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EXPERIMENTAL STUDY

Amlodipine inhibits doxorubicin-induced apoptosis in neonatal rat cardiac myocytes

Satoshi Yamanaka, MD^*, Tetsuya Tatsumi, MD, PhD^*,*, Jun Shiraishi, MD^*, Akiko Mano, MD^*, Natsuya Keira, MD^*, Satoaki Matoba, MD, PhD^*, Jun Asayama, MD, PhD, Shinji Fushiki, MD, PhD, Henry Fliss, PhD and Masao Nakagawa, MD, PhD^*

^* Second Department of Medicine, Kyoto, Japan
Department of Dynamic Pathology, Kyoto Prefectural University of Medicine, Kyoto, Japan
Department of Clinical Pharmacology, Kyoto Pharmaceutical University, Kyoto, Japan
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada

Manuscript received December 3, 2001; revised manuscript received July 17, 2002, accepted October 17, 2002.

^* Reprint requests and correspondence: Dr. Tetsuya Tatsumi, Second Department of Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.
tatsumi{at}koto.kpu-m.ac.jp

OBJECTIVES: 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.

Abbreviations and Acronyms   ATP   adenosine 5'-triphosphate   Bax   Bcl-2–associated X protein   DCF   2',7'-dichlorofluorescein   DMEM   Dulbecco’s modified Eagle’s medium   FITC   fluorescein isothiocyanate   H2DCFDA   2',7'-dichlorofluorescin diacetate   JC-1   5,5',6,6'-tetrachloro-1,1',3'tetraethylbenzimidazolylcarbocyanine iodide   MAPK   mitogen-activated protein kinase   NAC   N-acetylcysteine   PBS   phosphate-buffered saline   SOD   superoxide dismutase

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