Purification and substrate specificity of an angiotensin converting elastase-2 from the rat mesenteric arterial bed perfusate

Paula, Carmem Aparecida de; Sousa, Marcelo Valle de; Salgado, Maria Cristina de Oliveira; Oliveira, Eduardo B.

doi:10.1016/s0167-4838(98)00186-1

Cited by 39 publications

(67 citation statements)

References 46 publications

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“…Similarly, chymostatin inhibits both chymases (56,59) and elastase-2 (47), so the use of Ac-AAPL-CK and chymostatin cannot unequivocally indicate the relative contribution of different serine proteases in the generation of ANG II in the isolated rat MAB or elsewhere. However, as established in literature, rat chymase is mainly an angiotensinase (6,30,62), which argues in favor of elastase-2 being responsible for the ACE-independent pathway for ANG II generation in the rat MAB because it does not degrade ANG II (47,52 ]-ANG I are either homologous to human heart chymase (22,38,41,43,63) or rat elastase-2 (52), so this latter enzyme is the only known rat protease fitting the experimental evidence described for the ACE-independent pathway for ANG II generation in the rat MAB. ]-ANG I in the isolated MAB was abolished by the ANG II receptor antagonist saralasin (50 nM) in the perfusion solution (Fig.…”

Section: Discussionmentioning

confidence: 85%

“…All assays were carried out at 37°C by incubating the specified substrate with the enzyme samples in Tris-buffered saline (TBS; 0.03 M Tris ⅐ HCl containing 0.15 M NaCl; pH 8.1). Affinity-purified rat MAB elastase-2 was prepared as previously described (47), and human skin chymase was purchased from Calbiochem (San Diego, CA). To measure the enzymatic activity of samples toward the chromogenic substrates N-succinylAla-Ala-Pro-Leu-p-nitroanilide (N-suc-AAPL-pNA; 560 M) or N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (N-suc-AAPFpNA; 730 M), 100 l of each sample were incubated for a period ranging from 10 min to 20 h with the substrate in a clear 96-well plate in a final volume of 300 l. The absorbance of each reaction mixture resulting from the release of p-nitroaniline was read in a universal microplate reader (ELX 800, Bio-Tek Instruments; Winooski, VT) at 405 nm, and the amount of product formed was determined by comparison with a standard curve of p-nitroaniline ranging from 1 to 64 nmol in 300 l of the assay buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Conditioned media samples of cultured MEC were pooled and concentrated 24-fold by ultrafiltration (Centriplus YM-10, Millipore; Bedford, MA). Rat elastase-2 was chromatographically purified using a two-step procedure (47) by monitoring the ANG II-forming activity of the fractions using ANG I as the substrate, as described above. Briefly, 1 ml of the concentrated conditioned media was chromatographed over a Sephacryl S-300 column (10 ϫ 920 mm) equilibrated with TBS and developed at room temperature at a flow rate of 6.0 ml/h.…”

Section: Methodsmentioning

confidence: 99%

“…Rat elastase-2 was purified from MECconditioned media by the method previously described (47). Table 1 shows that the ANG II generation from ANG I by the enzyme purified from MEC-conditioned media was not affected by captopril (1 mM) but was inhibited in a concentration-dependent fashion by chymostatin and Ac-AAPL-CK (1-100 M).…”

Section: Functional Experiments Performed On the Isolated Ratmentioning

confidence: 99%

“…We (47,52) have recently described the biochemical, enzymatic, and inhibitory properties of a chymostatinsensitive ANG II-forming elastase-2 found in the perfusate of the isolated rat mesenteric arterial bed (MAB). One of the most interesting findings was that purified elastase-2 from the rat MAB perfusate was also capable of efficiently forming ANG II from [Pro 11 ,D-Ala 12 ]-ANG I, suggesting that in vivo formation of ANG II ascribed to chymases may have been overestimated in previous investigations of ANG II-forming pathways.…”

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Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme

Santos

Caprio²,

Oliveira³

et al. 2003

American Journal of Physiology-Heart and Circulatory Physiology

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. Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme. Am J Physiol Heart Circ Physiol 285: H775-H783, 2003. First published April 24, 2003 10.1152/ajpheart.00818. 2002We recently described a chymostatin-sensitive elastase-2 as the major angiotensin (ANG) II-forming enzyme in the perfusate of the rat mesenteric arterial bed (MAB) with the same cDNA sequence as rat pancreatic elastase-2. The role of this enzyme in generating ANG II was examined in the rat isolated and perfused MAB. The vasoconstrictor effect elicited by ANG I and the renin substrate tetradecapeptide was only partially inhibited by captopril but abolished by the combination of captopril and chymostatin or N-acetyl-AlaAla-Pro-Leu-chloromethylketone (Ac-AAPL-CK; inhibitor originally developed for human elastase-2). The effect induced by [Pro 11 ,D-Ala 12 ]-ANG I, an ANG I-converting enzyme (ACE)-resistant biologically inactive precursor of ANG II, was blocked by chymostatin or Ac-AAPL-CK. It was also demonstrated that cultured rat mesenteric endothelial cells synthesize elastase-2 and that mRNA for this enzyme can be detected in different rat tissues such as the pancreas, MAB, lung, heart, kidney, liver, and spleen. In conclusion, the demonstration of a functional alternative pathway to ACE for ANG II generation in the rat MAB and the fact that cultured MAB endothelial cells are capable of producing and secreting elastase-2 represent strong evidence of a physiological role for this enzyme in the rat vasculature. endothelium; chymostatin SEVERAL STUDIES HAVE POSTULATED the existence of alternative pathways to angiotensin (ANG)-converting enzyme (ACE) for ANG II generation in tissues of different species based on evidence derived from experiments carried out with a combination of protease inhibitors and ANG II receptor antagonists. In a pioneering study (9) in this area, it was demonstrated that the vasoconstrictor response induced by ANG I in blood vessels of the hamster cheek pouch was blocked only partially by ACE inhibitors but completely abolished by ANG II receptor antagonists, leading to the conclusion that this vascular bed converts significant amounts of ANG I to ANG II by a route that does not involve ACE. In other investigations, the partial or total blockade of ANG II formation by different combinations of ACE inhibitors with chymostatin or other protease inhibitors has provided clues as to the nature of the enzymes involved in ANG I conversion in a particular tissue or pharmacological preparation (23,45). Since the advent of [Pro 11 ,D-Ala 12 ]-ANG I, a biologically inactive precursor that selectively yields ANG II on incubation with chymases but not with ACE or carboxypeptidases (22), several reports (18,22,26,38,41,43,61,63) have described the relative contribution of chymases in ANG II formation in isolated preparations derived from various species.The vascular endothelium actively participates in the control of vascular tone through the synthesis and metabolism of seve...

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Section: Discussionmentioning

confidence: 85%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Functional Experiments Performed On the Isolated Ratmentioning

confidence: 99%

mentioning

confidence: 99%

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Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme

Santos

Caprio²,

Oliveira³

et al. 2003

American Journal of Physiology-Heart and Circulatory Physiology

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Microvascular angiogenesis and the renin-angiotensin system

Greene

Amaral

2002

Current Science Inc

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The renin-angiotensin system is one of the major regulatory mechanisms essential for maintaining cardiovascular homeostasis. Angiotensin II is a multifunctional hormone that plays a key role in regulating this system. The importance of the renin-angiotensin system in controlling sodium homeostasis and vascular resistance is well established, however, in the past decade, much attention has been focused on the importance of angiotensin II as a regulator of microvessel density, acting through the AT1 and AT2 receptors. In this review, we discuss the connections between the renin-angiotensin system and other growth factor pathways known to be involved in pathologic and physiologic angiogenesis and rarefaction.

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Elastase-2 Knockout Mice Display Anxiogenic- and Antidepressant-Like Phenotype: Putative Role for BDNF Metabolism in Prefrontal Cortex

et al. 2018

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Several pieces of evidence indicate that elastase-2 (ELA2; chymotrypsin-like ELA2) is an alternative pathway to the generation of angiotensin II (ANGII). Elastase-2 knockout mice (ELA2KO) exhibit alterations in the arterial blood pressure and heart rate. However, there is no data on the behavioral consequences of ELA2 deletion. In this study, we addressed this question, submitting ELA2KO and wild-type (WT) mice to several models sensitive to anxiety- and depression-like, memory, and repetitive behaviors. Our data indicates a higher incidence of barbering behavior in ELA2KO compared to WT, as well as an anxiogenic phenotype, evaluated in the elevated plus maze (EPM). While a decrease in locomotor activity was observed in ELA2KO in EPM, this feature was not the main source of variation in the other parameters analyzed. The marble-burying test (MBT) indicated increase in repetitive behavior, observed by a higher number of buried marbles. The actimeter test indicated a decrease in total activity and confirmed the increase in repetitive behavior. The spatial memory was tested by repeated exposure to the actimeter in a 24-h interval. Both ELA2KO and WT exhibited decreased activity compared to the first exposure, without any distinction between the genotypes. However, when submitted to the cued fear conditioning, ELA2KO displayed lower levels of freezing behavior in the extinction session when compared to WT, but no difference was observed during the conditioning phase. Increased levels of BDNF were found in the prefrontal cortex but not in the hippocampus of ELA2KO mice compared to WT. Finally, in silico analysis indicates that ELA2 is putatively able to cleave BDNF, and incubation of the purified enzyme with BDNF led to the degradation of the latter. Our data suggested an anxiogenic- and antidepressant-like phenotype of ELA2KO, possibly associated with increased levels of BDNF in the prefrontal cortex.

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Purification and substrate specificity of an angiotensin converting elastase-2 from the rat mesenteric arterial bed perfusate

Cited by 39 publications

References 46 publications

Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme

Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme

Microvascular angiogenesis and the renin-angiotensin system

Elastase-2 Knockout Mice Display Anxiogenic- and Antidepressant-Like Phenotype: Putative Role for BDNF Metabolism in Prefrontal Cortex

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