The angiotensin II C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl aminopeptidase (EC 22.214.171.124), a membrane-associated zinc-dependent metallopeptidase of the M1 family, has recently been found to display high affinity for Ang IV and it was proposed to represent the AT4 receptor. We present evidence for the presence of endogenous cystinyl aminopeptidase in membranes from Chinese hamster ovary (CHO-K1) cells by binding studies with [125I]Ang IV and by measuring the cleavage of L-leucine-p-nitroanilide. The equilibrium dissociation constant of [125I]Ang IV in saturation binding studies (KD= 0.90 nM) was similar to the value (KD= 0.70 nM) calculated from the association and dissociation rates. Binding was displaced with high potency by the "AT4 receptor" ligands (Ang IV > divalinal1-Ang IV approximately LVV-hemorphin-7 approximately LVV-hemorphin-6 > Ang (3-7) > Ang III > Ang (4-8)) but not by AT1/AT2 receptor antagonists. Enzymatic activity in CHO-K1 cell membranes was competitively inhibited upto 94% by Ang IV and other "AT4 receptor" ligands (Ang IV > Ang III approximately divalinal1-Ang IV approximately Ang (3-7) approximately LVV-hemorphin-7 > Ang (4-8) approximately LVV-hemorphin-6). High affinity binding of [125I]Ang IV required the presence of metal chelators and the ligands such as Ang IV and LVV-hemorphin-7 displayed higher potency in the binding studies as in the enzyme assay. This difference in potency varied from one peptide to another. These pharmacological properties match those previously reported for the recombinantly-expressed human cystinyl aminopeptidase in embryonal kidney cells.
"Determination of the aminopeptidase catalytic activity was based on the cleavage of the substrate L-Leu-pNA into L-leucine and p-nitroaniline (Demaegdt et al., 2004). This latter compound displays a characteristic light absorption maximum at 405 nm. "
[Show abstract][Hide abstract] ABSTRACT: The hexapeptide angiotensin IV (Ang IV) induces diverse biological effects such as memory enhancement and protection against ischemic stroke. Studies on the mechanism of Ang IV however are hampered by its instability and its lack of selectivity. The high-affinity binding site for Ang IV is the insulin-regulated aminopeptidase (IRAP, EC 126.96.36.199), but Ang IV also acts as a weak agonist for the Ang II-receptor (AT(1)), implying the need for stable and highly selective Ang IV-analogues. Here we present the screening of novel Ang IV-analogues, selected on basis of high affinity for IRAP, high selectivity (compared to aminopeptidase N and the AT(1) receptor) and resistance against proteases. The selected compound IVDE77 possesses a number of advantages compared to Ang IV: (i) it has a 40 times higher affinity for IRAP (K(i) 1.71nM), (ii) it does not activate the AT(1) receptor, (iii) it is easily radiolabeled with tritium and (iv) it is resistant to proteolysis, even in human plasma. In addition, pre-treatment of intact CHO-K1 cells with IVDE77 led to a virtually complete inhibition of subsequent intracellular accumulation of [(3)H]IVDE77-IRAP complexes. IVDE77 thus represents the first Ang IV-analogue able to abolish IRAP-availability completely at the cell surface in vitro. In summary, IVDE77 is a useful tool for the detection of IRAP under physiological conditions, and may contribute to elucidating the mechanism of Ang IV to ascertain which functions are IRAP-dependent.
European journal of pharmacology 01/2013; 702(1-3). DOI:10.1016/j.ejphar.2013.01.026 · 2.53 Impact Factor
"We hypothesized an occurrence of such an interaction because DA-related pro-cognitive effects of Ang IV and des-Phe 6 -angiotensin IV have convincingly been linked with the DA-releasing activity of both peptides via presynaptic i.e. autoreceptor-related mechanisms (Stragier et al., 2004, 2005, 2007). The parallel use of Ang IV and its des- Phe 6 -Ang IV derivative with a similar to Ang IV high affinity for the AT 4 receptor identified as insulin regulated aminopeptidase (IRAP) (Albiston et al., 2001) [Kd = 2.6 nM for Ang IV, Kd = 3.9 nM for des-Phe 6 -Ang IV (Sardinia et al., 1993)], as well as similar IRAP enzyme inhibiting activity [pKi = 6.90 for Ang IV, pKi = 6.37 for des-Phe 6 -Ang IV (Demaegdt et al., 2004)], allowed control for any cognitive effects mediated through the angiotensin AT 1 receptors of which Ang IV but not des-Phe 6 -Ang IV is a weak agonist (de Gasparo et al., 2000; Harding et al., 1992; Le et al., 2002; Noda et al., 1995). "
[Show abstract][Hide abstract] ABSTRACT: We have recently found that postsynaptic D3 dopamine (DA) receptors appear not to participate in the memory enhancing effects of the angiotensin AT4 receptor agonists angiotensin IV (Ang IV) and des-Phe(6)-Ang IV. In this study we evaluated role of the presynaptic DA D3 receptors in these effects. For that purpose effect of (+)-UH 232, a selective D3 DA receptors partial agonist preferring presynaptic sites, on the pro-cognitive action of intracerebroventricularly (icv) injected Ang IV and des-Phe(6)-Ang IV was examined. Male Wistar rats weighing 180-200 g were used. Both peptides given at the dose of 1 nmol facilitated recall of a passive avoidance (PA) behaviour, improved object recognition (OR), and increased apomorphine-induced stereotype behaviour. In the auxiliary tests performed to control for the unspecific influence of motor (open field, OF) and emotional ('plus' maze, PM) effects of our treatments on the results of the memory tests they had either no (OF) or negligible (PM) effects. Intraperitoneal pre-treatment of the animals with an ineffective on its own dose (1 mg/kg) of (+)-UH 232 abolished or markedly diminished effects of both peptides on PA and OR but did not influence enhancement of stereotypy caused by the peptides.
European neuropsychopharmacology: the journal of the European College of Neuropsychopharmacology 04/2010; 20(4):218-25. DOI:10.1016/j.euroneuro.2009.11.012 · 4.37 Impact Factor
"This suggests that under physiological conditions the affinity of Ang IV to IRAP may be in a similar range as its affinity for the AT 1 receptor. This low affinity contradicts the high affinity by which Ang IV competes with [ 125 I]Ang IV for binding to IRAP with K i values in the range 7.4–14 nM (Lew et al. 2003; Demaegdt et al. 2004b). However, this highaffinity binding is likely to be an artefact since it has only been observed in the presence of chelators, which remove the Zn 2+ ion from the catalytic site of IRAP to produce its non-physiological apo-form (Demaegdt et al. 2004a,b; Laeremans et al. 2005). "
[Show abstract][Hide abstract] ABSTRACT: J. Neurochem. (2010) 112, 1223–1234.
Intracerebroventricular (i.c.v.) administration of angiotensin IV (Ang IV) or Leu–Val–Val-haemorphin 7 (LVV-H7) improves memory performance in normal rats and reverses memory deficits in rat models for cognitive impairment. These memory effects were believed to be mediated via the putative ‘AT4 receptor’. However, this binding site was identified as insulin-regulated aminopeptidase (IRAP). Correspondingly, Ang IV and LVV-H7 were characterised as IRAP inhibitors. This study investigates whether and how IRAP may be involved in the central effects of Ang IV and LVV-H7. We determined the effects of i.c.v. administration of Ang IV or LVV-H7 on hippocampal neurotransmitter levels using microdialysis in rats. We observed that Ang IV modulates hippocampal acetylcholine levels, whereas LVV-H7 does not. This discrepancy was reflected in the observation that Ang IV binds with micromolar affinity to the AT1 receptor whereas no binding affinity was observed for LVV-H7. Correspondingly, we demonstrated that the AT1 receptor is involved in the effects of Ang IV on hippocampal neurotransmitter levels and on spatial working memory in a plus maze spontaneous alternation task. However, the AT1 receptor was not involved in the spatial memory facilitating effect of LVV-H7. Finally, we demonstrated that Ang IV did not diffuse to the hippocampus following i.c.v. injection, suggesting an extrahippocampal site of action. We propose that AT1 receptors are implicated in the neurochemical and cognitive effects of Ang IV, whereas LVV-H7 may mediate its effects via IRAP.
Journal of Neurochemistry 12/2009; 112(5):1223-34. DOI:10.1111/j.1471-4159.2009.06547.x · 4.28 Impact Factor
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