Rapid, stimulation-induced reduction of C12-resorufin in motor nerve terminals: Linkage to mitochondrial metabolism

Janet D. Talbot, John N. Barrett, Ellen F. Barrett, Gavriel David

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)807-819
Number of pages13
JournalJournal of neurochemistry
Volume105
Issue number3
DOIs
StatePublished - May 2008

Keywords

  • Calcium
  • Mitochondria
  • Mitochondrial membrane potential
  • Motor nerve terminal
  • Resazurin
  • Resorufin

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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