Cu2+ and acute thermal stress induce protective eventsviathe p38-MAPK signalling pathway in the perfusedRana ridibundaheart

Abstract

SUMMARYIn the present study, we investigated the induction of the p38-MAPK signalling pathway by copper, as exemplified by CuCl2, in the isolated perfused heart of the amphibian Rana ridibunda. We found that p38-MAPK phosphorylation by CuCl2 occurs in a dose-dependent manner, with maximum activation (8.73±1.43-fold relative to control values) attained by perfusion with 500 μmol l–1CuCl2 for 15 min, while this activation sustained even after 60 min of reperfusion with normal bicarbonate buffer. CuCl2 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 CuCl2 had a synergistic effect on p38-MAPK activation. Furthermore,perfusions with the potent antioxidant L-ascorbic acid (100 μmol l–1), the antioxidant enzymes catalase (CAT) (150 U ml–1) or superoxide dismutase (SOD) (30 U ml–1) in the presence of 500 μmol l–1CuCl2 did not attenuate the CuCl2-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 CuCl2 and hyperthermia was partially inhibited by catalase, indicating that hyperthermia possibly activates the kinase through the production of H2O2. Caspase-3, an effector protease of apoptosis,remained inactive in hearts perfused at normal or hyperthermic conditions, in the absence or presence of 500 μmol l–1 CuCl2. All the above results suggest that, in the amphibian Rana ridibundaheart, p38-MAPK activation by copper has a possible protective role through the small Hsp27.