Tricyclic antidepressant inhibition of depolarization-induced uptake of calcium by synaptosomes from rat brain.
Tricyclic antidepressant inhibition of depolarization-induced uptake of calcium by synaptosomes from rat brain.
复制标题
三环类抗抑郁药抑制大鼠脑突触体去极化诱导的钙吸收。
DOI：
10.1016/0006-2952(85)90775-0
复制
发表时间：
1985
影响因子：
5.8
通讯作者：
Hoss,W
中科院分区：
文献类型：
--
作者：
Aronstam,RS;Hoss,W
Methods. Two buffered salt solutions were used: S buffer contained 130 mM NaCI, 5 mM KCI, 1.2 mM NazHPO4, 0.1 mM CaCI2, 10 mM glucose, and 20 mM Tris-Cl, pH 7.4. In K buffer, the NaCI was omitted and the KCI concentration was raised to 135 mM. Synaptosomes were prepared by homogenizing the forebrains (telencephalon and diencephalon with the olfactory lobes removed) of 200-250 g male Wistar rats in 10 vol. of ice-cold 0.32 M sucrose. After centrifuging at 1000g for 5 min, the supernatant fraction was centrifuged at 35,000 g for 25min and the pellet was resuspended in 0.32 M sucrose. This crude synaptosomal-mitochondrial fraction was layered over a density gradient comprised of 15 ml of 0.8 M sucrose and 15ml of 1.2 M sucrose and spun at 100,000 g for 1 hr in a swinging bucket rotor. The synaptosomes were recovered from the 0.8 to 1.2 M sucrose interface, slowly diluted with S buffer, spun at 30,000 g for 20 rain and resuspended in S buffer at a concentration of 0.4 to 0.5 mg synaptosomal protein/ml. This final suspension was allowed to sit at room temperature for 60 rain before uptake measurements were performed. To measure calcium uptake, 500/ul of S buffer (for resting uptake) or K buffer (for stimulated uptake) containing 0.1 pCi 45Ca2+(New England Nuclear) was added to synaptosomes (0.2 to 0.3 mg protein) suspended in 500pl S buffer. After an appropriate time (generally 20 sec), the samples were filtered through Whatman GF/B glass fiber filters. The filters were washed once with 2 ml of S buffer and their radioactivity content was determined by liquid scintillation counting. All uptake values were determined in triplicate. To assess the influence of various drugs on calcium uptake, the synaptosomes were preincubated with the drugs for 15 min in S buffer before the 45Ca2÷-containing influx medium was added.Results and discussion. Dilution of synaptosomes with medium containing depolarizing concentrations of K+ 45 2+ approximately doubled Ca uptake. This uptake reached 50% of the final level in about 15 sec and was 90% complete within 1 min. After 2 min, the calcium content of depolarized synaptosomes slowly decreased so that after 45 min the 45Ca2+ content of synaptosomes diluted with S or K uptake medium was the same (data not shown). K~-stimulated Ca 2÷ uptake was inhibited 50% by 3 mM Mn 2+ and 0.28 mM Co 2+(not shown). The extent of 45Ca2+ uptake at 2 min increased with K÷ concentration up to about 60 mM K. K+-stimulated uptake was linearly related to tissue concentration up to at least I mg synaptosomal protein/ml. The initial rate of K+-stimulated Ca 2" uptake was estimated by measuring 45Ca2~ uptake at 5 sec, at which time calcium uptake was 15-20% of the level reached by 1 rain. A Lineweaver-Burk plot of this data was linear (Fig. 1) and indicated a maximum rate of uptake of 0.30 nmole Ca 2÷.(mg protein)-1, sec-t and an apparent K,, of 0.37 mM Ca 2-'
