Characterization of angiotensin-converting enzymes 1 and 2 in the soleus and plantaris muscles of rats

Fernandes, Tiago; Hashimoto, Nara Yumi; Oliveira, Edilamar Menezes de

doi:10.1590/s0100-879x2010007500088

Cited by 30 publications

(23 citation statements)

References 18 publications

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“…In contrast to our work, other groups have reported nearcomplete inhibition of mouse and rat ACE2 activity at DX600 concentrations of 1 M or lower (12,19). However, despite our attempts, we have not been able to reproduce these data and effectively inhibit rodent ACE2 with 1 M DX600, suggesting that additional divergences in experimental condi- tions may contribute to these differences.…”

Section: Discussioncontrasting

confidence: 99%

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

Pedersen

Sriramula

Chhabra

et al. 2011

American Journal of Physiology-Regulatory, Integrative and Comp

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Pedersen KB, Sriramula S, Chhabra KH, Xia H, Lazartigues E. Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays. Am J Physiol Regul Integr Comp Physiol 301: R1293-R1299, 2011. First published August 31, 2011 doi:10.1152/ajpregu.00339.2011.-Angiotensin-converting enzyme 2 (ACE2) is a component of the reninangiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 M DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC50) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 M DX600 (Ͼ95% inhibition) but not with 1 M DX600 (Ͻ75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed. DX600; quenched fluorescent substrate; (7-methoxycoumarin-4-yl) acetyl-Ala-Pro-Lys(2,4-dinitrophenyl)-OH; enzyme inhibition ANGIOTENSIN-CONVERTING ENZYME 2 (ACE2) is a metallocarboxypeptidase that hydrolyzes the octapeptide ANG II, as well as several other small peptides (6,26,28). ANG II signaling promotes vasoconstriction and elevation of blood pressure (32). By decreasing the concentration of ANG II and increasing the concentration of the heptapeptide ANG-(1-7), whose signaling tends to be opposite of that of ANG II, ACE2 can diminish the effects of ANG II, while loss of ACE2 exacerbates ANG II-driven processes (11,14). To assess how ACE2 abundance affects biological processes, it is essential to be able to accurately quantify the enzymatic activity of ACE2, especially since there can be discordance in the regulation of ACE2 at the levels of mRNA, protein, and enzymatic activity (24,29,30). ACE2 activity is commonly quantified using the quenched fluorescent substrates (7-methoxycoumarin-4-yl)acetylTyr-Val-Ala-Asp-Ala-Pro-Lys(2,4-dinitrophenyl)-OH [Mca-YVADAPK(Dnp)] and (7-methoxycoumarin-4-yl)acetyl-AlaPro-Lys(2,4-dinitrophenyl)-OH [Mca-APK(Dnp)] (28). However, these fluorogenic substrates are not specific for ACE2. Mca-YVADAPK(Dnp) can, f...

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Section: Discussioncontrasting

confidence: 99%

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

Pedersen

Sriramula

Chhabra

et al. 2011

American Journal of Physiology-Regulatory, Integrative and Comp

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“…This is consistent with the finding that adenovirus-mediated overexpression of ACE2 in the pancreas of db/db mice improves glycemic control by increasing islet cell insulin content [7]. Nevertheless, since ACE2 is expressed in the liver, adipose tissue, and skeletal muscle [8], it could also influence peripheral glucose utilization and insulin sensitivity, although these actions are less well understood [9].…”

Section: Introductionsupporting

confidence: 88%

ACE2 deficiency shifts energy metabolism towards glucose utilization

et al. 2015

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“…ACE activity was determined in serum, and in skeletal soleus and plantaris muscle by using fluorescent substrates [32]. Frozen skeletal muscle samples were homogenized in 0.1 M Tris-HCl buffer pH 7.0, containing 50 mM NaCl and centrifuged at 1,000×g for 10 minutes.…”

Section: Methodsmentioning

confidence: 99%

Effects of Exercise Training on Circulating and Skeletal Muscle Renin-Angiotensin System in Chronic Heart Failure Rats

et al. 2014

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BackgroundAccumulated evidence shows that the ACE-AngII-AT1 axis of the renin-angiotensin system (RAS) is markedly activated in chronic heart failure (CHF). Recent studies provide information that Angiotensin (Ang)-(1–7), a metabolite of AngII, counteracts the effects of AngII. However, this balance between AngII and Ang-(1–7) is still little understood in CHF. We investigated the effects of exercise training on circulating and skeletal muscle RAS in the ischemic model of CHF.Methods/Main ResultsMale Wistar rats underwent left coronary artery ligation or a Sham operation. They were divided into four groups: 1) Sedentary Sham (Sham-S), 2) exercise-trained Sham (Sham-Ex), sedentary CHF (CHF-S), and exercise-trained CHF (CHF-Ex). Angiotensin concentrations and ACE and ACE2 activity in the circulation and skeletal muscle (soleus and plantaris) were quantified. Skeletal muscle ACE and ACE2 protein expression, and AT1, AT2, and Mas receptor gene expression were also evaluated. CHF reduced ACE2 serum activity. Exercise training restored ACE2 and reduced ACE activity in CHF. Exercise training reduced plasma AngII concentration in both Sham and CHF rats and increased the Ang-(1–7)/AngII ratio in CHF rats. CHF and exercise training did not change skeletal muscle ACE and ACE2 activity and protein expression. CHF increased AngII levels in both soleus and plantaris muscle, and exercise training normalized them. Exercise training increased Ang-(1–7) in the plantaris muscle of CHF rats. The AT1 receptor was only increased in the soleus muscle of CHF rats, and exercise training normalized it. Exercise training increased the expression of the Mas receptor in the soleus muscle of both exercise-trained groups, and normalized it in plantaris muscle.ConclusionsExercise training causes a shift in RAS towards the Ang-(1–7)-Mas axis in skeletal muscle, which can be influenced by skeletal muscle metabolic characteristics. The changes in RAS circulation do not necessarily reflect the changes occurring in the RAS of skeletal muscle.

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Characterization of angiotensin-converting enzymes 1 and 2 in the soleus and plantaris muscles of rats

Cited by 30 publications

References 18 publications

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

ACE2 deficiency shifts energy metabolism towards glucose utilization

Effects of Exercise Training on Circulating and Skeletal Muscle Renin-Angiotensin System in Chronic Heart Failure Rats

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