Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes

Objectives: Recent evidence indicates that reactive oxygen species (ROS) ma y act as second messengers in receptor-mediated signaling pathways. The pos sible role of ROS during Et-1 stimulation in cardiomyocytes was therefore i nvestigated. Methods: Intracellular ROS levels were measured with fluoresce nce probe 2',7'-dichlorofluorescin diacetate by confocal microscopy in cult ured neonatal rat cardiomyocytes. The ROS-inducible c-fos expression was an alyzed by Northern blotting and promoter activity. Results: Et-1 applied to cardiomyocytes dose-dependently increased intracellular ROS levels. The in crease of ROS levels was attenuated by pretreating cardiomyocytes with Et-A receptor antagonist-BQ485, but not with Et-B receptor antagonist. Cardiomy ocytes pretreated with catalase or an antioxidant N-acetylcysteine (NAC) re duced Et-1-induced ROS levels. Et-1 or H2O2 treatment of cardiomyocytes rap idly induced the expression of an immediate early gene c-fos. Et-1-treated cardiomyocytes enhanced the c-fos gene expression as revealed by functional analysis using a reporter gene construct containing c-fos promoter region (-2.25 kb) and reporter gene chloramphenicol acetyltransferase. The inducti on of mRNA levels and the promoter activities of c-fos gene by Et-1 or H2O2 were abolished by pretreating cardiomyocytes with catalase or NAC.Cells tr ansiently transfected with the dominant positive mutant of p21(ras) (RasL61 ) led to a significant increase in intracellular ROS. Concomitantly, the mR NA levels and the promoter activities of c-fos were also induced. In contra st, cells transfected with the dominant negative mutant of Ras (RasN17) inh ibited Et-1-induced ROS. Consistently, the increase of c-fos mRNA levels an d promoter activities by Et-1 were also inhibited. Conclusions: These findi ngs clearly indicate that Et-1 treatment to cardiomyocytes fan induce ROS v ia Ras pathway and the increased ROS are involved in the increase of c-fos expression. Our studies thus emphasize the importance of ROS as second mess engers in Et-1-induced responses in cardiomyocytes. (C) 1999 Published by E lsevier Science B.V. All rights reserved.