15-Lipoxygenase metabolites contribute to age-related reduction in acetylcholine-induced hypotension in rabbits

Abstract

Arachidonic acid (AA) metabolites from the 15-lipoxygenase-1 (15-LO-1) pathway, trihydroxyeicosatrienoic acids (THETAs) and hydroxy-epoxyeicosatrienoic acids (HEETAs), are endothelium-derived hyperpolarizing factors (EDHFs) and relax rabbit arteries. Rabbit vascular 15-LO-1 expression, THETA and HEETA synthesis, and nitric oxide and prostaglandin-independent relaxations to acetylcholine (ACh) and AA decreased with age (neonates to 16-wk-old). We characterized age-dependent ACh-hypotensive responses in vivo in 1-, 4-, 8-, and 16-wk-old rabbits and the contribution of THETAs and HEETAs to these responses. In anesthetized rabbits, blood pressure responses to ACh (4-4,000 ng/kg) were determined in the presence of vehicle or various inhibitors. ACh responses decreased with age (P > 0.001). In the absence or presence of N(omega)-nitro-l-arginine methyl ester (l-NAME) and indomethacin (Indo), maximum responses in 1 (-54.7 +/- 7.4 and -37.9 +/- 3.9%)- and 4 (-48.8 +/- 2.4 and -35.5 +/- 7.8%)-wk-old rabbits were higher than 8 (-30.0 +/- 2.8 and -26.6 +/- 4.4%)- and 16 (-36.7 +/- 3.5 and -27.3 +/- 10%)-wk-old rabbits. A lipoxygenase inhibitor, BW755C, reduced THETA and HEETA synthesis in mesenteric arteries. In the presence of Indo and N(omega)-nitro-l-arginine, ACh relaxations were reduced by BW755C to a greater extent in the mesenteric arteries from the younger rabbits. In 4-wk-old rabbits treated with l-NAME and Indo, the maximum ACh hypotension was reduced by the potassium channel inhibitors apamin and charybdotoxin to -6.9 +/- 0.9%, by apamin alone to -19.5 +/- 1.4%, and by BW755C to -18.8 +/- 3.5%. The present study indicates that the age-related decrease in ACh-induced hypotension is mediated by the decreased synthesis of the 15-LO-1 metabolites THETAs and HEETAs.

Fig. 1.

Effect of acetylcholine (ACh) on mean arterial pressure (MAP) in 1 (n = 6)-, 4 (n = 9)-, 8 (n = 7)-, and 16 (n = 7)-wk-old rabbits. Rabbits were anesthetized and increasing doses of ACh were injected intravenously. Decreases in MAP are expressed as percent decrease from baseline. Each value is mean ± SE. MAP changes to ACh in 1- and 4-wk-old rabbits are significantly different from 8- and 16-wk-old rabbits (**P < 0.001; ***P < 0.0001).

Fig. 2.

Effects of nitric oxide synthase and cyclooxygenase inhibition on the MAP responses to ACh in 1 (A; n = 5)-, 4 (B; n = 7)-, 8 (C; n = 5)-, and 16 (D; n = 5)-wk-old rabbits. Rabbits were treated with vehicle or N^ω-nitro-l-arginine methyl ester (l-NAME; 20 mg/kg; 5 mg·kg⁻¹·h⁻¹) and indomethacin (Indo; 6 mg/kg). After 30 min, ACh responses were determined. Decreases in MAP are expressed as percent decrease from baseline. Each value is mean ± SE (*P < 0.05; **P < 0.001; ***P < 0.0001). All symbols are as defined in A.

Fig. 3.

Metabolism of [¹⁴C]arachidonic acid (AA) by mesenteric arteries of 1 (A)-, 4 (B)-, 8 (C)-, and 16 (D)-wk-old rabbits. Mesenteric arterial rings with intact endothelium were incubated with [¹⁴C]AA in the presence of Indo (10⁻⁵ M). Media were extracted, and eicosanoids were resolved by HPLC. The production of ¹⁴C metabolites is normalized by tissue weight. Migration times of known standards are shown above chromatogram in A. THETA, 11,12,15-trihydroxyeicosatrienoic acid; HEETA, 15-hydroxy-11,12-epoxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid.

Fig. 4.

Effect of BW755C on [¹⁴C]AA metabolism in rabbit mesenteric arteries. Mesenteric arterial rings from 4-wk-old rabbits were incubated with [¹⁴C]AA in the presence of Indomethacin (10⁻⁵ M) without (A) and with (B) BW755C (10⁻⁴ M). Media were removed and extracted, and the metabolites were resolved by HPLC. Migration times of known standards are indicated above chromatograms. Data represent 3 sets of experiments on arteries from different rabbits.

Fig. 5.

Effects of the lipoxygenase (LO) inhibitor BW755C (BW) and the IK_Ca channel inhibitor charybdotoxin (CTX) on the vascular activity in rabbit mesenteric arteries. Arterial rings (n = 8–12) were pretreated with Indo (10⁻⁵ M) and N^ω-nitro-l-arginine (l-NNA; 3 × 10⁻⁵ M) with vehicle, or BW755C (10⁻⁴ M), or CTX (2 × 10⁻⁷ M), or both CTX and BW755C; they were precontracted with phenylephrine (10⁻⁷ to 10⁻⁶ M), and relaxations to cumulative concentrations of ACh were determined. Data are expressed as percent relaxations, and each value represents mean ± SE. All symbols are as defined in A.

Fig. 6.

Effects of potassium channel inhibitors and the LO-inhibitor BW755C on the MAP response to ACh in l-NAME- and Indo-treated 4-wk-old (n = 6) rabbits. Rabbits were treated with l-NAME (20 mg/kg; 5 mg·kg⁻¹·h⁻¹) and Indo (6 mg/kg) for 30 min, and ACh responses were determined. Rabbits were then injected with (A) apamin (Apa; 50 μg/kg) and CTX (50 μg/kg), Apa (B; 50 μg/kg), or BW755C (C; 20 mg/kg) and, after 30 min the ACh responses were repeated. Decreases in MAP are expressed as percent decrease from baseline. Each value represents mean ± SE (**P < 0.001; ***P < 0.0001).