Pharmacological Research 66 (2012) 383–391 Contents lists available at SciVerse ScienceDirect Pharmacological Research jo ur n al hom epage: www.elsevier.com/locate/yphrs Endothelium-dependent nitroxyl-mediated relaxation is resistant to superoxide anion scavenging and preserved in diabetic rat aorta C.H. Leo a,1,2 , A. Joshi a,b,1 , J.L. Hart a , O.L. Woodman a,∗ a School of Medical Sciences, Health Innovations Research Institute, RMIT University, Bundoora, Victoria, Australia b Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia a r t i c l e i n f o Article history: Received 25 May 2012 Received in revised form 30 July 2012 Accepted 30 July 2012 Keywords: Diabetes Endothelium-dependent relaxation Nitroxyl Nitric oxide Superoxide anion a b s t r a c t The aim of the study was to investigate whether diabetes-induced oxidant stress affects the contribu- tion of nitroxyl (HNO) to endothelium-dependent relaxation in the rat aorta. Organ bath techniques were employed to determine vascular function of rat aorta. Pharmacological tools (3 mM l-cysteine, 5 mM 4-aminopyridine (4-AP), 200 M carboxy-PTIO and 100 M hydroxocobalamin, HXC) were used to distinguish between NO and HNO-mediated relaxation. Superoxide anion levels were determined by lucigenin-enhanced chemiluminescence. In the diabetic aorta, where there is increased superoxide anion production, responses to the endothelium-dependent relaxant ACh were not affected when the contribution of NO to relaxation was abolished by either HXC or carboxy-PTIO, indicating a preserved HNO-mediated relaxation. Conversely, when the contribution of HNO was inhibited with l-cysteine or 4-AP, the sensitivity and maximum relaxation to ACh was significantly decreased, suggesting that the con- tribution of NO was impaired by diabetes. Furthermore, whereas HNO appears to be derived from eNOS in normal aorta, in the diabetic aorta it may also arise from an eNOS-independent source, perhaps derived from nitrosothiol stores. Similarly, exposure to the superoxide anion generator, pyrogallol (100 M) significantly reduced the sensitivity to the NO donor, DEANONOate and ACh-induced NO-mediated relax- ation but had no effect on responses to the HNO donor, Angeli’s salt and ACh-induced HNO-mediated relaxation in the rat aorta. These findings demonstrate that NO-mediated relaxation is impaired during oxidative stress but the HNO component of relaxation is preserved under those conditions. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction The endogenous production of nitric oxide (NO) is well recog- nized as an important contributor to vascular homeostasis. The vascular action of NO predominately involves the activation of soluble guanylate cyclase (sGC), which catalyses the conversion Abbreviations: ACh, acetylcholine; AS, Angeli’s salt; 4-AP, 4-aminopyridine; BH4, tetrahydrobiopterin; cGMP, cyclic guanosine monophosphate; DEA, DEANONOate; DPI, diphenylene iodonium; EA, ethacrynic acid; EDHF, endothelium-derived hyperpolarizing factor; eNOS, endothelial nitric oxide synthase; GTP, guanosine triphosphate; HXC, hydroxocobalamin; HNO, nitroxyl; l-NNA, N-nitro-l-arginine; NO, nitric oxide; ODQ, 1H-[1,2,4]-oxadiazolo[4,2-a]quinoxalin-1-one; pEC50, neg- ative logarithm of the half maximal effective concentration; Rmax, maximum relaxation; PHMBA, p-hydroxymercuribenzoic acid; SNP, sodium nitroprusside; sGC, soluble guanylate cyclise; SOD, superoxide anions dismutase. ∗ Corresponding author at: School of Medical Sciences, Health Innovations Research Institute, RMIT University, PO Box 71, Bundoora, Victoria 3083, Australia. Tel.: +61 3 9925 7305; fax: +61 3 9925 7063. E-mail address: owen.woodman@rmit.edu.au (O.L. Woodman). 1 Co-first authors. 2 Present address: Department of Zoology, University of Melbourne, Parkville, Victoria, Australia. of guanosine triphosphate (GTP) to cyclic guanosine monophos- phate (cGMP), leading to vasodilatation [1]. NO may not be the only endothelium-derived nitrogen species, with nitroxyl (HNO), the one electron reduced and protonated form of NO, likely to have a role in modulating vascular tone as there is evidence that it is formed endogenously and contributes to endothelium-dependent relaxation in both conduit [2,3] and resistance vessels [4]. HNO can be produced by several distinct pathways in the vas- cular endothelium. Biochemical studies indicate that HNO can be synthesized via endothelial NO synthase (eNOS)-dependent and independent pathways [5]. HNO can be synthesized by eNOS itself as an intermediate product during the conversion of l-arginine to NO. Subsequently, HNO is oxidized to NO by superoxide dis- mutase [6]. Moreover, the production of HNO by eNOS could occur under conditions of reduced levels of the eNOS cofactor, tetrahydrobiopterin (BH 4 ) [7] or where there is oxidation of NOS intermediates, N-hydro-l-arginine [8] and hydroxylamine [9]. Fur- thermore, HNO can be produced from NOS-independent sources, including the reduction of NO by mitochondrial cytochrome c [10] and xanthine oxidase [11]. Finally, S-nitrosothiols are also known to generate HNO via S-thiolation, a reaction between S-nitrosothiols and other thiol species [12]. 1043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2012.07.010