Direct imaging of dehydrogenase activity within living cells using enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP).
Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q(10) of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD(+)) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.
Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1061, USA. combsc@zeus.nhlbi.nih.gov
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D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D003999:Dichloroacetic Acid / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D008956:Models, Chemical; D009206:Myocardium / Q000166:cytology; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry*; D010477:Perfusion; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D003999:Dichloroacetic Acid; D000242:Cyclic AMP; D010088:Oxidoreductases; D005969:Glutamate Dehydrogenase