A cuvette-based fluorometric analysis of mitochondrial membrane potential measured in cultured astrocyte monolayers.
Mitochondrial membrane potential (Deltapsi(M)) plays a key role in coordinating mitochondrial function and cell biology in general. In astrocytes, Deltapsi(M) is an important indicator of the health of these brain cells and their response to traumatic and hypoxic injury. We have shown previously how fluorescent signals can be measured from cells attached to a coverslip in a standard cuvette with a fluorometer and modulated using a cuvette perfusion system (Pflugers Arch-Eur. J. Physiol. 421 (1992) 400). Here we report on how this method can be employed to characterize the actions of a number of potentiometric fluorescent cationic dyes, including JC-1, Rh123 and TMRM, for their ability to monitor Deltapsi(M) in primary cultures of intact astrocytes. All dyes detected the reversible depolarization produced by brief exposure to the mitochondrial uncoupler protonophore FCCP, which short circuits and dissipates Deltapsi(M). Qualitatively similar responses were measured after treatment with either azide, an inhibitor of complex IV in the mitochondrial respiratory chain, or the oxidant H(2)O(2). Cell depolarization with high potassium modified the responses to FCCP. The time courses of these responses differed in a manner dependent on the particular dye used and in a way that correlated with expected permeation rate. The merits and pitfalls of these different potentiometric dyes for monitoring Deltapsi(M) are discussed.
Feeney
Chris J
CJ
Institute of Medical Science, University of Toronto, Ont., M5S1A8, Toronto, Canada.
Pennefather
Peter S
PS
Gyulkhandanyan
Armen V
AV
D000818:Animals; D000831:Animals, Newborn; D000968:Antimycin A / Q000031:analogs & derivatives* / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects / Q000502:physiology*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D006652:Histological Techniques; D006851:Hydrochloric Acid / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D010784:Photomicrography / Q000379:methods; D051381:Rats; D017208:Rats, Wistar; D019810:Sodium Azide / Q000494:pharmacology; D013997:Time Factors
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007476:Ionophores; C032456:antimycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000968:Antimycin A; D019810:Sodium Azide; D006861:Hydrogen Peroxide; D006851:Hydrochloric Acid