Nitrosothiol stores in vascular tissue: Modulation by ultraviolet light, acetylcholine and ionomycin Ella S.M. Ng a , Zhong-Jian Cheng a , Anthie Ellis b , Hong Ding b , Yanfen Jiang a , Yang Li a , Morley D. Hollenberg a , Chris R. Triggle a,b, ⁎ a Smooth Muscle Research Group, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Alberta, Canada b School of Medical Sciences, RMIT University, Bundoora West Campus, Plenty Road, Bundoora, Melbourne, Victoria, 3083 Australia Received 28 August 2006; received in revised form 4 January 2007; accepted 8 January 2007 Available online 19 January 2007 Abstract Our previous studies demonstrated that light-induced vascular relaxation (photorelaxation) was mediated by a tissue source of nitric oxide that was independent of endothelial nitric oxide synthase (eNOS), but sensitive to inhibitors of soluble guanylate cyclase, extracellular nitric oxide scavengers and possessed the properties of a nitrosothiol. In the present study we describe High Performance Liquid Chromatography and spectrofluorometric techniques that allowed us to measure tissue levels of the nitrosothiol, S-nitrosoglutathione and its modulation in mouse aortic tissues, smooth muscle cells and human umbilical vein endothelial cells (HUVECs) following exposure to exogenous S-nitrosoglutathione, light and chemical stimuli. Basal levels of S-nitrosoglutathione were similar in control mouse aortae and HUVECs and the store size could be enhanced by exposure of tissues/cells to nitric oxide solution. No basal S-nitrosoglutathione was detected in tissue from diabetic db/db mice; however, ultraviolet light was still able to elicit relaxation of aortic tissues. Ultraviolet light induced the release of nitric oxide from the S-nitrosoglutathione store with an associated increase in the concentration of nitrite. The release of nitric oxide from the store in HUVECs was modulated by extracellular oxidative stress induced by xanthine/xanthine oxidase and also, in an atropine-sensitive process, by acetylcholine, as well as by the calcium ionophore, ionomycin. These interventions resulted in a reduced S-nitrosoglutathione store and elevated levels of nitrite. These data suggest that endothelial and vascular smooth muscle cells possess stores of nitric oxide that, in part, exist in the form of S-nitrosoglutathione. Furthermore, these stores, albeit small, may provide an additional mechanism for the regulation of vascular tone, especially under conditions, such as diabetes, in which nitric oxide generation or bioavailability is compromised; however, additional studies are required to determine not only whether there are additional chemical storage forms of nitric oxide, but also the location of such stores. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. Keywords: S-nitrosothiols; Endothelium; Vascular smooth muscle; Photorelaxation; Acetylcholine 1. Introduction The endothelium plays a key role in the regulation of vascular tone with endothelium-dependent vasodilatation determined, in a vessel and stimulus-dependent manner, by the contributions of three components: nitric oxide, prostacyclin and endothelium- derived hyperpolarizing factor (EDHF). Although it is generally accepted that the endothelium-dependent release of nitric oxide results from the de novo generation of nitric oxide following the activation of endothelial nitric oxide synthase (eNOS), an increasing body of evidence also supports the concept that preformed nitric oxide-stores are associated with vascular tissue and contribute to the regulation of vascular tone (Alencar et al., 2003; Andrews et al., 2003; Chauhan et al., 2003; Batenburg et al., 2004a; Danser et al., 1998; Davisson et al., 1996; Furchgott et al., 1985; Lovren and Triggle, 1998). Similarly in gastric muscle (Ergun and Ögulener, 2005) thus suggesting that such stores may be ubiquitous — at least in smooth muscle. Interest in the existence and location of these putative stores stems from studies of a phenomenon termed photorelaxation, or ultraviolet light activated vasorelaxation, that was first described by Furchgott et al. (1961), and a potential role for preformed nitric oxide stores in contributing to nitric oxide endothelium- European Journal of Pharmacology 560 (2007) 183 – 192 www.elsevier.com/locate/ejphar ⁎ Corresponding author. School of Medical Sciences, RMIT University, Bundoora West Campus, Plenty Road, Bundoora, Melbourne, Victoria, 3083 Australia. Tel.: +61 3 9925 6537; fax: +61 3 9925 6555. E-mail address: chris.triggle@rmit.edu.au (C.R. Triggle). 0014-2999/$ - see front matter Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.01.016