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Mitochondrial membrane potential and hydroethidine‐monitored superoxide generation in cultured cerebellar granule cells
Author(s) -
Budd Samantha L.,
Castilho Roger F.,
Nicholls David G.
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)01088-0
Subject(s) - protonophore , superoxide , antimycin a , ethidium bromide , mitochondrion , biophysics , membrane potential , fluorescence , chemistry , depolarization , rotenone , granule (geology) , biochemistry , biology , dna , paleontology , physics , quantum mechanics , enzyme
Mitochondrial depolarisation has been reported to enhance the generation of superoxide anion (O ⋅− 2 ) in a number of cell preparations while an inhibition has been observed with isolated mitochondria. Cerebellar granule cells equilibrated with >1 μM hydroethidine (dihydroethidium) which is oxidised to the fluorescent ethidium cation by O ⋅− 2 showed a large increase in fluorescence on protonophore addition. However, controls showed the fluorescent enhancement to be a consequence of release of unbound preformed ethidium from the mitochondrial matrix within the cell with resultant fluorescent enhancement. This ambiguity was removed by the use of low (1 μM) concentrations of dye in which case generated ethidium remained bound within the mitochondria. Under these conditions antimycin A, but not protonophore addition, produced an increase in fluorescence. It is concluded that excess ethidium acts as an indicator of mitochondrial membrane potential obscuring the monitoring of O ⋅− 2 and that certain experiments employing this indicator in cells may require re‐evaluation.