Hydrogen peroxide is an endothelium-dependent contracting factor in rat renal artery

Abstract

1 In addition to endothelium-derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone by releasing endothelium-derived contracting factor(s) (EDCF), but the identity of EDCF remains obscure. We studied here the involvement of hydrogen peroxide (H 2O 2) in endothelium-dependent contraction (EDC) of rat renal artery to acetylcholine (ACh). 2 ACh (10 -6, 10 -5, and 10 -4 M) induced a transient contraction of rat renal artery with intact endothelium in a concentration-related manner, but not in the artery with endothelium removed. In phenylephrine-precontracted renal arteries, ACh induced an endothelium-dependent relaxation response at lower concentrations (10 -8-10 -6 M), and a relaxation followed by a contraction at higher concentrations (10 -5 M). Inhibition of nitric oxide synthase by N ω-nitro-L-arginine (10 -4 M) enhanced the EDC to ACh. 3 Catalase (1000 U ml -1) reduced the EDC to ACh. H 2O 2 (10 -6, 10 -5, and 10 -4 M) induced a similar transient contraction of the renal arteries as ACh, but in an endothelium-independent manner. Inhibition of NAD(P)H oxidase and cyclooxygenase by diphenylliodonium chloride and diclofenac greatly attenuated ACh-induced EDC, while inhibition of xanthine oxidase (allopurinol) and cytochrome P450 monooxygenase (17-octadecynoic acid) did not affect the contraction. Antagonist of thromboxane A2 and prostaglandin H 2 receptors (SQ 29548) and thromboxane A 2 synthase inhibitor (furegrelate) attenuated the contraction to ACh and to H 2O 2. 4 In isolated endothelial cells, ACh (10 -5 M) induced a transient H 2O 2 production detected with a fluorescence dye sensitive to H 2O 2 (2′,7′- dichlorofluorescein diacetate). The peak concentration of H 2O 2 was 5.1 × 10 -4 M at 3 min and was prevented by catalase. 5 Taken together, these results show that ACh triggers H 2O 2 production through NAD(P)H oxidase activation in the endothelial cells, and that ACh and H 2O 2 share the same signaling pathway in causing smooth muscle contraction. Therefore, H 2O 2 is most likely the EDCF in rat renal artery in response to ACh stimulation. © 2005 Nature Publishing Group. All rights reserved.