| Research Abstract |
Mitochondrial K_<ATP> channel (mitoK_<ATP>) has been implicated as end effectors and/or triggers of ischemic preconditioning (IP). Although a mitoK_<ATP> opener, diazoxide, mimics IP, the mechanisms for the cardioprotective action remain unclear. We measured Ca^<2+> transients (CaT) and mitochondrial inner membrane potential (Δφ_m) with confocal microscopy and fluorescent probes, fluo-4 and tetramethylrhodamine ethyl ester perchlorate (TMRE), in rat ventricular myocytes. Diazoxide increased the amplitudes and diastolic levels of CaT dose-dependently. The effects of diazoxide on CaT were inhibited by the mitoK_<ATP> antagonist, sodium 5-hydroxydecanoic acid (5-HD ; 100μmol/L) whereas the application of diazoxide caused little change in Δφ_m. After SR function was disabled with ryanodine and thapsigargin, the effects of diazoxide on CaT were still observed. The opening of the mitochondrial permeability transition pore (mPTP) was monitored with fluorescent calcein. Diazoxide accelerated the leakage of calcein from mitochondrial matrix (16 % of control, P<0.05), and this effect was inhibited by cyclosporin A (CsA ; 2 μmol/L). CsA also abolished the effect of diazoxide on CaT. Diazoxide oxidized flavoprotein fluorescence reversibly and this effect was partially blunted by CsA (by 24%, P<0.05). We conclude that in rat ventricular myocytes, diazoxide modulates the opening of mPTP, resulting in the increase in CaT, independently of the changes in Δφ_m. The action of diazoxide on mPTP also affects mitochondrial redox state.
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