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. 2014 Apr 25;3(2):e000731. doi: 10.1161/JAHA.113.000731

Figure 3.

Figure 3.

Ang II reduces NO bioavailability. A, Effect of Ang II on basal and ACh‐stimulated NO‐sensitive DAF2DA fluorescence in HUVECs. Cell nuclei were counterstained with DAPI (blue). Changes in the DAF2DA signal (df) were measured after stimulation with ACh (1 μmol/L) for 20 minutes (n=4; cells from 4 randomly chosen areas of interest in each group were included in the analysis). B, NO measurement by EPR with Fe(DETC)2 in control, in vitro Ang II‐treated (n=9), and in vivo captopril‐treated rabbit aortic segments (n=10 control and 6 captopril‐treated rabbits). Amplitude of NO‐Fe(DETC)2 signal was determined as the perpendicular height between the top of the first low‐field signal (g1=3297 G) and the valley of the third high‐field signal (g2=3323 G; arrows). Traces from the standard spermine NONOate in DMSO solution and endothelium‐denuded aorta are also shown. C, Effect of Ang II on NO‐sensitive DAF‐FM diacetate fluorescence in HUVECs overexpressing the WT or the double Cys mutant (Δ) eNOS in the presence of ACh. Statistical comparison was made in each cell type between conditions with and without Ang II (n=3). AU=arbitrary unit. Results are shown as means±SEM. Statistical significance (P<0.05) is indicated by asterisk (*). DAF indicates 4‐amino‐5‐methylmino‐2′,7′‐difluorofluorescein; DAPI, 4′,6‐diamidino‐2‐phenylindole; DHE, dihydroethidium; DTT, dithiotreitol; eNOS, endothelial nitric oxide synthase; EPR, electron paramagnetic resonance; HUVECs, human umbilical vein endothelial cells; l‐NAME, l‐NG‐nitroarginine methyl ester; NO, nitric oxide.