title: Stimulation of oxidative phosphorylation by calcium in cardiac  mitochondria is not influenced by cAMP and PKA activity.
abstract: Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 μM free Ca2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca2 + increased the reduction of NADH (8%), and of cytochromes bH (3%), c1 (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 μM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 μM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca2 + stimulation of oxidative phosphorylation
authors.affiliation: Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Dr, Room B1D416, Bethesda, MD 20892, USA. Electronic address: raul.coviangarcia@nih.gov.
authors.forename: Raul
authors.initials: R
authors.lastname: Covian
authors.affiliation: Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Dr, Room B1D416, Bethesda, MD 20892, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: French
authors.affiliation: Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Dr, Room B1D416, Bethesda, MD 20892, USA.
authors.forename: Heather
authors.initials: H
authors.lastname: Kusnetz
authors.affiliation: Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Dr, Room B1D416, Bethesda, MD 20892, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Balaban
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D020134:Catalytic Domain / Q000187:drug effects; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003563:Cyclic CMP / Q000031:analogs & derivatives / Q000494:pharmacology; D003573:Cytochrome b Group / Q000378:metabolism; D004579:Electron Transport / Q000187:drug effects; D042967:Electron Transport Complex I / Q000378:metabolism; D014450:Electron Transport Complex III / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D010101:Oxygen Consumption / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013449:Sulfonamides / Q000494:pharmacology; D013552:Swine
chemical_list: D003573:Cytochrome b Group; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D009243:NAD; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003563:Cyclic CMP; D000244:Adenosine Diphosphate; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate; D000242:Cyclic AMP; D003576:Electron Transport Complex IV; D017868:Cyclic AMP-Dependent Protein Kinases; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium