Inhibition by singlet molecular oxygen of the vascular reactivity in rabbit mesenteric artery

Abstract

1. The effects of reactive oxygen intermediates derived from photoactivated rose bengal on the vascular reactivity have been evaluated in rabbit mesenteric artery ring preparations. The artery rings were exposed to xanthene dye rose bengal (50 nM) illuminated (6,000 lux) at 560 nm for 30 min. Spin trapping studies with 2,2,6,6-tetramethylpiperidine (TEMP) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with electron spin resonance spectrometry were also conducted in solution (and not within tissues) to determine quantitatively the reactive oxygen species generated from photoactivated rose bengal.

2. Contraction of the ring preparations induced by noradrenaline (10⁻⁸ to 10⁻⁴ M) was attenuated by previous exposure to photolysed rose bengal; the observation that the pD₂ decreased without a significant reduction in maximum tension generation is consistent with the view that receptor dysfunction may be involved in the effect of photolysed rose bengal.

3. Prior exposure to photolysed rose bengal of the ring preparations inhibited the endothelium-dependent relaxation evoked by acetylcholine (10⁻⁶ M) and calcium ionophore A23187 (10⁻⁷ M), but not the endothelium-independent relaxation evoked by nitroglycerin (10⁻⁶ M).

4. A variety of scavengers, superoxide dismutase (33 units ml⁻¹), catalase (32 units ml⁻¹) and 1,3-dimethyl-2-thiourea (DMTU, 10 mM), which should eliminate the superoxide anion radical, H₂O₂ and the hydroxyl radical, had no effect on the attenuated responses to noradrenaline and acetylcholine induced by photolysed rose bengal. In contrast, the inhibition of the observed effect of photolysed rose bengal was obtained with addition of histidine (25 mM), a singlet molecular oxygen quencher.

5. It was found that photolysis of rose bengal from a 1 : 2 : 2 : 1 quartet, characteristic of the hydroxyl radical-DMPO spin adduct, which was effectively blunted by DMTU, superoxide dismutase and catalase whereas histidine was ineffective. The results of the electron spin resonance study also showed that a singlet molecular oxygen was produced by photoactivation of rose bengal; this was detected as singlet oxygen-TEMP product (TEMPO; 2,2,6,6-tetramethylpiperidine-N-oxyl). The formation of the TEMPO signal was strongly inhibited by histidine, but not by DMTU, superoxide dismutase and catalase.

6. It is suggested that the superoxide anion radical, H₂O₂ and hydroxyl radical are formed in addition to singlet molecular oxygen, and the data obtained from the present study indicate that singlet molecular oxygen is one of the most destructive oxygen species. Endothelium-dependent relaxation is quite vulnerable to singlet molecular oxygen. Singlet oxygen also depresses noradrenaline-induced contraction possibly via α-adrenoceptor dysfunction. This, in turn, may lead to vascular incompetence.

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