Clinical and zyxwvutsrqpon Experimental Pharmacology and Physiology zyxwvut (1998) 25, 908-912 OXYGEN RADICALS AND NITRIC OXIDE IN RAT MESENTERIC ISCHAEMIA-REPERFUSION: MODULATION BY L-ARGININE AND NG-NITRO-~-ARGININE METHYL ESTER zy G Haklar," C Ulukaya-Durakbasa: M Yuksel," T D&li and AS Yalpn" "Departments of Biochemistry and ?Pediatric Surgery, School of Medicine, Marmara University, istanbul, Turkey SUMMARY 1. The aims of the present study were to detect changes in superoxide anion zyxwvutsrq (02.3, nitric oxide (NO) and other reactive oxygen species (ROS) directly by measurement of chemilumin- escence (CL) and to investigate the role of L-arginine, a nitric oxide synthase (NOS) substrate, and NG-nitro-L -arginine methyl ester (L-NAME), a NOS inhibitor, together with their molecular enantiomers D-arginine and D -NAME, in a rat mesenteric ischaemia-reperfusion zyxwvuts (VR) model. 2. Seventy-nine female Wistar albino rats were divided into eight groups. The first three groups underwent sham operation; group 1 was the control group, group 2 received L-arginine and group 3 received L-NAME. Ischaemia was produced in the remaining five groups by ligation of the superior mesenteric artery for 30 min followed by 60 min reperfusion. Group zyxwvu 4 rats were control I/R rats and groups 5-8 received either L-arginine, L-NAME, D-argiuiue or D-NAME, respectively. 3. Both lumiuol and lucigenin CL was significantlyincreased in I/R groups compared with sham-operated groups. L-Arginine significantly reduced CL measurements. D-Arginine was also protective, but not as much as L-arginine. Both L zyxwvutsrq - and D-arginine had in vitro 02'--scavenging potential, as tested by the xanthine- xanthine oxidase system. NG-Nitro-L -arginine methyl ester decreased lipid peroxidation values in addition to reducing CL measurements. Nitric oxide concentrations were significantly increased in zyxwvutsr UR groups in comparison with sham-operated groups. Peroxynitrite formation was increased by I/R. Treatment with L-NAMEwas beneficial by reducing NO concentrations in the reperfused ileum. 4. In our I/R model, 02'-, NO and other ROS were increased. Although NOS inhibitors were effective in reducing oxidative damage, increasing NO concentrations with L-arginine was also beneficid, presumably due to the ability of L-arginine to inhibit phagocyte adherence and its radical scavenging potential. In fact, NO may have different effects in terms of tissue injury or protection depending on the concentration of oxygen and the haemodynamic state of the tissue. - Correspondence: Assistant Professor Goncagiil Haklar, Department of Biochemistry, School of Medicine, M m a r a University, 8 1326 Haydarpasa Istanbul, Turkey. Email: <ayalcin@marun.edu.tr> Received 7 October 1997; revision 25 March 1998; accepted 9 June 1998. Key words: chemiluminescence,mesenteric ischaemidreper- fusion, nitric oxide, reactive oxygen species, superoxide radical, thiobarbituric acid-reactive substances. INTRODUCTION Reactive oxygen species (ROS) are involved in various pathologies, including gastrointestinal injuries after ischaemia-reperfusion (VR) and haemorrhagic shock.' The sources and possible actions of ROS during reperfusion of the ischaemic intestine involve conversion of xanthine dehydrogenase to xanthine oxidase, which is responsible for the production of superoxide anions (Ot-) after reperfusion. In the presence of metal ions, 02'- can be further metabolized to more ROS, such as hydrogen peroxide (H202) and hydroxyl radical ('OH). Membrane lipids are particularly vulnerable to ROS and peroxi- dation of lipids is proposed to lead to the production of additional chemoattractants for phagocytes, in addition to bacterial toxins.2 Following activation, phagocytes will produce both 02'- and NO and 02'- will be converted to either hypochlorous acid, through the action of myeloperoxidase, or to other ROS through metal-catalysed reactions. Reactive oxygen species are known to be responsible for disturbances of intestinal barrier function and mucosal permeability.' Nitric oxide (NO) is endogenously produced by a variety of mam- malian cells. It is an important regulator of intestinal blood flow and mucosal barrier function. Moreover, it inhibits phagocyte adherence to the vessel walL3 Several studies have shown NO to be protective against VR by acting as a vasodilator and anti-aggregat~r.~~' Other studies suggest that NO can react with 02'- and can contribute to VR injury via the production of peroxynitrite.6 In view of this con- troversy, the first aim of the present study was to directly detect changes in ROS, including 02'- and NO, in ileal tissue by measuring chemiluminescence (CL). The second aim of the present study was to investigate the protective roles of L-arginine, a sub- strate for nitric oxide synthase (NOS), and @-nitro+ -arginine methyl ester (L-NAME), an inhibitor of NOS, together with their molecular enantiomers D-arginine and D-NAME, in a rat mesenteric I/R model. METHODS Lucigenin (bis-N-methylacridiniumnitrate), luminol(5-amino-2,3-dihydro- 1,4-phthalazinedione), HEPES, L-arginine, D-arginine, xanthine, xanthine oxidase (grade I from butter milk), sulphanilic acid and N-(I-naphthy1)- ethylene diamine were all obtained from Sigma Chemical Co. (St Louis, MO, USA). Both L-NAME and D-NAME were obtained from Bachem (CA, USA). Carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-l-oxyl-3-oxide