Mitochondrial K(ATP) channel opening protects a human atrial-derived cell line by a mechanism involving free radical generation.
OBJECTIVE
The mechanism by which the mitochondrial K(ATP) channel openers confer protection against ischemia/reperfusion injury is debated. Evidence suggests that rather than solely being an end effector, opening of these channels may act by a trigger mechanism. We examined the effects of the mitochondrial K(ATP) channel opener, diazoxide on parameters of mitochondrial function with specific reference to reactive oxygen species (ROS) generation in a human atrial derived cell line model of simulated ischemia/reperfusion (LSI/R).


RESULTS
Propidium iodide (PI) exclusion was used to assess survival. Diazoxide treatment conferred protection against LSI/R (13.9+/-0.9% vs. 36.9+/-4.5% controls) that was abolished by pre-treatment with the mitoK(ATP) channel blocker, 5-hydroxydecanoate (5-HD) (33.3+/-3.6%) and with the free radical scavenger, 2-mercaptopropionylglycine (MPG) (29+/-4.0%). Diazoxide caused increased oxidation of the ROS probe, reduced mitotracker orange (1.3 vs. 1.0 arbitrary units for control; P<0.01 vs. control) that was abrogated by either 5-HD or MPG (1.07 and 1.07 arbitrary units, respectively). At the same time there was no change in orange fluorescent signal from the membrane potential sensitive probe, JC-1 indicating no change in mitochondrial membrane potential. Changes in light scattering, reflecting changes in mitochondrial volume, occurred during treatment with diazoxide.


CONCLUSIONS
These results demonstrate for the first time that the mitoK(ATP) channel opener diazoxide can act as a trigger of preconditioning by a mechanism involving mitochondrial swelling and the generation of ROS.
Carroll
R
R
The Hatter Institute and Centre for Cardiology, University College Hospital and Medical School, Grafton Way, London WC1E 6DB, UK. hatter-institute@ucl.ac.uk
Gant
V A
VA
Yellon
D M
DM
D000704:Analysis of Variance; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D002460:Cell Line; D003652:Decanoic Acids / Q000494:pharmacology; D003981:Diazoxide / Q000494:pharmacology*; D005434:Flow Cytometry; D016166:Free Radical Scavengers / Q000494:pharmacology; D006325:Heart Atria; D006801:Humans; D006880:Hydroxy Acids / Q000494:pharmacology; D015640:Ion Channel Gating; D019157:Ischemic Preconditioning, Myocardial; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015221:Potassium Channels / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D008625:Tiopronin / Q000494:pharmacology
D000889:Anti-Arrhythmia Agents; D003652:Decanoic Acids; D016166:Free Radical Scavengers; D006880:Hydroxy Acids; D015221:Potassium Channels; D017382:Reactive Oxygen Species; C052853:5-hydroxydecanoic acid; D008625:Tiopronin; D003981:Diazoxide