Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines.
Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines.
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DOI：
10.1054/ceca.1999.0043
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发表时间：
1999-06
期刊：
影响因子：
4
通讯作者：
S. Yagodin;N. Pivovarova;S. Andrews;D. B. Sattelle
中科院分区：
文献类型：
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作者：
S. Yagodin;N. Pivovarova;S. Andrews;D. B. Sattelle
用胞内游离钙([Ca~(2+)]i)和胞内pH(Phi)显像法结合细胞内总钙浓度的测定，研究了酸性细胞内钙储备在果蝇Schneider细胞系2(S2)细胞内钙稳态中的作用。弱碱(NH4Cl，15 mM)和Na+/H+离子载体(莫能菌素，10微米)都能引起胞浆碱化，然后从酸性的细胞内钙库中释放钙离子。用内质网Ca(2+)-ATPase抑制剂thapsigargin(1微米)或钙离子载体离子载体离子霉素(10微米)处理S2细胞，对碱化引起的钙释放幅度无影响。胆碱能激动剂氨甲酰胆碱(100微米)应用于表达果蝇M胆碱受体(DM1)的S2-DM1细胞，可排空InsP3敏感的钙库，但不影响碱化诱导的钙释放的幅度。甘氨酰-L-苯丙氨酸-β-萘胺(200微米)是一种弱疏水性碱，可通过渗透肿胀使溶酶体通透性，它能引发溶酶体内钙的释放，而应用分散高尔基复合体的抗生素布雷菲尔丁A(10微米)可使[Ca~(2+)]i的增加较小。这些结果表明，碱诱导的钙释放主要是由溶酶体引起的，直接测量S2细胞器的总钙含量证实了这一结论。溶酶体和内质网是仅有的细胞器，其总钙浓度明显高于胞浆。然而，15 mM的NH4Cl只降低了溶酶体内的总钙水平。酸性钙库的耗尽不会引起耗竭操作的钙离子进入。当细胞再次暴露于生理盐水([Ca~(2+)]o=2 mM)时，它们会被重新灌流，但不会因thapsigargin诱导的无钙生理盐水中的[Ca~(2+)]i升高而恢复。
The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.