Ouabain binding to renal tubules of the rabbit.
It is well known that ouabain, a specific inhibitor of Na-K ATPase-dependent transport, interferes with renal tubular salt reabsorption. In this study, we employed radiochemical methods to measure the kinetics of [3H]ouabain binding to slices of rabbit renal medulla and high resolution quantitative autoradiography to determine the location and number of cellular binding sites. The kinetics obeyed a simple bimolecular reaction with an association constant of 2.86 +/- 0.63 SD x 10(3) M-1 min-1 and a dissociation constant of 1.46 x 10(-3) min-1, yielding an equilibrium binding constant of 0.51 x 10(-6) M. Binding was highly dependent upon temperature. At a concentration of 10(-6) M, the rate of accumulation between 25 degrees C and 35 degrees C exhibited a Q10 of 1.8. At 0 degree C the rate of ouabain dissociation was negligible. The specificity of binding was demonstrated with increasing potassium concentrations. At a concentration of 1 microM, 6 mM, and 50 mM K+ produced a 2.5- and 7-fold decrease, respectively, in the rate of ouabain accumulation observed at zero K+. Binding was completely inhibited by 1 mM strophanthin K. The major site of ouabain binding was the thick ascending limb; little or no binding was observed in thin limbs and collecting ducts. Moreover, binding was confined to the basolateral membranes. From autoradiographic grain density measurements, it was estimated that each cell contains over 4 x 10(6) ouabain binding sites or Na-K ATPase molecules. These results taken together with physiological and biochemical observations suggest that Na-K ATPase plays a key role in salt reabsorption by this segment.
Shaver
J L
JL
Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle 98195, USA.
Stirling
C
C
D000818:Animals; D001345:Autoradiography / Q000379:methods; D005260:Female; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D010042:Ouabain / Q000378:metabolism*; D011817:Rabbits; D014316:Tritium
D014316:Tritium; D010042:Ouabain