Angiotensin (1-7)

Kinase experiment:

Competition assays using purified canine ACE are determined using a fixed concentration of the substrate Hip-His-Leu (1 mM) and varying the concentrations of the competing agents [Lisinopril (0.1 to 100 nM), Angiotensin (1-7) (10 nM to 10 μM), or Sar1, Thr8-Ang II (10 nM to 10 μM)]. Inhibitory constants (IC50) are determined from the respective competition curves. To study the effect of Angiotensin (1-7) on BK metabolism in intact coronary rings, 125I-[Tyr0]-BK (final concentration of 1 nM) is added to the tubes containing three rings preincubated with 1 mL Krebs' buffer and aerated with 95% O2 and 5% CO2 at 37°C. Lisinopril (2 μM), Angiotensin (1-7) (2 μM), or Krebs' buffer as control are added to the rings 10 minutes before addition of the radiolabeled BK. Aliquots of the incubation medium are removed at 5, 10, and 20 minutes and diluted with 1% HFBA to inhibit peptidase activity[1].

Cell experiment:

500 μM Methylglyoxal is incubated with 100 μM BSA dissolved in phosphate buffered saline (PBS) for 24 hours, then washed on 10 kDa filters to remove excess methyl glyoxal, reconstituted with DMEM/F12 serum free media and passed through a 0.2 μmicron filter. TGF-β (5 ng/mL) is prepared to treat cells in a subset of experiments. Cells are co-treated with one or combinations of the following: Angiotensin (1-7) (100 nM), D-Ala7-Ang-(1-7) (10 μM), ERK1/2 kinase inhibitor, PD 98059 (1 μM), TGF-β receptor kinase inhibitor; SB525334 (1 μM), the AT1 receptor antagonist Losartan (1 μM), the renin inhibitor Aliskerin (1 μM) and the ACE inhibitor Lisinopril (1 μM)[2].

Animal experiment:

Mice[3] Male and female BALB/c mice (1:1 ratio, 6-10 weeks old, mean weight 20 g.) are used. Angiotensin fragment 1-7 acetate salt hydrate (Ang 1-7) is dissolved in 0.9% saline (vehicle) at 1 mg/mL and stored at -80°C. Various doses (0.01, 0.06, 0.1, 0.3 and 1 mg/kg) are freshly prepared from the stock each day of the experiment, and administered to mice by daily intra-peritoneal (i.p) injections in a volume of 500 μL per injection, either before (prophylactic approach) or after (treatment approach) DSS treatment. A779 (MAS-1 R antagonist) is similarly dissolved in distilled water at 1 mg/mL and stored at -80°C. A freshly prepared dose of 1 mg/kg is administered to a second group of mice by daily i.p injections in a volume of 500 μL daily (for 4 days) along with colitis induction (prophylactic approach). A third group of mice receive DSS containing water and daily i.p injections of 0.9% saline (vehicle). The fourth group receive DSS containing water along with daily i.p injections with Dexamethasone (DEX) at doses of 0.01-1.0 mg/kg or its vehicle (0.9% saline) (prophylactic approach). Rats[4] Twenty six ovariectomized female Wistar rats weighing 200±20 g are used. Angiotensin (1-7) is administered intravenously by a microsyringe pump at two different continuous doses of 100 and 300 ng/kg/min after antagonist/saline infusion. Each dose is infused for 15 min; and MAP, RPP, and RBF are recorded during Angiotensin (1-7) infusion and the last 3-5 min of each dose measured as “response to Angiotensin (1-7) infusion”. During Angiotensin (1-7) infusion, RPP is sustained at pre-Ang1-7 infusion levels via an adjustable aortic clamp. At the end of the experiment, the rats are humanely killed by anesthetic overdose, and the left kidneys are removed and weighed immediately.

References:

[1]. Vaz-Silva J, et al. The vasoactive peptide angiotensin-(1-7), its receptor Mas and the angiotensin-converting enzyme type 2 are expressed in the human endometrium. Reprod Sci. 2009 Mar;16(3):247-56.
[2]. Li P, et al. Angiotensin-(1-7) augments bradykinin-induced vasodilation by competing with ACE and releasing nitric oxide. Hypertension. 1997 Jan;29(1 Pt 2):394-400.
[3]. Khajah MA, et al. Anti-Inflammatory Action of Angiotensin 1-7 in Experimental Colitis. PLoS One. 2016 Mar 10;11(3):e0150861.
[4]. Alzayadneh EM, et al. Angiotensin-(1-7) abolishes AGE-induced cellular hypertrophy and myofibroblast transformation via inhibition of ERK1/2. Cell Signal. 2014 Sep 19. pii: S0898-6568(14)00314-3.