Abstract
Cocaine induces apoptosis in fetal rat myocardial cells (FRMCs). However, the mechanisms are not clear. The present study examined the role of p38 mitogen-activated protein kinase (MAPK) and cytochrome c release in the cocaine-induced apoptosis in primary culture of FRMCs prepared from the fetal heart of gestational age of 21 days. Cocaine induced time-dependent, concurrent increases in cytochrome c release and activities of caspase-9 and caspase-3, which preceded apoptosis. Caspase-8 was not activated. In accordance, cyclosporin A and the inhibitors of caspase-9 and caspase-3 inhibited cocaine-induced caspase activation and apoptosis. Cocaine stimulated a transient increase in the p38 MAPK activity at a time point of 15 min but reduced the extracellular signal-regulated kinase (ERK) activity at 5 and 15 min in FRMCs. The p38α MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] inhibited cocaine-induced activation of caspases and apoptosis. In contrast, the p38β MAPK and mitogen-activated protein kinase kinase/ERK inhibitors SB 202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] and PD98059 (2′-amino-3′-methoxyflavone), respectively, increased apoptosis in the absence of cocaine and potentiated cocaine-induced apoptosis. Consistent with its inhibition of apoptosis, SB203580 inhibited cocaine-induced cytochrome c release and activation of caspase-9 and caspase-3. In addition, cocaine induced a decrease in Bcl-2 protein levels, with no effect on Bax levels. The cocaine-mediated reduction of Bcl-2 levels was not affected with SB203580 and the caspase inhibitors. The results suggest that in FRMCs, p38α MAPK plays an important role in the cocaine-induced apoptosis by promoting cytochrome c release, downstream or independent of Bcl-2 protein-mediated regulation. In contrast, p38β MAPK and ERK protect fetal myocardial cells against apoptosis.
Footnotes
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This work was supported in part by National Institutes of Health Grants HL67745, HL57787, and HD31226 and by the Loma Linda University School of Medicine.
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doi:10.1124/jpet.104.073494.
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ABBREVIATIONS: FRMC, fetal rat myocardial cell; MAPK, mitogen-activated protein kinase; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; ERK, extracellular signal-regulated kinase; Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-CHO; Z-LEHD-FMK, N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethyl ketone; SB 202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole; PD98059, 2′-amino-3′-methoxyflavone.
- Received June 30, 2004.
- Accepted September 13, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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