Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron

Journal Article · Sun Jul 01 04:00:00 EDT 1990 · American Journal of Physiology; (USA)

OSTI ID:6532528

Cheval, L; Doucet, A ^[1]

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1. Centre National de la Recherche Scientifique, Paris (France)

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.

• OSTI ID: 6532528
• Journal Information: American Journal of Physiology; (USA), Journal Name: American Journal of Physiology; (USA) Vol. 259; ISSN 0002-9513; ISSN AJPHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ALKALI METAL ISOTOPES
ALKALI METALS
ANIMAL CELLS
ANIMALS
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MARKERS
BODY
CELL CONSTITUENTS
CELL MEMBRANES
DAYS LIVING RADIOISOTOPES
ELEMENTS
ENZYME ACTIVITY
ENZYMES
HYDROLASES
INTERMEDIATE MASS NUCLEI
ISOMERIC TRANSITION ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KIDNEYS
KINETICS
MAMMALS
MEMBRANE TRANSPORT
MEMBRANES
METALS
MINUTES LIVING RADIOISOTOPES
NERVE CELLS
NUCLEI
ODD-ODD NUCLEI
ORGANS
PHOSPHOHYDROLASES
POTASSIUM
RADIOISOTOPES
RATS
REACTION KINETICS
RODENTS
RUBIDIUM
RUBIDIUM 86
RUBIDIUM ISOTOPES
SODIUM
SOMATIC CELLS
TRACER TECHNIQUES
TUBULES
VERTEBRATES