Di Virgilio F, Fasolato C, Steinberg T H
C.N.R. Unit for the Study of the Physiology of Mitochondria, Padova, Italy.
Biochem J. 1988 Dec 15;256(3):959-63. doi: 10.1042/bj2560959.
The neuroblastoma-like cell line N2A and the pheochromocytoma-like cell line PC12 excrete about 20-25% of the intracellular fluorescent Ca2+ indicator fura-2 during 10 min of incubation at 37 degrees C. The drug probenecid, known to inhibit membrane systems for the transport of organic anions [Cunningham, Israili & Dayton (1981) Clin. Pharmacol. 6, 135-151], inhibited fura-2 excretion in both cell types. However, probenecid also had untoward effects on intracellular Ca2+ homeostasis in N2A and PC12 cells. We therefore tested the drug sulphinpyrazone, another known inhibitor of organic-anion transport systems. Sulphinpyrazone fully inhibited excretion of fura-2 at 250 microM, a concentration one order of magnitude lower than that of probenecid. At this concentration and for incubation times up to 20 min, sulphinpyrazone had no untoward effects on cell viability and metabolic functions. Fura-2 was also loaded into the cytoplasm of N2A cells by permeabilization of the plasma membrane with extracellular ATP. In this case as well, the dye was rapidly released from the cells and the efflux was blocked by sulphinpyrazone. These findings suggest that N2A and PC12 cells possess a membrane system for the transport of the free-acid form of fura-2. This transport system is probably responsible for the excretion of fura-2 from these cells. Incubation of N2A and PC12 cells with sulphinpyrazone may help overcome problems arising in the investigation of [Ca2+]i homeostasis in these cell types.
神经母细胞瘤样细胞系N2A和嗜铬细胞瘤样细胞系PC12在37℃孵育10分钟期间，会排出约20 - 25%的细胞内荧光Ca2+指示剂fura-2。已知药物丙磺舒可抑制有机阴离子转运的膜系统[坎宁安、伊斯雷利和代顿（1981年）《临床药理学》6，135 - 151]，它能抑制这两种细胞类型中fura-2的排出。然而，丙磺舒对N2A和PC12细胞内的Ca2+稳态也有不良影响。因此，我们测试了另一种已知的有机阴离子转运系统抑制剂药物磺吡酮。磺吡酮在250微摩尔浓度时能完全抑制fura-2的排出，该浓度比丙磺舒低一个数量级。在此浓度下且孵育时间长达20分钟时，磺吡酮对细胞活力和代谢功能没有不良影响。通过用细胞外ATP使质膜通透，fura-2也被加载到N2A细胞的细胞质中。同样在这种情况下，染料会迅速从细胞中释放出来，且流出被磺吡酮阻断。这些发现表明，N2A和PC12细胞拥有一个用于转运fura-2游离酸形式的膜系统。这个转运系统可能负责这些细胞中fura-2的排出。用磺吡酮孵育N2A和PC12细胞可能有助于克服在研究这些细胞类型中[Ca2+]i稳态时出现的问题。
