title: Mitochondrial handling of excess Ca2+ is substrate-dependent with implications for reactive oxygen species generation.
abstract: The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca(2+) uptake. Here we sought to elucidate the effects of extramitochondrial Ca(2+) (e[Ca(2+)]) on ROS production (measured as H(2)O(2) release) from complexes I and III. Mitochondria isolated from guinea pig hearts were preincubated with increasing concentrations of CaCl(2) and then energized with the complex I substrate Na(+) pyruvate or the complex II substrate Na(+) succinate. Mitochondrial H(2)O(2) release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (ΔΨ; assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca(2+)] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial ΔΨ depolarization with succinate was accompanied by a large release in H(2)O(2) (assessed using Amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca(2+)], adding cyclosporin A to inhibit mitochondrial permeability transition pore opening restored ΔΨ and significantly decreased antimycin A-induced H(2)O(2) release. Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized ΔΨ under excess e[Ca(2+)] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane, which enhances H(2)O(2) emission from complex III during ischemia.
authors.affiliation: Department of Anesthesiology, The Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Aldakkak
authors.forename: David F
authors.initials: DF
authors.lastname: Stowe
authors.forename: Ranjan K
authors.initials: RK
authors.lastname: Dash
authors.forename: Amadou K S
authors.initials: AK
authors.lastname: Camara
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D006168:Guinea Pigs; D006861:Hydrogen Peroxide / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D002118:Calcium