TY - JOUR
T1 - Rapid, stimulation-induced reduction of C12-resorufin in motor nerve terminals
T2 - Linkage to mitochondrial metabolism
AU - Talbot, Janet D.
AU - Barrett, John N.
AU - Barrett, Ellen F.
AU - David, Gavriel
N1 - Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2008/5
Y1 - 2008/5
N2 - 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 Ca2+ influx into mitochondria, and provides an assay for rapid changes in motor terminal metabolism.
AB - 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 Ca2+ influx into mitochondria, and provides an assay for rapid changes in motor terminal metabolism.
KW - Calcium
KW - Mitochondria
KW - Mitochondrial membrane potential
KW - Motor nerve terminal
KW - Resazurin
KW - Resorufin
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U2 - 10.1111/j.1471-4159.2007.05176.x
DO - 10.1111/j.1471-4159.2007.05176.x
M3 - Article
C2 - 18205748
AN - SCOPUS:42449122375
VL - 105
SP - 807
EP - 819
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
SN - 0022-3042
IS - 3
ER -