Mitochondrial Ca2+-activated K+ channels in cardiac myocytes: a mechanism of the cardioprotective effect and modulation by protein kinase A.
BACKGROUND
The large-conductance Ca2+-activated K+ (BK(Ca)) channel in the cardiac inner mitochondrial membrane (mitoK(Ca) channel) has been shown to protect the heart against ischemic injury. However, questions about the cardioprotective mechanism and the kinase-mediated regulation of mitoK(Ca) channels remain to be answered.


RESULTS
Flavoprotein fluorescence in guinea pig ventricular myocytes was measured to assay mitoK(Ca) channel activity. The mitochondrial Ca2+ concentration ([Ca2+]m) and membrane potential (DeltaPsi(m)) were measured by loading cells with rhod-2 and JC-1, respectively. Cell death was assessed by trypan blue permeability. The BK(Ca) channel opener NS1619 reversibly increased the flavoprotein oxidation in a concentration-dependent manner. NS1619 (30 micromol/L) attenuated the ouabain (1 mmol/L)-induced elevation of [Ca2+]m with accompanying depolarization of DeltaPsi(m). These effects of NS1619 were completely antagonized by the BK(Ca) channel blocker paxilline (2 micromol/L) but not by the mitochondrial ATP-sensitive K+ (mitoK(ATP)) channel blocker 5-hydroxydecanoate (500 micromol/L). Paxilline, however, failed to block the oxidative effect of diazoxide (100 micromol/L), a mitoK(ATP) channel opener. The combined application of submaximally effective concentrations of NS1619 (10 micromol/L) and diazoxide (30 micromol/L) produced additive effects. NS1619 (30 micromol/L) blunted the rate of cell death during exposure to ouabain; this cardioprotective effect was prevented by paxilline. Activation of cAMP-dependent protein kinase by 8-bromoadenosine 3'5'-cyclic monophosphate (0.5 mmol/L) and forskolin (10 micromol/L) potentiated the NS1619-induced flavoprotein oxidation.


CONCLUSIONS
Opening of mitoK(Ca) channels, which is modulated by cAMP-dependent protein kinase, depolarizes the DeltaPsi(m) and attenuates the mitochondrial Ca2+ overload. Our study further indicates that mitoK(Ca) channel activation confers cardioprotection in a manner similar to but independent of mitoK(ATP) channel activation.
Sato
Toshiaki
T
Department of Pharmacology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. tsato@faculty.chiba-u.jp
Saito
Tomoaki
T
Saegusa
Noriko
N
Nakaya
Haruaki
H
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles / Q000493:pharmacokinetics / Q000494:pharmacology; D002232:Carbocyanines / Q000493:pharmacokinetics; D016923:Cell Death; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D003652:Decanoic Acids / Q000494:pharmacology; D003981:Diazoxide / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006168:Guinea Pigs; D006352:Heart Ventricles; D006575:Heterocyclic Compounds, 3-Ring; D006880:Hydroxy Acids / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects / Q000502:physiology*; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001562:Benzimidazoles; D002232:Carbocyanines; D003652:Decanoic Acids; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D006880:Hydroxy Acids; D007211:Indoles; D024681:Potassium Channels, Calcium-Activated; C068483:rhod-2; C086491:NS 1619; D005576:Colforsin; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D015124:8-Bromo Cyclic Adenosine Monophosphate; C048220:paxilline; D010042:Ouabain; C052853:5-hydroxydecanoic acid; D017868:Cyclic AMP-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D003981:Diazoxide; D011188:Potassium