438 Introduction Mammalian cardiac muscle is an obligate aerobic organ and under anaerobic conditions cannot produce enough energy to maintain essential cellular processes; thus, a constant supply of oxygen is indispensable for sustaining cardiac function and viability (Giordano, 2005). On the other hand, ectothermic vertebrates, like amphibians, face a wide range of seasonal variations in their natural environment (Hoffman and Katz, 1997; Hutchison and Dupr, 1992), such as low oxygen pressure or temperature imbalances (Hermes-Lima and Storey, 1996; Hermes-Lima and Storey, 1998; Greenway and Storey, 2000), which are related to increased production of reactive oxygen species (ROS), while their skin is a site for gas exchange and high permeability to water (Katz et al., 1986). As a result, these organisms have developed effective antioxidant defences so as to adapt to the adverse environmental conditions (Hermes- Lima and Storey, 1996; Hermes-Lima and Storey, 1998). Copper, a heavy transition metal, can exist in both an oxidized, cupric (Cu 2+ ), or reduced, cuprous (Cu + ) state (Linder and Hazegh-Azam, 1996) and is able to move across cell membranes or through ion channels (Handy et al., 2002) into the cells. As a redox-active metal, copper plays important catalytic roles as a cofactor in many enzymes such as copper- zinc superoxide dismutase and cytochrome oxidase, which renders it an essential trace element for all aerobic organisms (Solomon and Lowery, 1993; Gaetke and Chow, 2003). At elevated cellular copper levels, rapid generation of ROS is induced via a Haber-Weiss or Fenton-like reaction (Gutteridge, 1985; Shi and Dalal, 1992; Gaetke and Chow, 2003). Interestingly, copper catalyzes the formation of hydroxyl radicals, which lead to oxidative damage of lipids, proteins and nucleic acids (Gaetke and Chow, 2003). In addition, this metal can be toxic by direct binding to protein sulfhydryl groups, which results in enzyme inactivation or altered protein conformation (Jeon et al., 2000). Recent studies have demonstrated that copper accumulates in the tissues of the In the present study, we investigated the induction of the p38-MAPK signalling pathway by copper, as exemplified by CuCl 2 , in the isolated perfused heart of the amphibian Rana ridibunda. We found that p38-MAPK phosphorylation by CuCl 2 occurs in a dose-dependent manner, with maximum activation (8.73±1.43-fold relative to control values) attained by perfusion with 500·mol·l –1 CuCl 2 for 15·min, while this activation sustained even after 60·min of reperfusion with normal bicarbonate buffer. CuCl 2 also induced the phosphorylation of the small heat shock protein 27 (Hsp27) in a p38-MAPK dependent manner, as revealed by experiments using the p38-MAPK inhibitor SB203580. p38-MAPK and Hsp27 phosphorylations were also strongly induced by hyperthermia (42°C), while the simultaneous use of hyperthermia and CuCl 2 had a synergistic effect on p38- MAPK activation. Furthermore, perfusions with the potent antioxidant L-ascorbic acid (100·mol·l –1 ), the antioxidant enzymes catalase (CAT) (150·U·ml –1 ) or superoxide dismutase (SOD) (30·U·ml –1 ) in the presence of 500·mol·l –1 CuCl 2 did not attenuate the CuCl 2 -induced p38-MAPK activation, implying that at least the reactive oxygen species (ROS) scavenged by these agents are not implicated in this kinase activation. The p38-MAPK phosphorylation induced by the combined action of CuCl 2 and hyperthermia was partially inhibited by catalase, indicating that hyperthermia possibly activates the kinase through the production of H 2 O 2 . Caspase-3, an effector protease of apoptosis, remained inactive in hearts perfused at normal or hyperthermic conditions, in the absence or presence of 500·mol·l –1 CuCl 2 . All the above results suggest that, in the amphibian Rana ridibunda heart, p38- MAPK activation by copper has a possible protective role through the small Hsp27. Key words: oxidative stress, thermal stress, copper, antioxidants, p38- MAPK, Hsp27, amphibian heart, Rana ridibunda, signal transduction. Summary The Journal of Experimental Biology 210, 438-446 Published by The Company of Biologists 2007 doi:10.1242/jeb.02680 Cu 2+ and acute thermal stress induce protective events via the p38-MAPK signalling pathway in the perfused Rana ridibunda heart Catherine Gaitanaki, Maria Pliatska, Konstantina Stathopoulou and Isidoros Beis* Department of Animal and Human Physiology, School of Biology, Faculty of Sciences, University of Athens, Panepistimioupolis, Athens 157 84, Greece *Author for correspondence (e-mail: ibeis@biol.uoa.gr) Accepted 5 December 2006 THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY