3Hbumetanide binding to membranes isolated from dog kidney outer medulla. Relationship to the Na,K,Cl co-transport system.

We have synthesized the radiolabeled "loop" diuretics 3Hbumetanide and 3Hbenzmetanide (3-benzylamino-4-phenoxy-5-sulfamoylbenzoic acid) and have tested their potential as reversible labels of the Na,K,Cl co-transport system. These compounds bind with high affinity (Kd less than or equal to 30 nM, under optimal conditions) to membranes isolated from dog kidney; we found approximately 2 pmol/mg of sites in crude membranes from the outer medulla, and less than or equal to 0.5 pmol/mg in a similar preparation from kidney cortex. On sucrose gradient centrifugation, a peak of 3Hbumetanide binding activity (30 pmol/mg) is obtained at 37% (w/v) sucrose, distinct from the basolateral membranes in outer medulla and from brush borders in proximal tubule; our hypothesis is that this peak contains luminal membranes from the thick ascending limb of the loop of Henle. 3HBumetanide is displaced from its binding sites by various unlabeled loop diuretics at concentrations that have previously been shown to inhibit co-transport. Na+, K+, and Cl- (K1/2 congruent to 2, 1, and 1 mM, respectively) are required for 3Hbumetanide binding, and Cl- inhibits at higher concentrations. We interpret these data to demonstrate that the Na,K,Cl cotransport system is the site involved in 3Hbumetanide binding in kidney membranes.