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A Fluorometric Method of Measuring Carboxypeptidase Activities for Angiotensin II and Apelin-13.

ABSTRACT: Degradation of the biologically potent octapeptide angiotensin Ang II-(1-8) is mediated by the activities of several peptidases. The conversion of Ang II to the septapeptide Ang-(1-7) is of particular interest as the latter also confers organ protection. The conversion is catalyzed by angiotensin-converting enzyme 2 and other enzymes that selectively cleave the peptide bond between the proline and the phenylalanine at the carboxyl terminus of Ang II. The contribution of various enzyme activities that collectively lead to the formation of Ang-(1-7) from Ang II, in both normal conditions and in disease states, remains only partially understood. This is largely due to the lack of a reliable and sensitive method to detect these converting activities in complex samples, such as blood and tissues. Here, we report a fluorometric method to measure carboxypeptidase activities that cleave the proline-phenylalanine dipeptide bond in Ang II. This method is also suitable for measuring the conversion of apelin-13. The assay detects the release of phenylalanine amino acid in a reaction with the yeast enzyme of phenylalanine ammonia lyase (PAL). When used in cell and mouse organs, the assay can robustly measure endogenous Ang II and apelin-13-converting activities involved in the renin-angiotensin and the apelinergic systems, respectively.

SUBMITTER: Liu P

PROVIDER: S-EPMC5381230 | biostudies-literature |

REPOSITORIES: biostudies-literature

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MicroRNA-503 promotes angiotensin II-induced cardiac fibrosis by targeting Apelin-13.

Project description:Cardiac fibrosis is a major cause of heart failure. MicroRNAs (miRs) are important epigenetic regulators of cardiac function and cardiovascular diseases, including cardiac fibrosis. This study aimed to explore the role of miR-503 and its mechanisms in regulating cardiac fibrosis. miR-503 was found up-regulated in the mouse LV tissues subjected to transverse aortic constriction (TAC) and in neonatal cardiac fibroblasts (CFs) cultured with Angiotension II. The role of miR-503 in regulating CF cell proliferation and/or collagen production in mice neonatal CFs were determined using an MTT assay and RT-PCR respectively. Forced expression of miR-503 increased the cellular proliferation and collagen production in mice neonatal CFs. The effects were abrogated by cotransfection with AMO-503 (a specific inhibitor of miR-503). Injection of antagomiR-503 elevated cardiac function and inhibited the expression of connective tissue growth factor (CTGF) and transforming growth factor (TGF)-? in the TAC mice. Additional analysis revealed that Apelin-13 is a direct target of miR-503, as the overexpression of miR-503 decreased the protein and mRNA expression levels of Apelin-13. In the CFs with pre-treatment of AngII, we transfected AMO-503 into the cells treated with siRNA-APLN. siRNA-APLN abolished the effects of AMO-503 on the production of collagen I and III and the expression of TGF-? and CTGF. Furthermore, pre-treatment of CFs with Apelin-13 (1-100 nmol/l) inhibited angiotensin II-mediated collagen production and activation of CTGF and TGF-?. So we conclude that miR-503 promotes cardiac fibrosis via miR-503-Apelin-13-TGF-?-CTGF-collagen production pathway. Thus, miR-503 is a promising therapeutic target for reducing cardiac fibrosis.

| S-EPMC4759464 | biostudies-literature

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Prolyl Carboxypeptidase Mediates the C-Terminal Cleavage of (Pyr)-Apelin-13 in Human Umbilical Vein and Aortic Endothelial Cells.

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Project description: Not available

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Loss of Apelin Augments Angiotensin II-Induced Cardiac Dysfunction and Pathological Remodeling.

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| S-EPMC6358887 | biostudies-literature

Balancing apelin and angiotensin-II signaling to reduce heart injury during weightlessness

Project description:In order to further develop gene expression methods for simulated weightlessness, genome-wide microarray expression profiles were used as a discovery platform to identify genes differentially expressed in mice after simulated weightlessness. After 28 days of tail suspension simulated weightlessness in 10-week-old male C57BL/6 mice, 706 genes were identified to distinguish tail suspension from control samples.

2019-01-18 | GSE125274 | GEO

The apelin receptor inhibits the angiotensin II type 1 receptor via allosteric trans-inhibition.

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| S-EPMC3594671 | biostudies-literature

Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production.

Project description:To test the hypothesis that apelin protects against angiotensin II (Ang II)-induced cardiovascular fibrosis and vascular remodeling.Wild-type mice administered apelin or apelin along with Ang II exhibited less cardiovascular fibrosis and decreased plasminogen activator inhibitor type-1 (PAI-1) gene expression than mice receiving Ang II, N-nitro-L-arginine methyl ester (L-NAME), apelin plus L-NAME or apelin plus Ang II plus L-NAME. In-vitro analysis using a luciferase construct driven by 3.1 kb of the human PAI-1 promoter revealed that apelin blocked Ang II-mediated PAI-1 gene expression. Immunoblotting for phosphorylated myosin phosphatase subunit and myosin light chain revealed that apelin blocked Ang II activation of the Rho kinase pathway, which is associated with induction of PAI-1 gene expression by Ang II. In addition, treatment of human aortic smooth muscle cells with apelin reduced PAI-1 mRNA and protein production in the presence and absence of Ang II. Conversely, L-NAME treatment attenuated the downregulation of PAI-1 by apelin in cells.Apelin protects against cardiac fibrosis and vascular remodeling through direct regulation of PAI-1 gene expression. This protective effect is mediated through the synergistic inhibition of Ang II signaling and increased production of nitric oxide by apelin. Our data extend previous findings and provide new insight into the molecular mechanisms by which apelin elicits a cardioprotective effect.

| S-EPMC3982221 | biostudies-literature

Apelin Inhibits Angiotensin II-Induced Atrial Fibrosis and Atrial Fibrillation via TGF-β1/Smad2/α-SMA Pathway.

Project description:BackgroundAngiotensin II (Ang II) could promote the development of atrial fibrosis in atrial fibrillation (AF). Apelin can inhibit the occurrence of myocardial fibrosis. However, the effect of apelin on Ang II-induced atrial fibrosis and subsequent AF still remains unknown.ObjectiveIn the present study, we examined the effect of apelin on the suppression of atrial fibrosis and subsequent AF, and investigated its underlying mechanisms.MethodsSprague-Dawley rats were treated for 2 weeks with Ang II (1080 μg/kg/24 h) and apelin-13 (140 μg/kg/24 h) using implantable mini-pumps. The incidence of AF induced by atrial pacing was determined. Atrial electrophysiological mapping was recorded by a 32-electrode microelectrode array. Blood was collected to measure the levels of Ang II and apelin. Atrial tissue samples were preserved to assess the pathohistological changes, DDR2 and α-SMA co-staining were performed, and the protein expression of Smad2 phosphorylation was evaluated.ResultsApelin significantly inhibited Ang II-induced atrial fibrosis (HE:1.45 ± 0.11 vs 6.12 ± 0.16, P < 0.001; Masson:1.49 ± 0.25 vs 8.15 ± 0.23, P < 0.001; Picrosirius Red:1.98 ± 0.64 vs 9.59 ± 0.56, P < 0.001, respectively) and decreased the vulnerability of AF (inducibility of AF: z = -4.40, P < 0.001; total AF duration: z = -4.349, P < 0.001). Left atrial epicardial mapping studies demonstrated preservation of atrial conduction homogeneity by apelin. The protective effects of apelin from fibrotic remodeling were mediated by suppression of Smad2-dependent fibrosis.ConclusionApelin potently inhibited Ang II-induced atrial fibrosis and subsequent vulnerability to AF induction via suppression TGF-β/Smad2/α-SMA pathway. Our results indicated that apelin might be an effective up-stream therapy for atrial fibrosis and AF.

| S-EPMC7711167 | biostudies-literature

NMR method for measuring carbon-13 isotopic enrichment of metabolites in complex solutions.

Project description:Isotope-based methods are commonly used for metabolic flux analysis and metabolite quantification in biological extracts. Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for these studies because NMR can unambiguously identify compounds and accurately measure (13)C enrichment. We have developed a new pulse sequence, isotope-edited total correlation spectroscopy (ITOCSY), that filters two-dimensional (1)H-(1)H NMR spectra from (12)C- and (13)C-containing molecules into separate, quantitatively equivalent spectra. The ITOCSY spectra of labeled and unlabeled molecules are directly comparable and can be assigned using existing bioinformatics tools. In this study, we evaluate ITOCSY using synthetic mixtures of standards and extracts from Escherichia coli . We show that ITOCSY has low technical error (6.6% for metabolites ranging from 0.34 to 6.2 mM) and can detect molecules at concentrations less than 10 muM. We propose ITOCSY as a practical NMR strategy for metabolic flux analysis, isotope dilution experiments, and other methods that rely on carbon-13 labeling.

| S-EPMC2878175 | biostudies-literature

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