Rapid, stimulation-induced reduction of C12-resorufin in motor nerve terminals: linkage to mitochondrial metabolism.
The Alamar blue (resazurin) assay of cell viability monitors the irreversible reduction of non-fluorescent resazurin to fluorescent resorufin. This study focused on the reversible reduction of C12-resorufin to non-fluorescent C12-dihydroresorufin in motor nerve terminals innervating lizard intercostal muscles. Resting C12-resorufin fluorescence decreased when the activity of the mitochondrial electron transport chain (ETC) was accelerated with carbonyl cyanide m-chloro phenyl hydrazone, and increased when ETC activity was inhibited with cyanide. Trains of action potentials (50 Hz for 20-50 s), which reversibly decreased NADH fluorescence and partially depolarized the mitochondrial membrane potential, produced a reversible decrease in C12-resorufin fluorescence which had a similar time course. The stimulation-induced decrease in C12-resorufin fluorescence was blocked by inhibitors of ETC complexes I, III, and IV and by carbonyl cyanide m-chloro phenyl hydrazone, but not by inhibiting mitochondrial ATP synthesis with oligomycin. Mitochondrial depolarization and the decreases in C12-resorufin and NADH fluorescence depended on Ca2+ influx into the terminal, but not on vesicular transmitter release. These results suggest that the reversible reduction of C12-resorufin in stimulated motor nerve terminals is linked, directly or indirectly, to the reversible oxidation of NADH and to Ca(2+) influx into mitochondria, and provides an assay for rapid changes in motor terminal metabolism.
Talbot
Janet D
JD
Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, Florida 33101, USA.
Barrett
John N
JN
Barrett
Ellen F
EF
David
Gavriel
G
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D001681:Biological Assay / Q000379:methods; D020013:Calcium Signaling / Q000502:physiology; D002247:Carbon Isotopes / Q000737:chemistry; D045222:Electron Transport Chain Complex Proteins / Q000187:drug effects / Q000378:metabolism; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology*; D005453:Fluorescence; D007202:Indicators and Reagents; D008116:Lizards; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009046:Motor Neurons / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009448:Neurochemistry / Q000379:methods; D009469:Neuromuscular Junction / Q000378:metabolism*; D010078:Oxazines / Q000737:chemistry*; D010084:Oxidation-Reduction; D017729:Presynaptic Terminals / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology
D002247:Carbon Isotopes; D045222:Electron Transport Chain Complex Proteins; D007202:Indicators and Reagents; D010078:Oxazines; D014475:Uncoupling Agents; D009243:NAD; C014180:resorufin