Endogenous cystinyl aminopeptidase in Chinese hamster ovary cells: Characterization by [125I]Ang IV binding and catalytic activity

Research Group on Experimental Pharmacology, Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel, Pleinlaan 2, Brussels 1050, Belgium.
Biochemical Pharmacology (Impact Factor: 5.01). 09/2004; 68(5):885-92. DOI: 10.1016/j.bcp.2004.05.047
Source: PubMed

ABSTRACT The angiotensin II C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl aminopeptidase (EC, 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.

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    • "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. "
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    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, 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
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    • "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). "
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    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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    • "This is in agreement with the enzyme profile of IRAP. The potency order for the enzyme with high affinity for 7B was: 7B > Ang IV > AL-11 >> L- M a n u s c r i p t 11 methionine, corresponding to the profile of APN (Demaegdt et al., 2004a; 2006; Stragier et al., 2006; Lukaszuk et al., 2008). The pK i values of these ligands for each enzyme, calculated according to the 'Cheng and Prusoff' equation (Cheng and Prusoff, 1973) on basis of their IC 50 values obtained either by single-or two-site analysis of the inhibition curves and the K m value of the substrate, are given in Table 4 "
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    ABSTRACT: 'AT(4) receptors' through which Angiotensin IV (Ang IV) improves memory acquisition, were recently identified as insulin regulated aminopeptidase (IRAP). Radioligand binding studies have hitherto been performed with iodinated Ang IV in the presence of divalent cation chelators EDTA and 1,10-phenanthrolin. Hence, they referred to the apo-form of IRAP. Presently, binding of [(3)H]Ang IV and [(3)H]AL-11, a stable Ang IV analog, was compared on Chinese hamster ovary (CHO-K1) and mouse hippocampal (P40H1) cell membranes. With chelators, their high affinity sites showed the same pharmacological profile as for [(125)I]Ang IV binding. Without chelators, only high affinity binding was perceived for [(3)H]AL-11. The same pharmacological profile was recorded in both membrane preparations; it was different from the one in the presence of chelators and corresponded to catalytically active IRAP (despite the concurrent presence of aminopeptidase N (APN) in P40H1 cell membranes). This confirms that the active and apo-forms of IRAP have a distinct pharmacological profile.
    Molecular and Cellular Endocrinology 08/2009; 311(1-2):77-86. DOI:10.1016/j.mce.2009.07.020 · 4.41 Impact Factor
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