Dx600

Heart tissue ACE2 activity was determined using the 10 μM of the fluorogenic substrate Mca-APK(Dnp)-OH (Aminotech Pesquisa e Desenvolvimento, SP, Brazil) in 0.2 M Tris-HCl buffer, 200 mM NaCl, and 10 mM ZnCl2, pH 7.5, [17 (link)]. The increase in fluorescence was followed in a 96-well fluorescence plate reader Hitachi F-7000 (λ excitation = 320 nm; λ emission = 420 nm). Measurements were performed in duplicate, and ACE2 activity values were obtained as fluorescence arbitrary units (AFU). Assay specificity was demonstrated by hydrolysis inhibition with 10 μM DX600 (Phoenix Pharmaceuticals, Burlingame, CA), a specific ACE2 inhibitor.

All treatments were performed in serum-free culture medium with penicillin and streptomycin. Cells were washed twice with serum-free culture medium and pre-incubated with saline or NaHS (50, 100, and 200 μM) for 24 h. Some cells were then stimulated with LPS (100 ng/ml) for 24 h in the continuous presence of NaHS. In the time course experiment, cells were pre-incubated with saline or NaHS (100 μM) for 0, 6, 24, or 48 h. For experiments using inhibitors, cells were pre-treated with DX600 (1 μM, an ACE2 inhibitor, Phoenix Pharmaceuticals; Pedersen et al., 2011 (link)) for 2 h before pre-incubation with NaHS (100 μM) for 24 h and subsequent stimulation with LPS (100 ng/ml) for 24 h.

The Mas receptor antagonist A779 (Asp-Arg-Val-Tyr-Ile-His-[D-Ala]) and the specific ACE2 inhibitor DX600 (Ac-Gly-Asp-Tyr-Ser-His-Cys-Ser-Pro-Leu-Arg-Tyr-Tyr-Pro-Trp-Trp-Lys-Cys-Thr-Tyr-Pro-Asp-Pro-Glu-Gly-Gly-Gly-NH2), were purchased from Phoenix Pharmaceuticals Inc. (St. Joseph, MO). Ang-(1–7) was purchased from Bachem Americas (Torrance, CA) and diphenyleneiodonium chloride (DPI) was purchased from Sigma (St. Louis, MO).

The ACE2 and ACE activity of the VSMCs exposed to cyclic mechanical stretch was determined as described previously (Liu et al., 2011 (link)). A reagent, 7-Mca-YVADAPK (Dnp) (R&D Systems, Minneapolis, United States), which is cleaved by ACE2, was used as a fluorogenic substrate. Ten μg total protein extracts were incubated with 1.0 μmol/L 7-Mca-YVADAPK (Dnp) in a final volume of 100 μL reaction buffer at room temperature. EDTA (1 mmol/L) and human ACE2 (25 ng) (R&D Systems, Minneapolis, MN, United States) were designed as negative and positive controls, respectively. Fluorescence kinetics was measured for 4 h by the use of Varioskan Flash (Thermo Fisher Scientific, Worcester, MA, United States) at an excitation wavelength of 320 nm and an emission wavelength of 400 nm. ACE2 activity was defined as the difference in fluorescence with or without the ACE2 inhibitor DX600 (1 μmol/L, Phoenix Pharmaceuticals, Belmont, CA, United States). Data were calculated from triplicate wells and presented as fluorescence unit per hour and normalized to milligram tissue protein.