BACKGROUND: Proton leak (H(+) leak) dissipates mitochondrial membrane potential (mΔΨ) through the re-entry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H(+) leak may affect reactive oxygen species (ROS) production. We measured H(+) leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production. MATERIALS AND METHODS: Isolated rat hearts (n = 6/group) were subjected to either control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2 mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H(+) leak is the respiratory rate required to maintain membrane potential at -150 mV in the presence of oligomycin-A. Mitochondrial complex III ROS production was measured by fluorometry using Amplex-red. RESULTS: IPC improved recovery of RPP at end reperfusion (63% ± 4% versus 21% ± 2% in control-IR, P < 0.05). Ischemia-reperfusion caused increased H(+) leak (94 ± 12 versus 31 ± 1 nmol O/mg protein/min in non-ischemic control, P < 0.05). IPC attenuates these increases (55 ± 9 nmol O/mg protein/min, P < 0.05 versus control-IR). IPC reduced mitochondrial ROS production compared with control-IR (31 ± 2 versus 40 ± 3 nmol/mg protein/min, P < 0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in control-IR for all mΔΨ and respiration rates. CONCLUSIONS: Increasing H(+) leak is not associated with decreased ROS production. IPC decreases both the magnitude of H(+) leak and ROS production after ischemia-reperfusion.
BACKGROUND: Proton leak (H(+) leak) dissipates mitochondrial membrane potential (mΔΨ) through the re-entry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H(+) leak may affect reactive oxygen species (ROS) production. We measured H(+) leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production. MATERIALS AND METHODS: Isolated rat hearts (n = 6/group) were subjected to either control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2 mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H(+) leak is the respiratory rate required to maintain membrane potential at -150 mV in the presence of oligomycin-A. Mitochondrial complex III ROS production was measured by fluorometry using Amplex-red. RESULTS: IPC improved recovery of RPP at end reperfusion (63% ± 4% versus 21% ± 2% in control-IR, P < 0.05). Ischemia-reperfusion caused increased H(+) leak (94 ± 12 versus 31 ± 1 nmol O/mg protein/min in non-ischemic control, P < 0.05). IPC attenuates these increases (55 ± 9 nmol O/mg protein/min, P < 0.05 versus control-IR). IPC reduced mitochondrial ROS production compared with control-IR (31 ± 2 versus 40 ± 3 nmol/mg protein/min, P < 0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in control-IR for all mΔΨ and respiration rates. CONCLUSIONS: Increasing H(+) leak is not associated with decreased ROS production. IPC decreases both the magnitude of H(+) leak and ROS production after ischemia-reperfusion.
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