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Endothelin-1 as a protective factor against beta-adrenergic agonist-induced apoptosis in cardiac myocytes

Makoto Araki, MD^*, Koji Hasegawa, MD^*, Eri Iwai-Kanai, MD^*, Masatoshi Fujita, MD, FACC, Tatsuya Sawamura, MD, Tsuyoshi Kakita, MD^*, Hiromichi Wada, MD^*, Tatsuya Morimoto, MD^* and Shigetake Sasayama, MD, FACC^*

^* Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan
College of Medical Technology, Kyoto University, Kyoto, Japan
National Cardiovascular Center, Osaka, Japan

View larger version (60K): [in a new window]   Figure 1 ET-1-mediated inhibition of Iso-induced apoptosis in cultured neonatal rat cardiac myocytes. TUNEL-stained myocytes. (A) In the absence of Iso or ET-1; (B and C) in the presence of Iso (10–5 mol/liter); (D) in the presence of Iso plus ET-1 (10–7 mol/liter). Terminal deoxytransferase was omitted in C. Arrows show cells with evidence of apoptosis, including chromatin condensation.

View larger version (28K): [in a new window]   Figure 2 Complete inhibition by ET-1 of Iso-induced apoptosis. Cultured cardiac myocytes were treated for 48 h in serum-free media in the presence or absence of Iso (10–5 mol/liter) and the indicated concentrations of ET-1. TUNEL-positive nuclei were counted and are expressed as the percentage of total nuclei. An average of 400 to 500 nuclei were counted from random fields in each slide. Results are the mean ± SE of three independent experiments.

View larger version (62K): [in a new window]   Figure 3 ET-1-mediated inhibition of genomic DNA fragmentation in cardiac myocytes. Genomic DNA was isolated from myocytes maintained for 48 h in serum-free media in the presence or absence of Iso (10–5 mol/liter) and ET-1 (10–7 mol/liter) as indicated and loaded on a 1.5% agarose gel. M, molecular marker.

View larger version (32K): [in a new window]   Figure 4 Effects of receptor subtype antagonist or agonist on ET-1-mediated inhibition of apoptosis. Cardiac myocytes were cultured in serum-free media in the presence or absence of Iso (10–5 mol/liter), ET-1 (10–7 mol/liter), an ETA receptor antagonist (FR139317), an ETB receptor antagonist (BQ788) or an ETB receptor agonist (IRL1620) as indicated for 48 h. The % TUNEL-positive nuclei values were calculated by counting an average of 400 to 500 nuclei in each slide. Values are mean ± SE of four independent experiments.

View larger version (30K): [in a new window]   Figure 5 Complete inhibition by ET-1 of 8-Br-cAMP-induced apoptosis. Cardiac myocytes were cultured in serum-free media in the presence or absence of 8-Br-cAMP (3 x 10–2 mol/liter) and ET-1 (10–7 mol/liter) as indicated for 48 h. The % TUNEL-positive nuclei values were calculated by counting an average of 400 to 500 nuclei in each slide. Values are mean ± SE of two independent experiments.

View larger version (32K): [in a new window]   Figure 6 The roles of MEK-1 and PI3'-kinase in the ET-1-mediated inhibition of apoptosis. Cardiac myocytes were cultured in serum-free media in the presence or absence of Iso (10–5 mol/liter), ET-1 (10–7 mol/liter), PD098059 (an MEK-1-specific inhibitor), wortmannin (PI3'-kinase inhibitor), or rapamycin (pp70 S6-kinase inhibitor) as indicated for 48 h. The % TUNEL-positive nuclei values were calculated by counting an average of 400 to 500 nuclei in each slide. Values are mean ± SE of four independent experiments.