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Figure 4 Angiotensin II (Ang II) increased ET-1 gene expression via extracellular signal-regulated kinase (ERK) in a redox-sensitive manner. (A to C) Angiotensin II-induced activation of ERK, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK) was mediated by reactive oxygen species-sensitive pathway. Cells were preincubated with either the catalase (350 U/ml) or N-acetylcysteine (NAC) (10 mM) for 30 min and stimulated with Ang II (100 nM) for 30 min. Phosphorylation of ERK, JNK, or p38MAPK was detected by Western blotting using anti-phospho-ERK, phospho-JNK, and phospho-p38MAPK antibodies. Both catalase and NAC inhibited Ang II-induced activation of ERK, JNK, or p38MAPK. Phosphorylations of ERK, JNK, or p38MAPK were detected, and densitometric analyses were performed. The results are shown as mean ± SEM (n = 4 per group). (D) Angiotensin II-induced ET-1 messenger RNA was attenuated by PD98059 in cardiac fibroblasts. Cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence of PD98059 (PD; 20 M) or SB203580 (SB; 20 M), and total RNA was isolated at 30 min. (E) Angiotensin II-increased ET-1 promoter activity was inhibited by PD98059 in cardiac fibroblasts. Cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence of PD98059 (PD; 20 M) or SB203580 (SB; 20 M), and chloramphenicol acetyltransferase (CAT) activity was assayed after 24 h. (F) Angiotensin II-increased ET-1 promoter activity via Ras/Raf/ERK pathway in cardiac fibroblasts. Cells, transfected with either pSR -empty vector (5 µg), or an expression plasmid encoding the dominant negative mutant mERK, Raf301, or RasN17 (5 µg), were co-transfected with 15 µg of ET-1 promoter-CAT plasmid. Cells co-transfected with ET-1 promoter-CAT plasmid and an expression plasmid encoding MEK1 (5 µg) or RasL61 (5 µg) were used as positive controls. The results are shown as mean ± SEM (n = 3 per group). *p < 0.05 vs. control (Student t test); #p < 0.05 versus Ang II alone (analysis of variance).
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