Angiotensin II relaxations of bovine adrenal cortical arteries: role of angiotensin II metabolites and endothelial nitric oxide

Abstract

Angiotensin (Ang) II regulates adrenal steroidogenesis and adrenal cortical arterial tone. Vascular metabolism could decrease Ang II concentrations and produce metabolites with vascular activity. Our goals were to study adrenal artery Ang II metabolism and to characterize metabolite vascular activity. Bovine adrenal cortical arteries were incubated with Ang II (100 nmol/L) for 10 and 30 minutes. Metabolites were analyzed by mass spectrometry. Ang (1-7), Ang III, and Ang IV concentrations were 146+/-21, 173+/-42 and 58+/-11 pg/mg at 10 minutes and 845+/-163, 70+/-14, and 31+/-3 pg/mg at 30 minutes, respectively. Concentration-related relaxations of U46619-preconstricted cortical arteries to Ang II (maximum relaxation=29+/-3%; EC(50)=3.4 pmol/L) were eliminated by endothelium removal and inhibited by the NO synthase inhibitor, nitro-L-arginine (30 micromol/L; maximum relaxation=14+/-7%). Ang II relaxations were enhanced by the angiotensin type-1 receptor antagonist losartan (1 micromol/L; maximum relaxation=41+/-3%; EC(50)=11 pmol/L). Losartan-enhanced Ang II relaxations were inhibited by nitro-L-arginine (maximum relaxation=18+/-5%) and the angiotensin type-2 receptor antagonist PD123319 (10 micromol/L; maximum relaxation=27+/-5%). Ang (1-7) and Ang III caused concentration-related relaxations with less potency (EC(50)=43 and 24 nmol/L, respectively) but similar efficacy (maximum relaxations=39+/-3% and 48+/-5%, respectively) as losartan-enhanced Ang II relaxations. Ang (1-7) relaxations were inhibited by nitro-L-arginine (maximum relaxation=16+/-4%) and the Ang (1-7) receptor antagonist 7(D)-Ala-Ang (1-7) (1 micromol/L; maximum relaxation=10+/-3%) and eliminated by endothelium removal. Thus, Ang II metabolism by adrenal cortical arteries to metabolites with decreased vascular activity represents an inactivation pathway possibly decreasing Ang II presentation to adrenal steroidogenic cells and limits Ang II vascular effects.

Figure 1

Effect of the endothelium on Ang II-induced relaxations of isolated bovine adrenal cortical arteries. Relaxations were performed under control conditions or after endothelial removal. n = 4–13. * p< 0.05 vs. control.

Figure 2

LC-MS/MS chromatograms of angiotensin peptides produced by bovine adrenal cortical arteries. Isolated arteries were incubated with Ang II (100 nmol/L) and the peptides extracted and analyzed. Synthetic ¹³C₅¹⁵N₁-Ang IV (top panel) was added as an internal standard (m/z 391.3→269.2, elution time = 11.92 min). Angiotensin peptides identified included Ang IV (m/z 388.8→263.4, elution time = 11.92 min), non-metabolized Ang II (m/z 349.6→255.2, elution time = 11.18 min), Ang III (m/z 311.3→228.4, elution time = 10.55) and Ang (1-7) (m/z 300.6→109.6, elution time = 4.77). The abundance of the internal standard, Ang IV, Ang III, and Ang (1-7) were amplified 10X.

Figure 3

Time course of Ang II metabolism by bovine adrenal cortical arteries. Isolated arteries were incubated with Ang II (100 nmol/L) for 10 and 30 min. The peptides were extracted and analyzed by LC-MS/MS. n=3–4. * p< 0.05 vs. other peptides.

Figure 4

Ang II-induced relaxations of isolated bovine adrenal cortical arteries. A. Effect of the NO synthase inhibitor, L-NA (30 μmol/L) and the AT1 receptor antagonist, losartan (1 μmol/L). Relaxations were performed under control conditions or after treatment with losartan or L-NA. B. Effect of L-NA on losartan-enhanced Ang II relaxations. Relaxations were performed on losartan-treated arteries before and after treatment with L-NA. C. Effect of the Ang (1-7) receptor antagonist, Ala7-Ang (1-7) (1 μmol/L) on losartan-enhanced Ang II relaxations. Relaxations were performed on losartan-treated arteries before and after treatment with Ala7-Ang (1-7). D. Effect of the AT2 receptor antagonist, PD123319 (1 and 10 μmol/L) on losartan-enhanced Ang II relaxations. Relaxations were performed on losartan-treated arteries before and after treatment with PD123319. n = 5–34, * p< 0.05 vs. control or losartan.

Figure 5

Concentration-dependent relaxations to Ang (1-7) and Ang III of isolated bovine adrenal cortical arteries. n = 7–10.

Figure 6

Ang (1-7)-induced relaxations of isolated bovine adrenal cortical arteries. A. Effect of the NO synthase inhibitor, L-NA (30 μmol/L) and endothelium removal. Responses to Ang (1-7) were performed before and after treatment with L-NA or after endothelial cell removal. B. Effect of the Ang (1-7) receptor antagonist, Ala7-Ang (1-7) (1 μmol/L), the AT1 receptor antagonist, losaratan (1 μmol/L) or the AT2 receptor antagonist, PD123319 (10 μmol/L). Responses were performed before and after receptor antagonist treatment. n = 6–21. * p< 0.05 vs. control.