114 INTRODUCTION Prolonged exposure of mammals to cold induces increases in heat production and enhances tolerance to cold through both shivering and non-shivering thermogenesis (NST) (Himms-Hagen, 1976). Shivering thermogenesis, achieved by increased skeletal muscle activity, occurs early in response to cold exposure (Jansky and Hart, 1968). A later decrease in shivering thermogenesis has been found to be accompanied by an increase in NST (Griggio, 1982). NST is achieved by the uncoupling of oxidative metabolism from ATP production, primarily in brown adipose tissue (Jansky, 1966; Foster and Frydman, 1979). In contrast to the clear role of skeletal muscle in shivering thermogenesis, the extent of NST in this tissue remains controversial, with some reports demonstrating NST in the skeletal muscle of pigeons (Skulachev and Maslov, 1960), ducklings (Barre et al., 1987) and rats (Mollica et al., 2005), whilst others found no NST in this tissue (Golozoubova et al., 2001). However, skeletal muscle, which represents a large percentage of body mass, significantly contributes to the intensification of metabolic activity of the whole organism during thermogenesis (Rolfe and Brand, 1996) due to its huge capacity for -oxidation and the oxidative capacity of mitochondria (Hoppeler and Fluck, 2003). Heat production increases metabolic rate and oxygen consumption in metabolically active tissues (Shiota and Masumi, 1988) that is implacably associated with the elevation of reactive oxygen species generation. Cellular homeostasis under conditions of increased reactive oxygen species production is achieved by a proportional increase in tissue antioxidative defence (AD) (Halliwell and Gutteridge, 1990; Buzadzic et al., 1997; Buzadzic et al., 1999; Korac and Buzadzic, 2001; Petrovic et al., 2006). AD consists of enzymes – copper, zinc and manganese superoxide dismutase (CuZnSOD and MnSOD, EC·1.15.1.1), catalase (CAT, EC·1.11.1.6), glutathione peroxidase (GSH-Px, EC·1.11.1.9), glutathione S-transferase (GST, EC·2.5.1.18), thioredoxin reductase (TR, EC·1.6.4.5), glutathione reductase (GR, EC·1.6.4.2) and low molecular mass antioxidants such as vitamins E and C, glutathione (GSH), etc. (Chance et al., 1979; Cadenas et al., 1989; Aruoma, 1996). In recent years, considerable progress has been achieved in the field of redox regulation and the concept of the protective effect of AD was extended to its regulatory role, since AD activity determines reactive species availability (Mugge et al., 1991; Korac and Buzadzic, 2000; Buzadzic et al., 2006). Reactive species in moderate concentrations, especially superoxide anion radical (O 2 · – ) and nitric oxide (NO), play an important role as regulatory mediators in biological processes (Dröge, 2002). In skeletal muscle, NO is produced by the activity of constitutively expressed endothelial and neuronal NO synthase SUMMARY Early in cold acclimation (1–7·days), heat is produced by shivering, while late in cold acclimation (12–45·days), skeletal muscle contributes to thermogenesis by tissue metabolism other than contractions. Given that both thermogenic phases augment skeletal muscle aerobic power and reactive species production, we aimed in this study to examine possible changes in skeletal muscle antioxidative defence (AD) during early and late cold acclimation with special emphasis on the influence of the L- arginine/nitric oxide (NO)-producing pathway on the modulation of AD in this tissue. Adult Mill Hill hybrid hooded rat males were divided into two main groups: a control group, which was kept at room temperature (22±1°C), and a group maintained at 4±1°C for 45·days. The cold-acclimated group was divided into three subgroups: untreated, L-arginine treated and N  -nitro-L-arginine methyl ester (L-NAME) treated. The AD parameters were determined in the gastrocnemius muscle on day 1, 3, 7, 12, 21 and 45 of cold acclimation. The results showed an improvement of skeletal muscle AD in both early and late cold acclimation. Clear phase- dependent changes were seen only in copper, zinc superoxide dismutase activity, which was increased in early cold acclimation but returned to the control level in late acclimation. In contrast, there were no phase-dependent changes in manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase, the activities of which were increased during the whole cold exposure, indicating their engagement in both thermogenic phases. L-Arginine in early cold acclimation accelerated the cold-induced AD response, while in the late phase it sustained increases achieved in the early period. L-NAME affected both early and late acclimation through attenuation and a decrease in the AD response. These data strongly suggest the involvement of the L-arginine/NO pathway in the modulation of skeletal muscle AD. Key words: skeletal muscle, antioxidative defence, nitric oxide, cold. The Journal of Experimental Biology 211, 114-120 Published by The Company of Biologists 2008 doi:10.1242/jeb.012674 Antioxidative defence alterations in skeletal muscle during prolonged acclimation to cold: role of L-arginine/NO-producing pathway Vesna Petrovic 1 , Biljana Buzadzic 1 , Aleksandra Korac 2 , Ana Vasilijevic 1 , Aleksandra Jankovic 1 , Ksenija Micunovic 2 and Bato Korac 1, * 1 Department of Physiology, Institute for Biological Research ʻSinisa Stankovicʼ, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia and 2 Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia *Author for correspondence (e-mail: koracb@ibiss.bg.ac.yu) Accepted 18 October 2007