Opuni K, Reeves J P
Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Graduate School of Biomedical Sciences, Newark, New Jersey 07103, USA.
J Biol Chem. 2000 Jul 14;275(28):21549-54. doi: 10.1074/jbc.M003158200.
Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger were subjected to two periods of 5 and 3 min, respectively, during which the extracellular Na(+) concentration (Na(+)) was reduced to 20 mm; these intervals were separated by a 5-min recovery period at 140 mm Na(+)(o). The cytosolic Ca(2+) concentration (Ca(2+)) increased during both intervals due to Na(+)-dependent Ca(2+) influx by the exchanger. However, the peak rise in Ca(2+) during the second interval was only 26% of the first. The reduced rise in Ca(2+) was due to an inhibition of Na(+)/Ca(2+) exchange activity rather than increased Ca(2+) sequestration since the influx of Ba(2+), which is not sequestered by internal organelles, was also inhibited by a prior interval of Ca(2+) influx. Mitochondria accumulated Ca(2+) during the first interval of reduced Na(+), as determined by an increase in fluorescence of the Ca(2+)-indicating dye rhod-2, which preferentially labels mitochondria. Agents that blocked mitochondrial Ca(2+) accumulation (uncouplers, nocodazole) eliminated the observed inhibition of exchange activity during the second period of low Na(+). Conversely, diltiazem, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, increased mitochondrial Ca(2+) accumulation and also increased the inhibition of exchange activity. We conclude that Na(+)/Ca(2+) exchange activity is regulated by a feedback inhibition process linked to mitochondrial Ca(2+) accumulation.
表达牛心脏钠/钙交换体的中国仓鼠卵巢细胞分别经历了两个时期，时长分别为5分钟和3分钟，在此期间细胞外钠浓度（[Na⁺]ₒ）降至20 mM；这两个时间段之间有一个5分钟的恢复期，恢复期时[Na⁺]ₒ为140 mM。由于交换体介导的钠依赖性钙内流，在这两个时间段内细胞质钙浓度（[Ca²⁺]ᵢ）均升高。然而，第二个时间段内[Ca²⁺]ᵢ的峰值升高仅为第一个时间段的26%。[Ca²⁺]ᵢ升高幅度降低是由于钠/钙交换活性受到抑制，而非钙螯合增加，因为未被内部细胞器螯合的钡离子（Ba²⁺）内流也受到先前钙内流时间段的抑制。通过优先标记线粒体的钙指示染料罗丹明-2（rhod-2）荧光增强测定，在第一个[Na⁺]ₒ降低的时间段内线粒体积累了钙。阻断线粒体钙积累的试剂（解偶联剂、诺考达唑）消除了在第二个低[Na⁺]ₒ时间段内观察到的交换活性抑制。相反，线粒体钠/钙交换体抑制剂地尔硫卓增加了线粒体钙积累，也增强了对交换活性的抑制。我们得出结论，钠/钙交换活性受与线粒体钙积累相关的反馈抑制过程调节。
