Department of Basic Animal and Veterinary Sciences, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark Direct Measurements of Nitric Oxide Release in Relation to Expression of Endothelial Nitric Oxide Synthase in Isolated Porcine Mitral Valves S. G. Moesgaard 1,4 , L. H. Olsen 1 , B. Aasted 2 , B. M. Viuff 2 , L. G. Pedersen 1 , H. D. Pedersen 3 and A. P. Harrison 1 Addresses of authors: Department of 1 Basic Animal and Veterinary Sciences and 2 Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark; 3 Safety Pharmacology, Novo Nordisk A/S, Maaloev, Denmark; 4 Corresponding author: Tel.: +45 35283884; fax: +45 35282525; E-mail: som@kvl.dk With 4 figures Received for publication May 10, 2006 Summary The aim of this study was to measure the direct release of nitric oxide (NO) from the porcine mitral valve using a NO micro- electrode. Furthermore, the expression and localization of endothelial nitric oxide synthase (eNOS) in the mitral valve was studied using immunohistochemistry, Western blotting and RT-PCR. Results show that bradykinin increases NO release from mitral valves (DBradykinin: 33.71 ± 10.41 nm NO, P < 0.001, n ¼ 10), whereas N-nitro-l-arginine methyl esther (l-NAME) decreases NO release when compared with basal level (Dl-NAME: 82.69 ± 15.66 nm NO, P < 0.005, n ¼ 4). Both protein and mRNA expression of eNOS in mitral valves and in isolated valvular endothelial cells suggest that the NO release is mainly associated with the mitral valve endo- thelium. It is concluded that direct NO release from porcine mitral valves coincides with eNOS expression. This study documents useful techniques for investigations into the role of local NO release in mitral valve diseases. Introduction Nitric oxide (NO) is a free-radical gas produced endogen- ously from l-arginine in a reaction catalysed by nitric oxide synthase (NOS) (Kiechle and Malinski, 1993). Nitric oxide diffuses freely across membranes but has a short half-life, and it is therefore difficult to measure directly. Whilst several indirect methods have been used to measure NO of more relevance, perhaps, are selective electrochemical NO micro- electrodes, which have the capability to measure NO directly, relying on the oxidation of NO and measurement of the redox current that is generated. Moreover, such microelec- trodes offer the advantages of speed and high levels of sensitivity (Berkels et al., 2001; Zhang, 2004). Nitric oxide is released from both vascular endothelial cells as well as the endocardium (Schulz et al., 1991; Simonsen et al., 1999), however, only a few studies have tried to assess the release of NO in heart valve endothelium. Two studies have measured the NO release in mitral valves from dogs and pigs using a bioassay technique. So far, however, there are no reported studies on direct measurements of NO release from mitral valves. The mitral valve is composed of an endothelial layer towards the atrium (the auricularis) overlying a thin layer of collagen fibers, elastic fibers, and fibroblasts. Below this layer is a loose collection of collagen fibers, fibroblasts, and elastic fibers in a mucopolysaccharide-rich substance, overlying a dense collagen layer, and another layer of endothelial cells (the ventricularis) (Buchanan, 1977; Veinot et al., 2001). Endothelial NOS (eNOS) expression in vasculature increases in association with exposure to mechanical factors, such as shear stress (Ziegler et al., 1998), a response that is thought to protect the endothelium against further damage e.g. platelet aggregation. Apart from endothelial cells the dom- inating cell type of the mitral valve is the valvular interstitial cell (VIC) which is believed to synthesize and remodel the connective tissue matrix (Mulholland and Gotlieb, 1997). These cells express inducible NOS and have been shown to be involved in in vitro heart valve repair (Durbin et al., 2005). The response to valve injury involves both endothelial cells as well as the underlying VICs, and some studies indicate that endothelial derived vasoactive substances inter- act with VICs promoting matrix secretion (Rabkin-Aikawa et al., 2004; Butcher and Nerem, 2006). The aim of this study was to measure the physiological presence and direct release of NO itself from porcine mitral valves. The study tests the hypotheses that (i) the basal NO release can be measured by a microelectrode sensitive to NO and can be stimulated by the addition of bradykinin, and inhibited by N-nitro-l-arginine methyl esther (l-NAME) addition and that; (ii) the same area of the mitral valve leaflet expresses eNOS both at the protein and mRNA level. Materials and Methods Tissue and buffer Porcine (sus domesticus) mitral valves were randomly collected over a period of 2 weeks post-mortem from slaughter pigs (approximately 6 months old) at a local abattoir (Danish Crown, Ringsted, Denmark). Pigs were all clinically healthy with macroscopically normal heart valves according to the Whitney classification (Whitney, 1974). Hearts were taken out approximately 20 min after the pigs were euthanased and the This work was carried out at Department of Basic Animal and Veterinary Sciences and Department of Veterinary Pathobiol- ogy, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark. www.blackwell-synergy.com J. Vet. Med. A 54, 156–160 (2007) Ó 2007 The Authors Journal compilation Ó 2007 Blackwell Verlag, Berlin ISSN 0931–184X