Distribution of ouabain-binding sites along the dog nephron

To quantify the relative amount of ouabain bound to different segments of the nephron after in vivo injection of the drug, an autoradiographic (ARG) study was carried out. After intrarenal injection of [3H]ouabain (120 nmol kg-1 body wt, 0.9-1.2 Ci mol-1) to intact kidneys of three anaesthetized dogs, 69-89% of renal Na,K-ATPase activity was inhibited. Sodium reabsorption decreased by 21-54%. Sections for ARG were obtained from tissue slices frozen in liquid Freon, freeze-dried and embedded in resin. Almost no loss of activity occurred during processing and background activity was negligible after 23-36 days' exposure. The density of [3H]ouabain grains per mu 2 of tubular walls was 3.8 times higher over medullary ascending limbs of Henle's loop (MAL) and distal cortical tubules (DT) as compared to proximal tubules (PT). In terms of tubular length, the grain density of MAL exceeded that of PT by merely 35% since the cross-sectional area of the MAL was only 25% of that of PT. In DT, grain density in terms of tubular length was lower than in PT by 10%. Based on previous estimate of the absolute ouabain-binding capacity in MAL of 60 fmol mm-1 tubule, the ouabain-binding capacity in PT and DT would equal 45 and 40 fmol mm-1, respectively. From composite microphotographs, the relative volume of PT was estimated to be 42% of the total renal volume. This means that 47% of the total renal ouabain-binding sites are localized to PT, whereas MAL and DT together contain 51%.