Abstract
6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone (cilostazol), a phosphodiesterase type 3 (PDE III) inhibitor, activates cAMP-dependent protein kinase A (PKA). The cAMP/PKA pathway potentiates the opening of mitochondrial Ca2+-activated K+ (mitoKCa) channels and confers cardioprotection. Although cilostazol has been reported to directly activate sarcolemmal large-conductance Ca2+-activated K+ channels, it remains unclear whether cilostazol modulates the opening of mitoKCa channels. Therefore, we tested the possibility that cilostazol opens mitoKCa channels and protects hearts against ischemia/reperfusion injury. Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoKCa channel activity. Infarct size in the isolated perfused rabbit hearts subjected to 30-min global ischemia and 120-min reperfusion was determined by triphenyltetrazolium chloride staining. Cilostazol (1, 3, 10, and 30 μM) oxidized flavoprotein in a concentration-dependent manner. The oxidative effect of cilostazol (10 μM) was antagonized by the mitoKCa channel blocker paxilline (2 μM). Activation of PKA by 8-bromoadenosine 3′5′-cyclic monophosphate (0.5 mM) potentiated the cilostazol-induced flavoprotein oxidation. Treatment with cilostazol (10 μM) for 10 min before ischemia significantly reduced the infarct size from 67.2 ± 1.3 (control) to 33.6 ± 5.3% (p < 0.05). This infarct size-limiting effect of cilostazol was abolished by paxilline (60.3 ± 4.9%) but not by the PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester (KT5720) (200 nM, 40.5 ± 3.5%). On the other hand, another PDE III inhibitor, milrinone (10 μM), neither oxidized flavoprotein nor reduced infarct size. Our results suggest that cilostazol exerts a cardioprotective effect via direct activation of mitoKCa channels.
Footnotes
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This study was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science, K. Watanabe Research Foundation, and the Vehicle Racing Commemoration Foundation.
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M.F. and H.Ni. contributed equally to this work.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.136218.
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ABBREVIATIONS: cilostazol, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone; PDE III, phosphodiesterase type 3; PKA, protein kinase A; mitoKca, mitochondrial Ca2+-activated K+; BKCa, sarcolemmal large-conductance Ca2+-activated K+; DNP, 2,4-dinitrophenol; LVEDP, left ventricular end-diastolic pressure; +dP/dt, positive change in pressure over time; LVDP, left ventricular developed pressure; CONT, control; CILO, cilostazol; KT5720, (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′, 1′-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester; KT, KT5720; PX, paxilline; DMSO, dimethyl sulfoxide; 8Br-cAMP, 8-bromoadenosine 3′5′-cyclic monophosphate; NS1619, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one; NO, nitric oxide.
- Received January 4, 2008.
- Accepted March 31, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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