Role of superoxide and thromboxane receptors in acute angiotensin II-induced vasoconstriction of rabbit vessels

Abstract

This study explored the hypothesis that a portion of angiotensin II-induced contractions is dependent on superoxide generation and release of a previously unidentified arachidonic acid metabolite that activates vascular smooth muscle thromboxane receptors. Treatment of rabbit aorta or mesentery artery with the thromboxane receptor antagonist SQ29548 (10 μM) reduced angiotensin II-induced contractions (maximal contraction in aorta; control vs. SQ29548: 134 ± 16 vs. 93 ± 10%). A subset of rabbits deficient in vascular thromboxane receptors also displayed decreased contractions to angiotensin II. The superoxide dismutase mimetic Tiron (30 mM) attenuated angiotensin II-induced contractions only in rabbits with functional vascular thromboxane receptors (maximal contraction in aorta; control vs. Tiron: 105 ± 5 vs. 69 ± 11%). Removal of the endothelium or treatment with a nitric oxide synthase inhibitor, nitro-l-arginine (30 μM) did not alter angiotensin II-induced contractions. Tiron and SQ29548 decreased angiotensin II-induced contractions in the denuded aortas by a similar percentage as that observed in intact vessels. The cyclooxygenase inhibitor indomethacin (10 μM) or thromboxane synthase inhibitor dazoxiben (10 μM) had no effect on angiotensin II-induced contractions indicating that the vasoconstrictor was not thromboxane. Angiotensin II increased the formation of a 15-series isoprostane. Isoprostanes are free radical-derived products of arachidonic acid. The unidentified isoprostane increased when vessels were incubated with the superoxide-generating system xanthine/xanthine oxidase. Pretreatment of rabbit aorta with the isoprostane isolated from aortic incubations enhanced angiotensin II-induced contractions. Results suggest the factor activating thromboxane receptors and contributing to angiotensin II vasoconstriction involves the superoxide-mediated generation of a 15-series isoprostane.

Fig. 1.

Contractile effects of U46619 (A) and the isoprostane 8-iso-PGF_2α (B) in aortas from rabbits with an increase in vascular smooth muscle cell TP receptor (vTP+) and those with a decrease (vTP−). Concentration-response curves were obtained by the cumulative addition of drugs. Values are means ± SE expressed as %response to KCl (40 mM).

Fig. 2.

Contractile responses to angiotensin II (ANG II). Concentration-response curves were obtained by the cumulative addition of angiotensin II in the presence or absence of SQ29548 (10 μM; A) in vTP+ or vTP− aortas (B). Values are means ± SE expressed as %response to KCl (40 mM). Each value represents means ± SE for n = 12. *P ≤ 0.05, SQ29548-treated compared with control (A) or vTP+ vs. vTP− (B).

Fig. 3.

Contractile responses to angiotensin II (ANG II). Concentration-response curves were obtained by the cumulative addition of angiotensin II in the presence or absence of SQ29548 (10 μM) in vTP+ or vTP− mesenteric arteries. Values are means ± SE expressed as %response to KCl (60 mM). Each value represents means ± SE for n = 12. *P ≤ 0.05, SQ29548-treated compared with control (A) or vTP+ vs. vTP− (B).

Fig. 4.

Metabolism of ¹⁴C-arachidonic acid (AA) in vTP+ aortas. Vessels with (B) and without (A) angiotensin II (0.1 μM) were incubated with HEPES buffer at 37°C for 15 min with ¹⁴C-AA. Metabolites of arachidonic acid were separated by reverse-phase HPLC as explained in the materials and methods. Migration time of known standard eicosanoids are shown above the chromatograms. TXB₂, thromboxane B₂; cpm, counts per minute.

Fig. 5.

Effect of Tiron (30 mM; B and C) and SQ29548 (10 μM; A) on contractile responses to angiotensin II in vTP+ aortas with (B) and without endothelium (A and C). Values are means ± SE expressed as %response to KCl (40 mM). Each value represents means ± SE for n = 8. *P ≤ 0.05, Tiron-treated compared with control.

Fig. 6.

Representative of liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) selected ion chromatogram (m/z = 353) of vTP+ vessels incubated in HEPES buffer with and without angiotensin II [0.1 μM; aorta (A) or mesentery artery (B)] or xanthine (100 μM)/xanthine oxidase (0.03 U/ml; aorta; C). Samples were extracted and analyzed by LC-ESI-MS as described in the materials and methods. Migration times of known isoprostane standards are shown. Arrow points to the 8.5-min peak increased by angiotensin II or xanthine/xanthine oxidase. These studies were repeated 3 separate times.

Fig. 7.

MS-MS analysis of 8.5 peak increased by angiotensin II in rabbit aorta. Inset: chemical structure of the 8-iso-PGF_2α isomer for reference only. m/z, Mass-to-charge ratio.

Fig. 8.

Effect of subthreshold concentration of 8-iso-PGF_2α (5 nM; B) and the 8.5-min isoprostane peak (A) on angiotensin II-induced contractions. Fractions corresponding to the 8.5-min peak were collected from the LC system shown in Fig. 6. Following extraction, the product was added to vTP+ rabbit aortic rings suspended in tissue baths before the administration of increasing concentration of angiotensin II (10⁻¹⁰–10⁻⁶ M). Data are expressed as percentage of the KCl contraction and are shown as means ± SE for n = 4. *P < 0.05, for 8-iso-PGF_2α or 8.5-min peak vs. vehicle control.