Lyu R M, Smith L, Smith J B
Department of Pharmacology, School of Medicine, University of Alabama, Birmingham 35294.
J Membr Biol. 1991 Oct;124(1):73-83. doi: 10.1007/BF01871366.
Kinetic properties of Na(+)-Ca2+ exchange in a renal epithelial cell line (LLC-MK2) were assessed by measuring cytosolic free Ca2+ with fura-2 and 45Ca2+ influx. Replacing external Na+ with K+ produced relatively small increases in free Ca2+ and 45Ca2+ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na+ and strongly potentiated the effect of replacing external Na+ with K+ on free Ca2+ and 45Ca2+ uptake. 45Ca2+ influx in 140 mM K+ or N-methyl-D-glucamine minus influx in 140 mM Na+ was used to quantify Na(+)-Ca2+ exchange activity of Na(+)-loaded cells. The dependence of exchange on cell Na+ was sigmoidal; the K0.5 was 26 +/- 3 mmol/liter cell water space, and the Hill coefficient was 3.1 +/- 0.2. The kinetic features of the dependence of exchange on cell Na+ partly account for the small increase in Ca2+ influx when all external Na+ is replaced by K+. Besides raising cell Na+ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg2+ was 8.2-fold lower with potassium instead of N-methyl-D-glucamine or choline as the replacement for external Na+. Potassium also increased the Vmax of exchange by 86% and had no effect on the Km for Ca2+. The exchanger does not cause detectable 22Na(+)-Mg2+ exchange and does not appear to require K+ or transport 86Rb+. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na(+)-Ca2+ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.
通过用fura - 2测量胞质游离Ca²⁺和⁴⁵Ca²⁺内流，评估了肾上皮细胞系（LLC - MK2）中Na⁺ - Ca²⁺交换的动力学特性。用K⁺替代细胞外Na⁺会使游离Ca²⁺和⁴⁵Ca²⁺摄取量相对小幅增加，除非细胞与哇巴因一起孵育。哇巴因显著增加细胞内Na⁺，并强烈增强用K⁺替代细胞外Na⁺对游离Ca²⁺和⁴⁵Ca²⁺摄取的影响。用140 mM K⁺中的⁴⁵Ca²⁺内流减去140 mM Na⁺中的内流来量化Na⁺负载细胞的Na⁺ - Ca²⁺交换活性。交换对细胞内Na⁺的依赖性呈S形；半最大效应浓度（K0.5）为26±3 mmol/升细胞水空间，希尔系数为3.1±0.2。当所有细胞外Na⁺被K⁺替代时，Ca²⁺内流的小幅增加部分可由交换对细胞内Na⁺的依赖性动力学特征来解释。除了增加细胞内Na⁺外，哇巴因似乎还激活了交换体。镁竞争性抑制交换活性。当用钾替代N - 甲基 - D - 葡糖胺或胆碱作为细胞外Na⁺的替代物时，Mg²⁺的效力低8.2倍。钾还使交换的最大反应速度（Vmax）增加86%，并且对Ca²⁺的米氏常数（Km）没有影响。该交换体不会引起可检测到的²²Na⁺ - Mg²⁺交换，似乎也不需要K⁺或转运⁸⁶Rb⁺。尽管猴肾上皮细胞中的交换活性丰富，但来自两栖动物、犬、负鼠和猪肾的其他细胞没有可检测到的交换活性。肾上皮细胞中Na⁺ - Ca²⁺交换的所有测量动力学特性与大鼠主动脉肌细胞中的交换体非常相似。
