Amlodipine inhibits doxorubicin-induced apoptosis in neonatal rat cardiac myocytes.
OBJECTIVE
We examined whether amlodipine, a calcium channel antagonist with potent antioxidant activity, inhibits doxorubicin-induced apoptosis in cultured neonatal rat cardiac myocytes.


BACKGROUND
Recent studies have shown that doxorubicin induces apoptosis as well as necrosis in myocytes through generation of reactive oxygen species.


METHODS
The effects of amlodipine and several other antioxidants on doxorubicin-induced oxidative stress and mitochondria-mediated apoptosis were examined.


RESULTS
Treatment of myocytes with doxorubicin (10(-6) mol/l) for 14 h increased the number of cells with elevated peroxides, as histochemically estimated by 2',7'-dichlorofluorescin (DCF) diacetate, and the percentage of apoptotic myocytes, as estimated by Hoechst 33258 nuclear staining, compared with control myocytes (25.0 +/- 1.6% vs. 5.2 +/- 1.2%). Moreover, doxorubicin-induced myocyte apoptosis was also confirmed by annexin V-fluorescein isothiocyanate binding assay. Doxorubicin induced a reduction in myocyte adenosine 5'-triphosphate content, a loss of mitochondrial membrane potential, cytochrome c release from the mitochondria into the cytosol, and caspase-3 activation to 1.9-fold of control. Amlodipine significantly attenuated increased DCF fluorescence, inhibited the mitochondria-mediated apoptotic responses described earlier, and decreased apoptosis in the doxorubicin-treated myocytes in a dose-dependent fashion. Amlodipine at 10(-6) mol/l significantly decreased apoptosis to 15.4 +/- 0.7%, and this antiapoptotic action was more effective than that seen with other antioxidants, including probucol, ascorbic acid, and alpha-tocopherol. In contrast, the calcium channel antagonist nifedipine (10(-6) mol/l) did not inhibit apoptosis. Catalase, glutathione, and N-acetylcysteine, but not mannitol or superoxide dismutase, significantly decreased DCF fluorescence and attenuated myocyte apoptosis induced by doxorubicin to 18.7 +/- 1.2%, 19.1 +/- 1.7%, and 18.7 +/- 0.6%, respectively.


CONCLUSIONS
Amlodipine significantly inhibits doxorubicin-induced myocyte apoptosis by suppressing the mitochondrial apoptotic pathway. This effect is attributed to the antioxidant properties of amlodipine, affecting mainly hydrogen peroxide.
Yamanaka
Satoshi
S
Second Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Tatsumi
Tetsuya
T
Shiraishi
Jun
J
Mano
Akiko
A
Keira
Natsuya
N
Matoba
Satoaki
S
Asayama
Jun
J
Fushiki
Shinji
S
Fliss
Henry
H
Nakagawa
Masao
M
D017311:Amlodipine / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004317:Doxorubicin / Q000494:pharmacology*; D004347:Drug Interactions; D023421:Models, Animal; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000502:physiology; D018384:Oxidative Stress / Q000187:drug effects; D011336:Probability; D051381:Rats; D012680:Sensitivity and Specificity
D017311:Amlodipine; D004317:Doxorubicin