Differential modulation of angiotensin II- and phenylephrine-induced vasoconstriction by nitric oxide derived from S-nitrosylated proteins in the aorta of rats

Background: Angiotensin II (AII) is described as a potent vasoconstrictor, but its contractile effects in endothelium-intact (E+) and endothelium-denuded (E-) arteries from healthy animals are lower than those induced by α1-adrenergic ligands such as phenylephrine (Phe). AII can stimulate nitric oxide synthases (NOS) in endothelial cells, which can directly counteract its vasoconstrictive effects in E+ arteries but not in E- arteries. We explored the hypothesis that nitric oxide (NO) derived from nitrosylated proteins is a physiological antagonist of AII-induced vasoconstriction in E- arteries. Methods: Male Wistar rats (3-4 months) were euthanized, the thoracic aorta was dissected, adjacent tissues were removed, and E+ and E- aortic rings were mounted in organ baths. The contractile response was assessed by adding cumulative concentrations of Phe and AII in vessels previously incubated with vehicle only or with N-ω-nitro-L-arginine methyl ester (L-NAME, 100 µM; 30 min), nitroglycerin (NTG, 100 nM; 5 min), DTNB (300 µM; for 10 min), 1400W (10 µM; for 30 min) S-methyl L-thiocitrulline (SMTC, 10 µM, 30 min), or carboxy-PTIO (100 µM, 30 min). Furthermore, the impact of a single concentration of Phe and AII on E-, in the presence or absence of L-NAME, NTG, and DTNB was investigated. NO was detected in E+ and E- aortic slices using the DAF2 fluorescent probe. Results are expressed as mean ± SEM (n = 6-7/group) and subjected to Student t-test or ANOVA followed by Bonferroni. Results: AII showed a lower maximal effect (Emax) in E+ (0.44 ± 0.07 g) arteries, compared with the Emax induced by Phe (2.1 ± 0.21 g). Phe- and AII-induced contraction were differentially enhanced in E- arteries by 52.3 ± 12.1% and 328.6 ± 60.2%, respectively. Similarly, pre-incubation of E+ rings with L-NAME (a non-selective NOS inhibitor) increased the Emax of Phe and AII by 78.1 ± 10.7 and 324.4 ± 19.1%, respectively. Moreover, the vasoconstrictor effect of AII was much more sensitive to NTG than Phe in both E+ and E- arteries. DAF-2 data showed no significant differences in fluorescence levels detected after stimulation by 10 µM Phe or 1 µM AII in E+ aortic samples. However, there was a ~40% increase in fluorescence detection in E- slices after exposure to AII, which was enhanced in samples incubated with NTG. Notably, Phe did not increase DAF-2 fluorescence in any experimental condition. DTNB, used to reduce protein S-nitrosylation, increased the Emax of AII and fully reversed NTG-induced hyporesponsiveness to AII in E-, without exerting any influence in Phe-induced vasoconstriction. Pre-incubation with carboxy-PTIO (a nitric oxide scavenger) in E- significantly increased the Emax of AII. Finally, pre-incubation with L-NAME and SMTC (a selective neuronal NOS inhibitor), but not with 1400W (a selective inducible NOS inhibitor), increased the Emax of AII (control/vehicle: 0.95 ± 0.13 g; L-NAME: 1.41 ± 0.25 g; SMTC: 1.51 ± 0.16 g) in E- arteries, without any influence in the contractile responses evoked by Phe. Conclusion: Our results indicate that in E- arteries, the vasoconstrictor effect of AII is attenuated by releasing NO from nitrosylated proteins in a process dependent on NO production by NOS, including the nNOS subtype. Although the signaling pathway has not been fully explored, understanding how this process is altered in diseases may provide important insights into cardiovascular pathophysiology. Financial Support : CAPES, 001; CNPq 405538/2021-9 e 403615/2023-2. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.