title: Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader.
abstract: Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the mitochondrial permeability transition (MPT) and leads to mitochondrial swelling, membrane depolarization, and release of intramitochondrial solutes. Here, our aim was to develop high-throughput assays using a fluorescence plate reader to screen potential inducers and blockers of the MPT. Isolated rat liver mitochondria (0.5 mg/ml) were incubated in multiwell plates with tetramethylrhodamine methyl ester (TMRM, 1 microM), a potential-indicating fluorophore, and Fluo-5N (1 microM), a low-affinity Ca(2+) indicator. Incubation led to mitochondrial polarization, as indicated by uncoupler-sensitive quenching of the red TMRM fluorescence. CaCl(2) (100 microM) addition led to ruthenium red-sensitive mitochondrial Ca(2+) uptake, as indicated by green Fluo-5N fluorescence. After Ca(2+) accumulation, mitochondria depolarized, released Ca(2+) into the medium, and began to swell. This swelling was monitored as a decrease in light absorbance at 620 nm. Swelling, depolarization, and Ca(2+) release were prevented by cyclosporin A (1 microM), confirming that these events represented the MPT. Measurements of Ca(2+), mitochondrial membrane potential, and swelling could be made independently from the same wells without cross interference, and all three signals could be read from every well of a 48-well plate in about 1 min. In other experiments, mitochondria were ester-loaded with carboxydichlorofluorescein (carboxy-DCF) during the isolation procedure. Release of carboxy-DCF after PT pore opening led to an unquenching of green carboxy-DCF fluorescence occurring simultaneously with swelling. By combining measurements of carboxy-DCF release, Ca(2+) uptake, membrane potential, and swelling, MPT inducers and blockers can be distinguished from uncouplers, respiratory inhibitors, and blockers of Ca(2+) uptake. This high-throughput multiwell assay is amenable for screening panels of compounds for their ability to promote or block the MPT.
authors.affiliation: Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Blattner
authors.forename: L
authors.initials: L
authors.lastname: He
authors.forename: J J
authors.initials: JJ
authors.lastname: Lemasters
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D005452:Fluoresceins; D005453:Fluorescence; D007425:Intracellular Membranes / Q000737:chemistry*; D007473:Ion Channels; D008297:Male; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000032:analysis*; D008930:Mitochondria, Liver / Q000737:chemistry*; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D008933:Mitochondrial Swelling; D010539:Permeability; D051381:Rats; D017207:Rats, Sprague-Dawley; D012235:Rhodamines
chemical_list: D005452:Fluoresceins; D007473:Ion Channels; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D012235:Rhodamines; C024098:6-carboxyfluorescein; C005358:tetramethylrhodamine; D002118:Calcium