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.

Effects of the organochlorine insecticides chlordecone, mirex, 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane on free intrasynaptosomal Ca2+ [( Ca++]i), synaptosomal 45Ca uptake and synaptosomal plasma and mitochondrial membrane potentials in vitro were studied. Chlordecone (10-50 microM) increased [Ca++]i from the resting level of 370 nM in a dose- and time-dependent manner to above 1.5 microM. This took place in the presence of 1 mM extrasynaptosomal Ca++ but not in nominally Ca++-free medium. Verapamil, a voltage sensitive Ca++ channel blocker, inhibited the initial increase of [Ca++]i caused by chlordecone, by 40%. Chlordecone also elevated [Ca++]i in synaptosomes in which mitochondrial Ca++ uptake had been abolished by valinomycin. Chlordecone depolarized partially the synaptosomal plasma membrane and, to a lesser extent, the potential of mitochondria within synaptosomes. However, chlordecone appeared to inhibit synaptosomal K+-stimulated and unstimulated 45Ca++ uptake by 20 to 30%. Inasmuch as chlordecone also stimulated release of 45Ca++ and the fluorescent dye fura-2 from preloaded synaptosomes, the apparent inhibition of uptake might be due to lysis of some synaptosomes by chlordecone. The effect of chlordecone on [Ca++]i decreased when the total amount of tissue in incubations was increased. [Ca++]i was only elevated marginally by mirex at the same concentration range. The results suggest that chlordecone increases free intrasynaptosomal Ca++ mainly by increasing influx of extrasynaptosomal Ca++. The principal mechanism appears to be a nonspecific leakage of Ca++ through the plasma membrane but some Ca++ may pass through voltage-sensitive Ca++ channels due to chlordecone-induced membrane depolarization.

The availability of techniques that allow the quantitation of levels of ionized calcium within intact cells and synaptosomes, allows a new approach to understanding the events underlying neurotoxicity. By use of various pharmacological agents, it is possible to dissect out vulnerable loci within the cell that account for the increases in cytosolic calcium which accompany many neurotoxic events. The relation between calcium and cell death can now be more concisely addressed. This article gives examples of the types of questions that this new methodology can resolve.