The estimation of free calcium within synaptosomes and mitochondria with fura-2; comparison to quin-2.
The utility of the acetoxymethyl esters of two tetracarboxylic acids, fura-2 and quin-2, in the determination of ionic calcium levels within synaptosomes and mitochondria was compared. Synaptosomes and isolated mitochondria both accumulated the esters but mitochondria had a much more limited capacity to hydrolyze them. Dye-loaded synaptosomes maintain their external membrane potential of magnitude similar to values for unloaded controls and do not accumulate radioactive Ca(2+) in excess with time. Both fluorescent compounds yielded similar values (about 300-400 nM) for free intrasynaptosomal calcium [Ca(2+)](i). Mitochondrial Ca(2+) could be measured only with fura-2. Isolated mitochondria contained 0.9-1 ?M free Ca(2+) in a similar extrasynaptosomal medium. Fura-2 tended to overestimate [Ca(2+)](i) while quin-2 tended to underestimate it due to chelation of these dyes with intrasynaptosomal trace elements. Fura-2 requiring the use of two excitation wavelengths was significantly superior to the single wavelength method using quin-2. Advantages included reduced danger of erroneous readings due to (i) synaptosomal sedimentation, (ii) photobleaching of the dye, (iii) underestimation of intrasynaptosomal calcium during correction for dye leakage by manganese entry into synaptosomes. Fura-2 interfered less with synaptosomal Ca(2+) transients than quin-2, probably due to lower intrasynaptosomal concentration of dye needed. Both unstimulated and K(+)-stimulated (45)Ca(2+) uptake were increased in quin-2-loaded synaptosomes but only K(+)-stimulated uptake in fura-2 loaded ones. This series of advantages makes fura-2 of superior utility in the determination of free intrasynaptosomal calcium.
Komulainen
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Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
Bondy
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SC