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

Abstract

We have synthesized the radiolabeled "loop" diuretics [3H]bumetanide and [3H]benzmetanide (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 [3H]bumetanide 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. [3H]Bumetanide 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 [3H]bumetanide 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 [3H]bumetanide binding in kidney membranes.