Mitochondrial K channel opening protects a human atrial-derived ATP cell line by a mechanism involving free radical generation

Objectives: The mechanism by which the mitochondrial K channel openers confer protection against ischemia / reperfusion injury is ATP 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 channel opener, diazoxide on parameters of mitochondrial function with specific reference ATP to reactive oxygen species (ROS) generation in a human atrial derived cell line model of simulated ischemia / reperfusion (LSI /R). Methods and results: Propidium iodide (PI) exclusion was used to assess survival. Diazoxide treatment conferred protection against LSI /R (13.960.9% vs. 36.964.5% controls) that was abolished by pre-treatment with the mitoK channel blocker, 5-hydroxydecanoate ATP (5-HD) (33.363.6%) and with the free radical scavenger, 2-mercaptopropionylglycine (MPG) (2964.0%). 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. Conclusion: These results demonstrate for the first time that the mitoK channel opener diazoxide can act as a trigger of preconditioning by a mechanism involving mitochondrial swelling ATP and the generation of ROS.  2001 Elsevier Science B.V. All rights reserved.