Endothelial nitric oxide and 15-lipoxygenase-1 metabolites independently mediate relaxation of the rabbit aorta

Abstract

Endothelial 15-lipoxygenase-1 (15-LO-1) metabolites of arachidonic acid (AA), 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and nitric oxide (NO) mediate relaxations to acetylcholine (ACH). However, interactions between NO and the 15-LO-1 pathway have not been explored. Therefore, the effect of physiological and pharmacological concentrations of NO on 15-LO activity and relaxation was studied in rabbit aorta. In indomethacin-treated aortic rings, maximal ACH relaxations of 91.3±4.0%, decreased to 54.5±3.0% by the NO synthase inhibitor, nitro-l-arginine (LNA), to 49.8±3% by the guanylate cyclase (GC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, to 63.7±4.9% by the lipoxygenase (LO) inhibitor, nordihydroguaiaretic acid (NDGA) and were completely inhibited by the combination of LNA and NDGA. AA relaxations were not affected by GC inhibition but were reduced by LO inhibition. The NO donor, dipropylenetriamine-NONOate (DPTA) caused concentration-related relaxations (EC(50)=4.7×10(-6)M). Aortic metabolism of (14)C-AA to THETA and HEETA was not altered by EC(50) concentrations of DPTA but were reduced 10-fold by 10(-3)M DPTA. In LNA-treated aorta, DPTA (3×10(-6)M) caused relaxations of 38.2.5±4%. Maximum relaxations to ACH did not differ in the presence and absence 3×10(-6)M DPTA (49.5±5% and 44.2±4%, respectively). These results indicate that NO and 15-LO-1 act in parallel to mediate ACH relaxations and NO does not alter 15-LO-1 activity.

Figure 1

Acetylcholine (ACH) or arachidonic acid (AA) induced aortic relaxations: Aortas were pretreated with indomethacin (10⁻⁵ M), precontracted with phenylephrine (10⁻⁷ to 10⁻⁶ M) and relaxations to cumulative concentrations of ACH or AA were determined. ACH-relaxations in the aortic rings treated with vehicle, nitro-L-arginine (LNA; 3 × 10⁻⁵ M), NDGA (3 × 10⁻⁵ M) or LNA and NDGA (A) and, vehicle, ODQ, or ODQ and NDGA (B). AA relaxations in the aortic rings treated with vehicle, ODQ or ODQ and NDGA (C). n = 8–16, ** = p<0.01, ***=p<0.0001.

Figure 2

Relaxations to the NO donor, DPTA NONOate (DPTA) in aortas: Aortas were precontracted with phenylephrine (10⁻⁷ to 10⁻⁶ M) and relaxations to cumulative concentrations of DPTA were determined. (A) Relaxations to cumulative concentrations of DPTA alone (EC₅₀=4.7 × 10⁻⁶ M). Aortas were treated with either vehicle or ODQ (B) or vehicle or NDGA (C) and relaxations to DPTA determined. n = 8–16, ** = p<0.01, ***=p<0.0001.

Figure 3

Effects of LNA and DPTA on 15-LO-1 metabolism of ¹⁴C-AA in aortic rings with endothelium: Aortic rings were treated with indomethacin (Indo, 10⁻⁵ M) and incubated with [¹⁴C]-AA in the presence of vehicle (A), nitro-L-arginine (LNA, 3 × 10⁻⁵ M) (B), LNA and 3 × 10⁻⁶ M DPTA (C), and LNA and 10⁻³ M DPTA (D). The media was removed, extracted and the metabolites resolved by HPLC. Migration times of known standards are indicated in each panel.

Figure 4

Effects of LNA and DPTA on the synthesis of 15-LO-1 metabolites in aortic rings with endothelium (see legend to Figure 3) in the presence of vehicle (A), nitro-L-arginine (LNA, 3 × 10⁻⁵ M) (B) or LNA treated rings treated with various concentrations of DPTA (C and D). The media was removed, extracted and metabolites resolved by HPLC. Percentage metabolite CPM synthesized per mg tissue is expressed as mean ± SEM and compared with values in the LNA only incubations. n = set of 3 experiments, ** = p<0.01, ***=p<0.0001

Figure 5

Effect of NO on acetylcholine (ACH)-induced relaxations: Aortas were pretreated with indomethacin (10⁻⁵ M) and LNA (3 × 10⁻⁵ M) and precontracted with phenylephrine (10⁻⁷ to 10⁻⁶ M). Rings were treated with vehicle or DPTA (3 × 10⁻⁶ M) and allowed to equilibrate to a stable baseline. Relaxations to cumulative concentrations of ACH were determined. (A) The percentage of relaxation was plotted against the concentrations of ACH. The addition of DPTA relaxed the aortas (shown by star on Y-axis). (B) The baseline for the two treatments was subtracted and the baseline corrected relaxations were plotted against the concentrations of ACH. n = 10–12.