Proposed network model showing all angiotensin (Ang) peptides down
to length 5 including bioactive peptides (red),
undetected peptides (yellow),
and all other peptides (blue).
Edges in black were supported in the literature and summarized in Velez.2 Edges in red were predicted in the first phase of our effort and confirmed by experiments reported here. Edges in blue were predicted in the revised model and found to significantly improve model fit (based on deviance information criterion [DIC]) in the dynamic systems model.
The histogram of the incoming peptide profile (left) is based on measured reported plasma values of Ang peptides.
The response profile is included for illustration purposes only.
ACEi indicates Ang-converting enzyme inhibitor;
APA, aminopeptidase A;
DP?, an unidentified dipeptidyl aminopeptidase;
and NEPi, neprilysin inhibitor.
Neprilysiini pilkkoo ( 2-10) angiotensiinin muotoon (2-7) angiotensiini. Myös konversiota (5-10)-angiotensiini muotoon (2-10) angiotensiinistä esiintyi neprilysiinillä, jos ei ollut inhibiittoria, mutta (5-10) angiotensiinin muodostuminen oli merkitsevästi vähempää. . Neprilysiini johtaa tuotteisiin, josta ei9 tule aktiiveja angiotensiinejä I,II,III tai IV
Confirmation of Ang(2–10) to Ang(2–7) Conversion by NEP
To confirm the proposed conversion of Ang(2–10) to
Ang(2–7), additional experiments were performed. Ang(2–10) was exposed
to human recombinant NEP untreated or after pretreatment with 1 μmol/L
SCH39370 (NEP inhibitor) or 70 nmol/L thiorphan (NEP inhibitor) for 15
minutes and subjected to MALDI MS (Figure 3).
For the conditions where inhibitor was omitted, the Ang(2–7) peak was
2.9±0.16-fold that of the Ang(2–10) area, at 15 minutes. Under SCH39370
treatment, none of the spectra contained Ang(2–7) peaks with R2
above our quality threshold indicating nearly complete inhibition
(0.009±0.002-fold, relative to Ang(2–10) peak area). Treatment with
thiorphan yielded a similar result with a relative Ang(2–7) peak area of
0.037±0.007. Together, these findings support NEP as the enzyme
responsible for Ang(2–10) conversion to Ang(2–7). Of note, the
conversion of Ang(2–10) to Ang(5–10) via NEP was also evident when
inhibitor was omitted, but the magnitude of the Ang(5–10) peak area was
significantly smaller. These data support the possibility of a direct
Ang(2–10) to Ang(5–10) conversion by NEP, in addition to the Ang(2–10)
to Ang(2–7) conversion.
We performed additional experiments adding the substrate Ang(2–10) with or without amastatin (100 μmol/L) or SCH39370 (10 μmol/L) pretreatment. Samples were collected immediately after the addition of substrate and hours postaddition. The peptides Ang(2–10), Ang(2–7), and Ang(3–10) were quantified using AQUA peptides, and Ang(4–10) and Ang(5–10) were normalized to the sum of the included AQUA peptide signals (Figure 4). These experiments indicated a significant decrease in Ang(2–7) production after pretreatment with SCH39370, thus providing additional evidence of the NEP-catalyzed Ang(2–10) to Ang(2–7) conversion. We additionally note that a significant decrease in net Ang(3–10) conversion after treatment with amastatin is accompanied by a significant increase (at 4 hours) in Ang(2–7). Also, Ang(2–10) levels at 2 and 4 hours in the presence of amastatin were significantly higher than control. Because amastatin can inhibit both AP A and AP N,48–50 this observation suggests that AP A/ AP N inhibition blocks the conversion of Ang(2–10) to Ang(3–10), providing additional substrate for conversion to Ang(2–7). Collectively, these data confirm NEP conversion of Ang(2–10) to Ang(2–7) in mouse podocytes.
We performed additional experiments adding the substrate Ang(2–10) with or without amastatin (100 μmol/L) or SCH39370 (10 μmol/L) pretreatment. Samples were collected immediately after the addition of substrate and hours postaddition. The peptides Ang(2–10), Ang(2–7), and Ang(3–10) were quantified using AQUA peptides, and Ang(4–10) and Ang(5–10) were normalized to the sum of the included AQUA peptide signals (Figure 4). These experiments indicated a significant decrease in Ang(2–7) production after pretreatment with SCH39370, thus providing additional evidence of the NEP-catalyzed Ang(2–10) to Ang(2–7) conversion. We additionally note that a significant decrease in net Ang(3–10) conversion after treatment with amastatin is accompanied by a significant increase (at 4 hours) in Ang(2–7). Also, Ang(2–10) levels at 2 and 4 hours in the presence of amastatin were significantly higher than control. Because amastatin can inhibit both AP A and AP N,48–50 this observation suggests that AP A/ AP N inhibition blocks the conversion of Ang(2–10) to Ang(3–10), providing additional substrate for conversion to Ang(2–7). Collectively, these data confirm NEP conversion of Ang(2–10) to Ang(2–7) in mouse podocytes.
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