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Title Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron
Creator/Author Cheval, L. ; Doucet, A. (Centre National de la Recherche Scientifique, Paris (France))
Publication Date1990 Jul 01
OSTI IdentifierOSTI ID: 6532528
Other Number(s)ISSN0002-9513; CODEN: AJPHA
Resource TypeJournal Article
Resource RelationAmerican Journal of Physiology ; Vol/Issue: 259
Subject550201 -- Biochemistry-- Tracer Techniques; ATP-ASE-- BIOCHEMICAL REACTION KINETICS;RUBIDIUM-- MEMBRANE TRANSPORT; BIOLOGICAL MARKERS;CELL MEMBRANES;ENZYME ACTIVITY;NERVE CELLS;POTASSIUM;RATS;RUBIDIUM 86;SODIUM;TRACER TECHNIQUES;TUBULES
Related SubjectACID ANHYDRASES;ALKALI METAL ISOTOPES;ALKALI METALS;ANIMAL CELLS;ANIMALS;BETA DECAY RADIOISOTOPES;BETA-MINUS DECAY RADIOISOTOPES;BODY;CELL CONSTITUENTS;DAYS LIVING RADIOISOTOPES;ELEMENTS;ENZYMES;HYDROLASES;INTERMEDIATE MASS NUCLEI;ISOMERIC TRANSITION ISOTOPES;ISOTOPE APPLICATIONS;ISOTOPES;KIDNEYS;KINETICS;MAMMALS;MEMBRANES;METALS;MINUTES LIVING RADIOISOTOPES;NUCLEI;ODD-ODD NUCLEI;ORGANS;PHOSPHOHYDROLASES;RADIOISOTOPES;REACTION KINETICS;RODENTS;RUBIDIUM ISOTOPES;SOMATIC CELLS;VERTEBRATES
Description/Abstract 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.
Country of PublicationUnited States
LanguageEnglish
FormatPages: F111-F121
System Entry Date2001 May 13

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