制备方法对大鼠肾刷状缘膜中Na⁺-H⁺交换及离子通透性的影响。

Effect of the preparation method on Na+-H+ exchange and ion permeabilities in rat renal brush-border membranes.

作者信息

Sabolić I, Burckhardt G

出版信息

Biochim Biophys Acta. 1984 May 16;772(2):140-8. doi: 10.1016/0005-2736(84)90037-3.

Abstract

The delta pH-dependent quenching of Acridine orange was used to characterize Na+-H+ exchange and K+ and H+ conductances in brush-border membrane vesicles isolated by precipitation with either CaCl2 or MgCl2 from rat kidney cortex. A transmembrane pH difference of 2.5 units (inside acidic) was imposed and the initial rate of its dissipation was followed after injecting a puls of tetramethylammonium gluconate (control) or sodium or potassium gluconate. In membranes isolated by CaCl2, the Na+-H+ exchange was partially electroneutral (45% to 77% of the total exchange) and the rest was due to electrically coupled Na+ and H+ movements through conductive pathways in the membranes. In membranes prepared by MgCl2, the rate of total Na+-H+ exchange was about twice as high as that in membranes obtained by CaCl2 precipitation. However, total and electroneutral exchanges were equal indicating negligible electrically coupled Na+ and H+ movements in these membranes. K0.5 for Na+ in all preparations was in the same range, being in average 30 mM. Amiloride was a competitive inhibitor of Na+-H+ exchange in membranes obtained with both preparations; Ki values ranged between 0.1 and 0.58 mM. The rates of delta pH-dissipation with K+ gradients (+/- valinomycin) were by 50% to 150% higher in membranes prepared with CaCl2 than in membranes isolated with MgCl2 indicating much higher H+ and K+ conductances in membranes obtained with CaCl2. Therefore, the rate of Na+-H+ exchange as well as the conductances for various ions in the isolated brush-border membranes depend on membrane preparation.

摘要

用吖啶橙的δpH依赖性猝灭来表征从大鼠肾皮质用氯化钙或氯化镁沉淀分离的刷状缘膜囊泡中的Na⁺-H⁺交换以及K⁺和H⁺电导。施加2.5个单位的跨膜pH差（内部呈酸性），在注入葡萄糖酸四甲基铵（对照）或葡萄糖酸钠或葡萄糖酸钾脉冲后，跟踪其消散的初始速率。在通过氯化钙分离的膜中，Na⁺-H⁺交换部分呈电中性（占总交换的45%至77%），其余部分归因于通过膜中传导途径的电耦合Na⁺和H⁺移动。在用氯化镁制备的膜中，总Na⁺-H⁺交换速率约为通过氯化钙沉淀获得的膜中的两倍。然而，总交换和电中性交换相等，表明这些膜中电耦合的Na⁺和H⁺移动可忽略不计。所有制剂中Na⁺的K0.5处于相同范围，平均为30 mM。氨氯地平是两种制剂获得的膜中Na⁺-H⁺交换的竞争性抑制剂；Ki值在0.1至0.58 mM之间。用氯化钙制备的膜中，K⁺梯度（±缬氨霉素）引起的δpH消散速率比用氯化镁分离的膜高50%至150%，表明用氯化钙获得的膜中H⁺和K⁺电导高得多。因此，分离的刷状缘膜中Na⁺-H⁺交换速率以及各种离子的电导取决于膜的制备方法。