Cardiovascular Pharmacology Arginine or citrulline associated with a statin stimulates nitric oxide production in bovine aortic endothelial cells Marie-Clotilde Berthe a, b, ⁎, Mélisande Bernard a , Carole Rasmusen a , Sylviane Darquy a , Luc Cynober a, c , Rémy Couderc a, b a Laboratoire de Biologie de la Nutrition, EA2498, Université Paris Descartes, France b Service de Biochimie, Hôpital Armand Trousseau, AP-HP, Paris, France c Service de Biochimie, Hôpitaux Hôtel-Dieu et Cochin, AP-HP, Paris, France abstract article info Article history: Received 10 June 2011 Received in revised form 29 July 2011 Accepted 17 August 2011 Available online 2 September 2011 Keywords: Arginine Citrulline eNOS Endothelial cell Nitric oxide Nitric oxide (NO) is an antiatherogenic vasodilator synthesized from arginine and, indirectly, from citrulline through argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). Hypercholesterolemia-induced atherosclerosis is usually treated by statins, which decrease cholesterolemia and increase endothelial NO synthase (eNOS) activity. Therefore, a treatment associating a statin with arginine or citrulline could be more efficient than statin alone. The aim of this study was to optimize NO production in bovine aortic endothelial cells (BAEC) by a combination of simvastatin with arginine or citrulline and to identify the molecular mechanisms involved. NO production was measured after stimulation of BAEC in different conditions (simvastat- in 0 to 10 μM associated with arginine or citrulline 0 to 5 mM) after 24-hour incubation. Intracellular levels of specific proteins were evaluated by Western-Blot analysis, and mRNA levels of eNOS, iNOS, caveolin-1, ASS and ASL were assessed by RT-PCR. Simvastatin co-administrated with arginine or citrul- line increased NO production, but at simvastatin 10 μM, 1 mM arginine-induced NO production was sig- nificantly (P b 0.01) higher than 1 mM citrulline-induced NO production. Simvastatin induced an increase in eNOS mRNA expression and protein levels in the presence of arginine or citrulline. ASS and ASL mRNA levels were increased by simvastatin, whereas a high substrate concentration (1 mM) strongly decreased ASL mRNA levels. Combining statin with arginine or citrulline increased NO produc- tion in endothelial cells by increasing eNOS protein levels. These results form a strong rationale to eval- uate the potential utilization of these in atherosclerosis prevention and treatment. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Nitric oxide (NO) has known antiatherogenic properties. In endo- thelial cells, NO is produced from arginine with simultaneous produc- tion of citrulline by endothelial NO synthase (eNOS). Endothelial NO decreases in atherosclerosis (Liu and Huang, 2008), leading to the endothelial dysfunction implicated in cardiovascular disease (Liu and Huang, 2008). Therefore, maintaining NO production in blood vessels represents a therapeutic target in atherosclerosis prevention (Gewaltig and Kojda, 2002). Several studies performed in animals and humans (Preli et al., 2002) have shown that L-arginine sup- plementation increases NO production and inhibited atherosclero- sis progression and leukocyte adhesion in hypercholesterolemia. The efficiency of arginine administered to produce NO stems from the fact that the CAT-1 transporter responsible for 60 to 80% of arginine transport into endothelial cells (McDonald et al., 1997) interacts directly with eNOS. Thus, this eNOS/CAT-1 interac- tion decreases the inhibitory interaction of caveolin-1 with eNOS which is activated (Li et al., 2005). Thus, NO production from arginine is tightly compartmentalized, which explains the “arginine paradox” (Husson et al., 2003; Shen et al., 2005). In addition, in endothelial cells, arginine recycling from citrulline via successive actions of argininosucci- nate synthase (ASS) and argininosuccinate lyase (ASL) is the main sub- strate source for NO production via eNOS (Shen et al., 2005) indicating a close relationship between eNOS activity and arginine recycling. Moreover, Goodwin et al. (2007) demonstrated that a decrease in ASS activity induced an 80% decrease in NO production. Indeed, Flam et al. (2001) showed that eNOS, ASS and ASL were colocalized in caveolae. Furthermore, from a therapeutic perspective, citrulline offers a number of advantages compared to arginine: better gastrointestinal tolerance (Grimble, 2007) and better whole-body bioavailability (Bode-Boger et al., 1998; Moinard et al., 2008; Tangphao et al., 1999). Statins are currently used to treat atherosclerosis in primary and secondary prevention (Vaughan et al., 2000). Cholesterol lowering is the predominant mechanism underlying the beneficial effects of statins in cardiovascular diseases (Igel et al., 2002) but several studies European Journal of Pharmacology 670 (2011) 566–570 ⁎ Corresponding author at: Hôpital Armand Trousseau, 26 avenue du Dr Arnold Netter, 75571 Paris Cedex 12, France. Tel.: +33 1 44 73 63 56; fax: +33 1 44 73 66 87. E-mail address: marie-clotilde.berthe@trs.aphp.fr (M.-C. Berthe). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.08.018 Contents lists available at SciVerse ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar