Cheval L, Doucet A
Laboratoire de Physiologie Cellulaire Collège de France Unité de Recherche Associée 219, Centre National de la Recherche Scientifique, Paris.
Am J Physiol. 1990 Jul;259(1 Pt 2):F111-21. doi: 10.1152/ajprenal.1990.259.1.F111.
To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5-30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20-30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.
为了测定大鼠肾单位中钠钾ATP酶的功能速率，开发了一种微量方法，用于测量从经胶原酶处理的肾脏中显微解剖出的单个肾单位节段对铷的摄取速率。由于钠钾ATP酶的水解活性对钾离子和铷离子表现出相同的表观亲和力，而钾离子引发的最大反应速度高于铷离子存在时的最大反应速度，因此实验在冷铷加86铷存在的情况下进行。在测定前，将肾小管在37℃预孵育10分钟，以恢复正常的跨膜阳离子梯度。通过在含Ba2+的冰冷氯化胆碱溶液中冲洗肾小管以洗去细胞外阳离子后，测量86铷的摄取量。在髓袢升支粗段，铷摄取量在孵育时间达30秒时呈准线性增加；在近端曲管中为1分钟；在集合管中为5分钟，并在5 - 30分钟后达到平衡。随着培养基中铷浓度从0.25 mM升至5 mM，铷摄取的初始速率以饱和方式增加。在髓质升支粗段，2.5 mM哇巴因和10(-5) M的代谢抑制剂羰基氰化物三氟甲氧基苯腙可使铷摄取的初始速率抑制超过90%。肾单位连续节段中钠钾ATP酶在最大反应速度时的水解活性与哇巴因敏感的铷摄取初始速率之间的相关性表明，在完整细胞中，该泵以其最大反应速度的约20 - 30%工作。通过在无铷和无钾的培养基中预孵育肾小管来增加细胞内钠浓度，可使铷摄取的初始速率增加至泵水解活性的最大反应速度。
