1 Experiments were designed to investigate whether or not oxygen-derived free radicals mediate endothelium-dependent contractions to acetylcholine in the aorta of spontaneously hypertensive rat (SHR). 2 Isometric tension was measured in aortic rings taken from adult male SHR and Wistar-Kyoto rat (WKY) in the presence of N G -nitro-L-arginine. 3 Endothelium-dependent contractions to acetylcholine were signi®cantly greater in rings from SHR compared to WKY. Oxygen-derived free radicals, generated from xanthine plus xanthine oxidase, induced contractions that were larger in aortas from SHR than from WKY. Contractions to acetylcholine and free radicals were abolished by a selective TP-receptor antagonist, S 18886, and a preferential inhibitor of cyclo-oxygenase-1, valeryl salicylate, but not by a preferential inhibitor of cyclo-oxygenase-2, NS-398. 4 Allopurinol, deferoxamine and the combination of superoxide dismutase plus catalase inhibited the contractions to oxygen-derived free radicals but did not signi®cantly aect those to acetylcholine. In contrast, diethyldithiocarbamic acid, an inhibitor of superoxide dismutase, or Tiron, a scavenger of superoxide anion, reduced endothelium-dependent contractions to acetylcholine in aortas from SHR. The eect of these two drugs was additive. 5 In SHR chronically treated with dimethylthiourea endothelium-dependent contractions to acetylcholine were decreased, and reduced further by acute in vitro exposure to deferoxamine or the combination of superoxide dismutase plus catalase. 6 These results suggest that in the SHR aorta acetylcholine-induced endothelium-dependent contractions involve endothelial superoxide anion production and the subsequent dismutation into hydroxyl radicals and/or hydrogen peroxide. The free radicals activate cyclo-oxygenase-1, most likely to produce endoperoxides. Activation of TP-receptors is required to observe endotheliumdependent contractions to acetylcholine or endothelium-independent contractions in response to free radical generation.

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Abstract-A modified bioassay system was designed to demonstrate the diffusible nature of endothelium-derived contracting factor(s) released by acetylcholine in the aorta of spontaneously hypertensive rat. In "sandwich"-like layered preparation, isometric tension was recorded from a bioassay strip (without endothelium) in the presence of N G -nitro-L-arginine and tetrahydrobiopterin to selectively potentiate endothelium-dependent contractions. A donor strip (with or without endothelium) was stitched on the bioassay tissue so that it did not directly contribute to the recorded contractions. Acetylcholine induced contractions that occurred only when the donor strip was with endothelium. Superoxide dismutase did not affect but catalase and the combination of superoxide dismutase plus catalase significantly decreased the endothelium-dependent contraction. The contractions in the layered preparations were abolished when the donor strip with endothelium was treated previously with valeryl salicylate, an irreversible cyclooxygenase-1 inhibitor, but remained unaffected when the bioassay strip was treated with the compound. Previous treatment of the bioassay strip alone with S 18886 abolished the contractile response, whereas treatment of the donor strip with endothelium by the selective TP receptor antagonist only produced a moderate inhibition. These results indicate that in the aorta of spontaneously hypertensive rats, endothelium-dependent contractions to acetylcholine involve a diffusible substance(s) released by the endothelium. The production of this contracting factor(s) requires the activation of endothelial cyclooxygenase-1, and its action the activation of TP receptors on the vascular smooth muscle cells. Key Words: cyclooxygenase Ⅲ endothelium-dependent contraction Ⅲ tetrahydrobiopterin Ⅲ free radicals Ⅲ rats, spontaneously hypertensive Ⅲ receptors, thromboxane T he endothelium-derived contracting factors (EDCF) identified so far include superoxide anions, endoperoxides, thromboxane A 2 , and endothelin-1. 1 In the aorta of the spontaneously hypertensive rat (SHR), endotheliumdependent contractions to acetylcholine involve reactive oxygen species that activate the cyclooxygenase-1 pathway with the production of endoperoxide(s), which stimulate TP receptors on the aortic vascular smooth muscle. 2 Of the products of the cyclooxygenase pathway, endoperoxide(s) (in particular prostaglandin H 2 ) are likely candidates as EDCF in the SHR aorta. 1,[3][4][5] This conclusion is supported by the observations that inhibitors of cyclooxygenase-1 and TP receptor antagonists abolish the endothelium-dependent contraction but that inhibitors of thromboxane synthase fail to do so. 2,3,5,6 The release of endoperoxides is larger in the aorta of SHR than in that of normotensive Wistar-Kyoto rat (WKY); this phenomenon is associated with a greater expression of cyclooxygenase-1 in the SHR aorta. 3 In addition, the aortas from SHR without endothelium show a higher sensitivity to exogenous prostaglandin H 2 than those of normotensive...