Antiapoptotic effect of endothelin-1 in rat cardiomyocytes in vitro.
Apoptosis of cardiac myocytes is thought to be a feature of many pathological disorders, including congestive heart failure (CHF) and ischemic heart disease (IHD). Because recent investigations indicate that endothelin-1 (ET-1) plays an important role in CHF and IHD, we investigated the effect of ET-1 on cardiomyocyte apoptosis. The presence of apoptosis in rat cardiomyocytes (H9c2 and neonatal) was evaluated by morphological criteria, electrophoresis of DNA fragments, 4',6'-diamidine-2'-phenylindole staining, and TUNEL analysis. ET-1, but not angiotensin II, prevented apoptosis induced by serum deprivation via ETA receptors in a dose-dependent manner (1 to 100 nmol/L). ET-1 also prevented cytochrome c release from mitochondria to the cytosol. The use of specific pharmacological inhibitors demonstrated that the antiapoptotic effect of ET-1 was mediated through a tyrosine kinase pathway (genistein and AG490) but not through protein kinase C (PKC; calphostin C), mitogen-activated protein kinases (PD98059 and SB203580), or PKA (KT5270) pathways. Adenovirus-mediated gene transfer of kinase-inactive (KI) c-Src reversed the antiapoptotic effect of ET-1. We further investigated whether Bcl-xL, an antiapoptotic molecule, would be upregulated by using a luciferase-based reporter system. ET-1 upregulated Bcl-xL, and this upregulation was inhibited by genistein or AG490 but not by calphostin C. The experiments with KI mutants for various tyrosine kinases revealed that c-Src and Pyk2 (but not JAK1, Jak2, Syk, and Tec) are involved in ET-1-induced upregulation of Bcl-xL expression. These findings suggest that ET-1 prevents apoptosis in cardiac myocytes through the ETA receptor and the subsequent c-Src/Bcl-xL-dependent pathway.
Ogata
Yukiyo
Y
Division of Cardiovascular Medicine, Department of Medicine, Jichi Medical School, Tochigi, Japan.
Takahashi
Masafumi
M
Ueno
Shuichi
S
Takeuchi
Koichi
K
Okada
Takashi
T
Mano
Hiroyuki
H
Ookawara
Shigeo
S
Ozawa
Keiya
K
Berk
Bradford C
BC
Ikeda
Uichi
U
Shimada
Kazuyuki
K
Kobayashi
Eiji
E
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1 / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010880:Piperidines / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology; D051020:bcl-X Protein; D019061:src-Family Kinases
C494813:Bcl2l1 protein, rat; D016895:Culture Media, Serum-Free; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D010456:Peptides, Cyclic; D010880:Piperidines; D019253:Proto-Oncogene Proteins c-bcl-2; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D050796:STAT3 Transcription Factor; C494088:Stat3 protein, rat; D015534:Trans-Activators; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D051020:bcl-X Protein; C086539:BQ 788; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C072247:cyclo(Trp-Asp-Pro-Val-Leu)