Komulainen H, Bondy S C
Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
Neurochem Int. 1987;10(1):55-64. doi: 10.1016/0197-0186(87)90172-0.
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+). 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+) 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.
比较了两种四羧酸（呋喃-2和喹啉-2）的乙酰氧基甲酯在测定突触体和线粒体中离子钙水平方面的效用。突触体和分离的线粒体都能积累这些酯，但线粒体水解它们的能力要有限得多。加载染料的突触体维持其外部膜电位的幅度与未加载对照的值相似，并且不会随着时间的推移过度积累放射性Ca(2+)。两种荧光化合物测得的突触体内游离钙Ca(2+)值相似（约300 - 400 nM）。线粒体Ca(2+)只能用呋喃-2测量。在类似的突触体外培养基中，分离的线粒体含有0.9 - 1 μM游离Ca(2+)。由于这些染料与突触体内微量元素的螯合作用，呋喃-2往往会高估Ca(2+)，而喹啉-2则往往会低估它。需要使用两个激发波长的呋喃-2明显优于使用喹啉-2的单波长方法。其优点包括减少因以下原因导致错误读数的风险：(i)突触体沉降，(ii)染料光漂白，(iii)在通过锰进入突触体校正染料泄漏过程中对突触体内钙的低估。呋喃-2对突触体Ca(2+)瞬变的干扰比喹啉-2小，这可能是因为所需的突触体内染料浓度较低。在加载喹啉-2的突触体中，未刺激和K(+)刺激的(45)Ca(2+)摄取均增加，但在加载呋喃-2的突触体中只有K(+)刺激的摄取增加。这一系列优点使得呋喃-2在测定突触体内游离钙方面具有更高的效用。
