail-flick test. Our findings showed that i.c.v. administration of neuronostatin (0.3 nmol/mouse i.c.v.) significantly enhanced the antinociceptive effect of morphine (2.5, 5 or 10 μg/kg) at the supraspinal level. Results of antagonism experiments suggested that the synergistic analgesia induced by morphine and neuronostatin was mediated by μ- and к-opioid receptors not δ-opioid receptor. In conclusion, there may be a cascade amplification phenomenon when morphine and neuronostatin were co-administered in acute pain model. The above results provide evidence for the potential use of neuronostatin in combination with morphine to control pain and addiction.     

Possible involvement of mu1-opioid receptors in the fentanyl- or morphine-induced antinociception at supraspinal and spinal sites

Fentanyl has been shown to be a potent analgesic with a lower propensity to produce tolerance and physical dependence in the clinical setting. The present study was designed to investigate the mechanisms of fentanyl- or morphine-induced antinociception at both supraspinal and spinal sites. In the mouse tail-flick test, the antinociceptive effects induced by both fentanyl and morphine were blocked by either the mu1-opioid receptor antagonist naloxonazine or the mu1/mu2-opioid receptor antagonist beta-funaltrexamine (beta-FNA) after s.c., i.c.v. or i.t. injection. In contrast, both fentanyl and morphine given i.c.v. or i.t. failed to produce antinociception in mu1-deficient CXBK mice. These findings indicate that like morphine, the antinociception induced by fentanyl may be mediated predominantly through mu1-opioid receptors at both supraspinal and spinal sites in mice. We also determined the ED50 values for s.c.-, i.c.v.- and i.t.-administered fentanyl- or morphine-induced antinociception in mice. The ED50 values for s.c.-, i.c.v.- and i.t.-administered fentanyl-induced antinociception were 73.7, 18.5 and 1.2-fold lower than that of morphine, respectively. The present data clearly suggest the usefulness of peripheral treatment with fentanyl for the control of pain.     

Lack of Intra-cellular Signalling by Angiotensin IV in IRAP Transfected Cells

A number of studies have suggested that angiotensin IV is able to mediate a range of signalling events through a receptor distinct to the well-characterised angiotensin AT₁ and AT₂ receptors. This receptor was termed the AT₄ receptor, but was subsequently identified to be the transmembrane enzyme, insulin regulated aminopeptidase, IRAP. Using HEK293T cells transfected with IRAP we investigated whether angiotensin IV was able to mediate signalling events via this aminopeptidase. No effect of the angiotensin IV analogue, Nle¹-Ang IV, on intracellular calcium or ERK phosphorylation was observed. In addition, the effect of Nle¹-Ang IV on IRAP internalization was investigated and, in contrast to classical ligand-mediated receptor endocytosis, Nle¹-Ang IV (10⁻⁶ M) extends the half-life of IRAP at the plasma membrane. Our results do not support a direct role for Ang IV signalling via IRAP in this system.     

Kinetic studies on aminopeptidase M-mediated degradation of human hemorphin LVV-H7 and its N-terminally truncated products.

The human hemorphin LVV-H7 belongs to the class of micro-opiod receptor-binding peptides, which also exhibits significant affinity to insulin-regulated aminopeptidase (IRAP) thereby affecting IRAP inhibition. The inhibitory potency towards IRAP is of pharmaceutical interest for the treatment of Alzheimer's disease. Consecutive N-terminal cleavage of the first two amino acid residues of LVV-H7 affects a drastic increase of the binding affinity (V-H7) but ultimately leads to its complete abolition after cleavage of the next amino acid residue (H7). Therefore, we investigated LVV-H7 truncation by aminopeptidase M (AP-M) identified as a LVV-H7 degrading enzyme potentially regulating hemorphin activity towards IRAP in vivo. Using a selective quantitative multi-component capillary zone electrophoretic method (CZE-UV), we analyzed the AP-M-mediated subsequent proteolysis of the hemorphins LVV-H7 (L32-F41), VV-H7 (V33-F41), and V-H7 (V34-F41) in vitro. Incubations were carried out with synthetic hemorphins applied as single substrates or in combination. Maximum velocities (V(max)), catalytic constants (turnover numbers, kcat), and specific enzyme activities (EA) were calculated. L32 cleavage from LVV-H7 happens more than two-times faster (kcat: 140 min(-1) +/- 9%, EA: 1.0 U/mg +/- 9%) than V33 cleavage from VV-H7 (kcat: 61 min(-1) +/- 10%, EA: 0.43 U/mg +/- 10%) or V32 deletion from V-H7 (kcat: 62 min(-1) +/- 8%, EA: 0.46 U/mg +/- 8%). In contrast, we showed that H7 (Y35-F41) was neither degraded by porcine AP-M nor did it act as an inhibitor for this enzyme. Determined turnover numbers were in the same dimension as those reported for dynorphin degradation. This is the first time that AP-M-mediated truncation of natural underivatized LVV-H7 and its physiological metabolites was analyzed to determine kinetic parameters useful for understanding hemorphin processing and designing hemorphin-derived drug candidates.     

Augmentation of analgesic effect of ibuprofen by alprazolam in experimental model of pain

The reports of analgesic effects of benzodiazepines are inconsistent. There is evidence of a hyperalgesic effect induced by activation of supraspinal GABAA receptors and an antinociceptive effect induced by activation of receptors located in the spinal cord (dorsal horns). The aim of the study was to discover whether the systemic administration of a benzodiazepine agent alprazolam increases the systemic analgesic efficacy of non-opioid analgesic ibuprofen. Experimental studies combining these agents have not yet been published. We used three experimental methods - writhing test (with acetic acid), tail-flick test and plantar test to assess analgesic action. The drugs were administered orally. Augmentation of the analgesic effect of ibuprofen by alprazolam was proved for the writhing test at a dose of 30 mg/kg of ibuprofen and alprazolam 1 mg/kg. The reaction time of the combination was significantly prolonged in comparison with ibuprofen alone. The results of the tail-flick test and plantar test were negative. The effect of ibuprofen was not enhanced by alprazolam in tests of acute thermal pain. Our results have demonstrated that the analgesic action of ibuprofen is only weakly enhanced by alprazolam.     

Contribution of spinal mu(1)-opioid receptors and dynorphin B to the antinociception induced by Tyr-d-Arg-Phe-Sar

The antinociceptive effect of Tyr-d-Arg-Phe-Sar (TAPS) at the spinal level was characterized with the mouse tail-flick test. Intrathecal (i.t.) administration of TAPS produced a dose-dependent antinociception. The antinociception induced by TAPS was completely blocked by i.t. pretreatment with the mu-opioid receptor antagonist beta-funaltrexamine, the mu(1)-opioid receptor antagonist naloxonazine or the kappa-opioid receptor antagonist nor-binaltorphimine, but not with the delta-opioid receptor antagonist naltrindole. Moreover, TAPS-induced antinociception was dose-dependently attenuated by i.t. pretreatment with an antiserum against dynorphin B, but not against dynorphin A, alpha-neo-endorphin, [Met(5)]enkephalin, or [Leu(5)]enkephalin. In mice lacking prodynorphin, TAPS-induced antinociception was significantly reduced compared to that in wild-type mice. These results suggest that TAPS mainly stimulates mu(1)-opioid receptors, which subsequently induce the release of dynorphin B, which then acts on kappa-opioid receptors to produce antinociception.     

Characterization of the AT(4) receptor in a human neuroblastoma cell line (SK-N-MC)

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II (Ang II), binds to a distinct receptor designated the AT(4) receptor. The peptide elicits a range of vascular and central actions including facilitation of memory retention and retrieval in several learning paradigms. The aim of this study was to characterize the AT(4) receptor in a human cell line of neural origin. Receptor binding studies indicate that the human neuroblastoma cell line SK-N-MC cells express a high-affinity Ang IV binding site with a pharmacological profile similar to the AT(4) receptor: (125)I]-Ang IV and (125)I]-Nle(1)-Ang IV bind specifically to the SK-N-MC cell membranes (K(d) = 0.6 and 0.1 nM) in a saturable manner (B(max) = 1.2 pmol/mg of protein). AT(4) receptor ligands, Nle(1)-Ang IV, Ang IV and LVV-haemorphin 7 (LVV-H7), compete for the binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to the SK-N-MC cell membranes with rank order potencies of Nle(1)-Ang IV > Ang IV > LVV-H7 with IC(50) values of 1.4, 8.7 and 59 nM ([(125)I]-Ang IV) and 1.8, 20 and 168 nM ([(125)I]-Nle(1)-Ang IV), respectively. The binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to SK-N-MC cell membranes was not affected by the presence of GTP gamma S. Both Ang IV and LVV-H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT(4) receptor in the SK-N-MC cells is a 180-kDa glycoprotein; under non-reducing conditions a 250-kDa band was also observed. In summary, the human neuroblastoma cell line, SK-N-MC, expresses functional AT(4) receptors that are responsive to Ang IV and LVV-H7, as indicated by an increase in DNA synthesis. This is the first human cell line of neural origin shown to express the AT(4) receptor.     

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ₁-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ₁-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH₂ (d-Pro²-endomorphin-2) but not by co-administration of the μ₂-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH₂ (d-Pro²-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH₂ (d-Pro²-Tyr-W-MIF-1). In CXBK mice whose μ₁-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ₁-opioid receptors.     

美普他酚及其同分异构体对角叉菜胶引起的炎症大鼠具有抗热痛敏作用

实验以清醒大鼠的缩腿潜伏期为指标,观察了腹腔注射美普他酚及其同分异构体112824和112825对角叉菜胶引起的热痛敏的影响.外周炎症由单侧足底注射角叉菜胶(2 mg/100 μl)引起.注射角叉菜胶3 h后,注射侧后肢局部红肿及热痛过敏反应达到高峰,持续数小时.腹腔注射0.1 mg/kg美普他酚对炎症和非炎症侧后肢的缩腿潜伏期无明显影响(P＞0.05,n=8).腹腔注射1mg/kg和10 mg/kg美普他酚对炎症和非炎症侧后肢产生明显的抗痛敏和抗伤害效应,且对炎症侧缩腿反应的抑制(抗痛敏)作用明显强于非炎症侧(抗伤害)(P＜0.05,n=8～11).预先腹腔注射1.5 mg/kg纳洛酮明显阻断美普他酚引起的抗伤害和抗痛敏效应.腹腔注射美普他酚的同分异构体112824(1 mg/kg)和112825(1.5 ms/kg)可产生与美普他酚类似的抗痛敏作用,该效应可被预先腹腔注射1.5 mg/kg纳洛酮完全阻断.提示美普他酚及其同分异构体具有明显抗伤害和抗痛敏作用,且以后者为强.该作用主要通过mu阿片受体介导.本研究为扩展美普他酚及其同分异构体在临床上的应用提供了依据.     

Cyclic insulin-regulated aminopeptidase (IRAP)/AT4 receptor ligands.

The angiotensin IV receptor (AT4 receptor) is the insulin-regulated aminopeptidase enzyme (IRAP, EC 3.4.11.3). This membrane-spanning enzyme belongs to the M1 family of zinc-dependent metallo-peptidases. It has been proposed that AT4 receptor ligands exert their physiological effects by binding to the active site of IRAP and thereby inhibiting the catalytic activity of the enzyme. The biological activity of a large series of linear angiotensin IV analogs was previously disclosed. Herein, the synthesis and biological evaluation of a series of angiotensin IV analogs, encompassing macrocyclic ring systems of different sizes, are presented. It is demonstrated that disulfide cyclizations of angiotensin IV can deliver ligands with high IRAP/AT4 receptor affinity. One ligand, with an 11-membered ring system (4), inhibited human IRAP and aminopeptidase N (AP-N) activity with similar potency as angiotensin IV but was considerably more stable than angiotensin IV toward enzymatic degradation. The compound provides a promising starting point for further optimization toward more drug-like derivatives. The cyclic constrained analogs allowed us to propose a tentative bioactive conformation of angiotensin IV and it seems that the peptide adopts an inverse gamma-turn at the C-terminal.     

The impact of ANG II and IV on INS-1 cells and on blood glucose and plasma insulin

The impact of angiotensin (ANG) for peripheral, global effects is well known. Local ANG systems including that of the insulin-releasing β cell are not well investigated. In insulin-secreting cell line (INS-1), AT₁ and AT₄ receptors for ANG II and IV were demonstrated by Western blots. Only small amounts of ANG II-binding sites of low affinity were observed. ANG II and SARILE displaced binding of ¹²⁵I-ANG II. ANG II and IV as well as their non-degradable analogs SARILE and Nle-ANG IV increased the glucose-induced insulin release in a bell-shaped way; the maximum effect was at ～1?nM. The increase was antagonized by 1 µM losartan or 10 µM divalinal (AT₁ and AT₄ receptor antagonists, respectively). The insulin release was accompanied by a ⁴⁵Ca²⁺ uptake in the case of ANG II and ANG IV. Divalinal abolished the effect of ANG IV and Nle-ANG IV on this parameter. ANG IV reduced the increase in blood glucose during a glucose tolerance test with corresponding, albeit smaller effects on plasma insulin. Using confocal laser scanning microscopy, transfected insulin-regulated aminopeptidase (IRAP) with AT₄ receptors was shown to be accumulated close to the nucleus and the cytosolic membrane, whereas GLUT4 was not detectable. IRAP was inhibited by ANG IV. In conclusion, AT₁ and AT₄ receptors may be involved in diabetic homeostasis. Effects are mediated by insulin release, which is accompanied by an influx of extracellular Ca²⁺. The impact of ANG IV/IRAP agonists may be worth being used as antidiabetics.     

Buprenorphine exerts its antinocicepttve activity via μ1-opioid receptors

The mechanism of the antinociceptive effect of buprenorphine was assessed by administering selective μ-, μ₁-, δ- and κ-opioid receptor antagonists in mice. Intraperitoneal administration of buprenorphine, at doses of 0.3 to 3 mg/kg, produced dose-dependent antinociception in the tail-flick test. The antinociceptive activity of buprenorphine did not result from the activation of κ- or δ-opioid receptors, since treatment with either nor-binaltorphimine, a selective κ-opioid receptor antagonist, or naltrindole, a selective δ-opioid receptor antagonist, was completely ineffective in blocking buprenorphine-induced antinociception. However, the antinociceptive effect of buprenorphine was significantly antagonized by β-funaltrexamine, a selective μ-opioid receptor antagonist. Moreover, selective μ₁-opioid receptor antagonists, naloxonazine and naltrexonazine, also significantly antagonized the antinociceptive effect of buprenorphine. Co-administration of κ- and δ-opioid receptor antagonists with the μ-opioid receptor antagonists had no significant effect on the antagonistic profiles of the μ-opioid receptor antagonists on the antinociceptive effect of buprenorphine. These results suggest that buprenorphine acts selectively at μ₁-opioid receptors to induce antinociceptive effects in mice.     

Enhanced recombinant expression and purification of human IRAP for biochemical and crystallography studies

Insulin-regulated aminopeptidase (IRAP) in humans is a membrane bound enzyme that has multiple functions. It was first described as a companion protein of the insulin-responsive glucose transporter, Glut4, in specialized vesicles. The protein has subsequently been shown to be identical to the oxytocinase/aminopeptidase or the angiotensin IV (Ang IV) receptor (AT₄ receptor). Some AT₄ ligand peptides, such as Ang IV and LVV-hemorphin-7, have been shown to act as IRAP inhibitors that exert memory-enhancing properties. As such IRAP has been a target for developing cognitive enhancers. To facilitate detailed mechanistic studies of IRAP catalysis and inhibition, and to pave the way for biophysical and structural studies of IRAP in complex with peptide inhibitors, we report here an optimized expression and purification system using High Five insect cells. We also report biochemical characterizations of the purified recombinant IRAP with a standard aminopeptidase substrate and an optimized IRAP peptide inhibitor with a Ki of 98 nM.     

Angiotensin receptors: form and function and distribution

The peptide hormone, angiotensin II, acts primarily via type I (AT(1)) and type II (AT(2)) angiotensin receptors. Proteolytic fragments of angiotensin II also have biological activity via these and other receptors, with actions that may mimic or antagonise angiotensin II. Most notably, a high affinity-binding site for angiotensin IV (the Val(3)-Phe(8) fragment of angiotensin II) has recently been identified as the insulin-regulated aminopeptidase (IRAP). While AT(1) and AT(2) receptors are seven transmembrane-spanning, G protein-coupled receptors with some well-established features of relevance to health and disease, the existence of separate receptor systems for angiotensin fragments offers exciting possibilities for new therapeutics to target the diverse actions of the angiotensin peptides.     

Evidence that the angiotensin IV (AT(4)) receptor is the enzyme insulin-regulated aminopeptidase.

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) receptor, by protein purification and peptide sequencing, to be insulin-regulated aminopeptidase (IRAP). HEK 293T cells transfected with IRAP exhibit typical AT(4) receptor binding characteristics; the AT(4) receptor ligands, angiotensin IV and LVV-hemorphin 7, compete for the binding of [(125)I]Nle(1)-angiotensin IV with IC(50) values of 32 and 140 nm, respectively. The distribution of IRAP and its mRNA in the brain, determined by immunohistochemistry and hybridization histochemistry, parallels that of the AT(4) receptor determined by radioligand binding. We also show that AT(4) receptor ligands dose-dependently inhibit the catalytic activity of IRAP. We have therefore demonstrated that the AT(4) receptor is IRAP and propose that AT(4) receptor ligands may exert their effects by inhibiting the catalytic activity of IRAP thereby extending the half-life of its neuropeptide substrates.     

Metabolism of angiotensin II is required for its in vivo effect on dopamine release in the striatum of the rat

The effect of angiotensin (Ang) IV, an inhibitor of insulin-regulated aminopeptidase (IRAP), on extracellular dopamine levels in the striatum of freely moving rats was examined using in vivo microdialysis. The Ang IV was administered locally in the striatum through the microdialysis probe. A concentration-dependent (10-100 microm) increase in extracellular striatal dopamine was observed. The effect of Ang II (10-100 microm), which has only a weak affinity for IRAP, was similar to that observed for Ang IV. The effects of both peptides could not be blocked by the AT1 antagonist candesartan (10 nm and 1 microm) nor by the AT2 antagonist S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenyl-acetyl)-4,5,6,7-tetrahydro-1H-amidazo(4,5-c) pyridine-6-carboxylic acid (1 microm), suggesting that the observed effects are both AT1 and AT2 independent. The effect of Ang II could be blocked by the aminopeptidase-A inhibitor (S)-3-amino-4-mercaptobutylsulphonic acid as well as the aminopeptidase-N inhibitor 2-amino-4-methylsulphonylbutane thiol, indicating that the effect of Ang II is mediated via metabolism into Ang IV. Other IRAP inhibitors, such as Divalinal-Ang IV and LVV-haemorphin-7, had similar effects on extracellular dopamine levels as compared with Ang IV. We propose a role for IRAP as mediator for the effects of Ang IV and related peptides on extracellular dopamine levels in the striatum of the rat.     

Small potent ligands to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor.

Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor. Displacement of the C-terminal of angiotensin IV with an o-substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (K(i) = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a gamma-turn in the C-terminal of its bioactive conformation.Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT(4) receptor.